WO2013165320A1 - Treating cancer by increasing expression of socs6 - Google Patents

Abstract

The present invention relates to a method of treating cancer in a patient (e.g. a human patient) by increasing expression of SOCS6 in a cancer cell by administration of a Raf/MAPK pathway inhibitor and a PI3K pathway inhibitor.

Description

TREATING CANCER BY INCREASING EXPRESSION OF SOCS6

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of US provisional application No. 61/642,847, filed May 4, 2012, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[002] The present invention relates to the fields of medicine and biochemistry. In particular, the present invention refers to a method of treating cancer.

BACKGROUND OF THE INVENTION

[003] The Epidermal Growth Factor Receptor family (ErBB) and downstream effectors including Mitogen-Activated Protein Kinases (MAPK)/ Phosphoinositide 3- Kinases (PI3K)/ Guanine Nucleotide Exchange Factors of the Ras-like (Ral) small GTPases (RalGEFs) are considered driver mutations in a variety of human cancers. Many clinically used drugs against the receptor tyrosine kinases often encounter resistance due to downstream effector mutations or activation of alternative pathways like Akt (also known as Protein Kinase B (PKB))/mammalian target of rapamycin (mTOR), a biological phenomenon called "oncogenic shift". A better understanding of the molecular cross-talk between Epidermal Growth Factor Receptor (EGFR)/Rat sarcoma (Ras) and other oncogenic signalling pathways could serve for better clinical treatment and development of combinatorial therapeutic strategies.

[004] The Hippo signal transduction pathway plays a critical role in organ size control and tumorigenesis. This pathway was first discovered in Drosophila and the core components and mechanisms of action are highly conserved in mammals. Hippo (Hpo; MST1/2 in mammals) is a member of the Ste-20 family of protein kinases. Hpo forms a complex with Salvador (Savl; WW45 in mammals) to phosphorylate and activate the protein kinase Warts (Wts; LATS1-2 in human). Savl is a WW domain- containing protein, that is, it contains an amino acids sequence in which a tryptophan and an invariant proline are highly conserved. Savl can be phosphorylated by Hpo and this Hpo-Sav interaction promotes phosphorylation of Wts. Hpo can also phosphorylate and activate Mob as tumor suppressor (Mats; MOBKL1A/B in _ mammals). The activation of Mats by Hpo allows Mats to bind to and strengthen the kinase activity of Wts.

[005] The activation of Wts/Mats can further phosphorylate the downstream effector Yorkie (Yki; two orthologs in mammals, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)) and lead to cytoplasmic sequestration and degradation of Yorkie. Yki is a transcriptional activator that when activated binds to the transcription factor Scalloped (Sd; Transcriptional enhancer factor TEF-1 (TEAD1-4) in mammals), thereby allowing expression of many genes involved in organ growth.

[006] Loss-of-function mutant clones in Hpo, Sav, Warts or Mats or overexpression of Yorkie/Y AP can induce a strong tissue overgrowth characterized by an increased cellular proliferation and inhibition of apoptosis. Oncogenic mutations of two different Hippo pathway components have been reported. Mutations in Merlin (Mer; NF2 in mammals), an upstream component of the core HpoAVts kinase cascade, have been shown to cause Neurofibromatosis 2; Mutations in TEAD1, a downstream transcription effector plays a critical role in Sveinsson's chorioretinal atrophy. Importantly, the downstream effector YAP has been shown to be activated in a variety of cancer types, including breast cancer, colorectal cancer, lung cancer, ovarian carcinoma and liver cancer. Hence, a better understanding of the underlying mechanism of Hippo pathway in tumorigenesis is crucial for cancer therapy.

[007] Compounds which targets receptor tyrosine kinases or downstream kinases, often have limited effectiveness for late-stage patients and must be used in conjunction with other therapeutic strategies. Current cancer therapies such as surgical therapy, radiotherapy, chemotherapy, and immunotherapy have either been of limited success or have been accompanied by serious side effects with high relapse rates. However, the presence of multiple pathways that promote oncogenic transformation of mammalian cells has been an obstacle for development of efficacious anti-cancer therapy.

[008] Many times after surgery and after some delay period, the original tumor is observed to have metastasized so that secondary sites of cancer invasion have spread throughout the body and the patient subsequently dies of the secondary cancer growth. Although chemotherapy is widely used in the treatment of cancer, it is a systemic treatment based usually on the prevention of cell proliferation. Accordingly, chemotherapy is a non-specific treatment modality affecting all proliferating cells, including normal cells, leading to undesirable and often serious side effects.

[009] Thus, a need exists for new compositions and methods for treating cancer, particularly cancer with rapidly proliferating cells. A need exist for new biomarkers to determine the susceptibility of patient suffering from cancer to chemotherapeutic treatment.

SUMMARY OF THE INVENTION

[0010] In a first aspect, there is provided a method of treating cancer in a patient or a patient suspected to suffer from cancer, wherein the method comprises increasing expression of SOCS6 protein in a cancer cell by administering to the patient an effective amount of at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor.

[0011] In a second aspect, there is provided a method of determining the susceptibility of a patient suffering or suspected to suffer from cancer to a treatment with at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor, wherein the method comprises comparing mRNA level and/or protein expression level and/or miRNA level results for SOCS6 protein and/or YAP protein and/or AREG protein (Locus: NM_001657) and/or Survivin (BRIC5; Locus: NM 001012271) and/or mir-17 and/or connective tissue growth factor (CTGF; Locus: NM_001901) and/or cysteine-rich, angiogenic inducer, 61 (CYR61; Locus: NM 001554) or any combination of transcriptional targets of YAP obtained from a patient suffering or suspected to suffer from cancer with the mRNA level and/or protein expression level results of a control group, wherein an mRNA level and/or protein expression level and/or miRNA level for SOCS6 protein and/or YAP protein and/or AREG protein (Locus: NM 001657) and/or Survivin (BRIC5; Locus: NM_001012271) and/or mir-17 and/or connective tissue growth factor (CTGF; Locus: NM_001901) and/or cysteine-rich, angiogenic inducer, 61 (CYR61; Locus: NM_001554) or any combination of transcriptional targets of YAP in a patient suffering or suspected to suffer from cancer that differs from the control expression level for SOCS6 protein and/or YAP protein and/or AREG protein (Locus: NM_001657) and/or Survivin (BRIC5; Locus: NM_001012271) and/or mir-17 and/or connective tissue growth factor (CTGF; Locus: NM_001901) and/or cysteine-rich, angiogenic inducer, 61 (CYR61; Locus: NM_001554) or any combination of transcriptional targets, of YAP indicates that the patient is susceptible to a treatment comprising the at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor.

[0012] In a third aspect, there is provided a method of treating a disease selected from the group consisting of cancer in a patient, wherein the method comprises sequestering a micro-RNA capable of decreasing SOCS6 protein levels.

[0013] In a fourth aspect, there is provided a kit for use in the method disclosed herein, wherein the kit comprises means to detect SOCS6 protein levels and/or mR A levels in a sample obtained from a patient suffering or suspected to suffer from cancer.

DESCRIPTION OF DRAWINGS

[0014] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0015] Fig. 1 A is a series of confocal microscopy images of genetically engineered human foreskin fibroblast BJ cells showing synergistic function of YAP and EGFR/RAS in promoting colonigenic growth in soft agar. The various distinct genetic modifications on this cell line were carried out using retroviral integration technology. GFP was expressed in a retroviral vector encoding a fusion protein of H2B-GFP- hTERT ( GFP was fused to C-terminal of Histone H2 protein and H2B-GFP to the N- terminal of human Telomere Reverse Transcriptase). The cells were transduced with retroviral constructs to direct expression of (1) hTERT, which encodes the catalytic subunit of the telomerase to allow replicative immortality; (2) Inducible oncogenic H- Ras^V12 fused to the ligand binding domain of the Estrogen Receptor (ER-H-Ras^V12), to activate Ras signaling upon addition of 4-hydroxy-tamoxifen (40HT; 4-hydroxy-(Z)- 2-[4-(l ,2-diphenylbut-l-enyl)phenoxy]-N,N-dimethylethanamine); (3) shRNAs to deplete p53 and pl6, to overcome Ras^vl2-induced growth arrest and confer colonigenic outgrowth; (4) SV40 small T antigen, which allows anchorage- independent growth in soft agar. The transgenic cells were either transduced to express YAP cDNA or empty vector control (EV) to compare the effect of YAP overexpression on soft agar colony formation. In control cells ,with normal YAP expression, addition of either EGF (10 ng/ml) or 40HT (10^"7M) give rise to colonies ranging from 50μηι to ΙΟΟμπι in size (cells labeled by GFP, incubated for 12 days). In comparison, YAP overexpression (YAP) alone without Ras activation could give rise to colonies of similar size to cells treated with EGF alone (EV+EGF). Surprisingly, overexpression of YAP together with EGF/40HT gives rise to bigger colonies (YAP+EGF; YAP+40HT).

[0016] Fig. 1 B is a bar graph showing quantitation of colonies of genetically engineered human fibroblast grown in conditions indicated in fig. 1 A (that is the cells were either transfected with an empty plasmidic vector (EV) or a plasmid encoding YAP (YAP). Cells were then treated either with 40HT or EGF/Ras^v12. After staining with (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 1 hour at 37°C, images were captured under light microscope (IX). The images were then used to count the colonies using Matlab R12.1 (2001b) software. Two independent soft agar experiments were performed.

[0017] Fig. 2 is a bar graph showing colony quantification as in Fig. IB. Genetically engineered human fibroblasts that were transduced with an empty vector (EV) or a retroviral small (or short) hairpin RNA (shRNA) vector targeting YAP, thereby silencing YAP gene expression by RNA interference (RNAi). The cells were subsequently treated either with EGF or 40HT and the colony quantified as in Fig. IB. Two independent soft agar experiments were performed.

[0018] Fig. 3 A and B are a pair of bar graphs showing quantification of colonies of genetically engineered human fibroblasts as described in Fig.l B. Genetically engineered human fibroblasts as described above were transfected with either an empty vector or a vector encoding for YAP. 40 000 cells were seeded and treated with 40HT, together with either (Fig. 3 A) 1 ,4-diamino-2,3 dicyano-l,4-bis[2- aminophenylthio] butadiene (U0126; a selective inhibitor of MEKl/2 (also called MAPK or Erk kinases) or (Fig. 3B) 2-morpholin-4-yl-8-phenylchromen-4-one (LY294002; a highly selective inhibitor of phosphatidylinositol 3 (PI3) kinase) at 10μΜ or 30μΜ for 12 days.

[0019] Fig. 4 A is a series of image of western blots analysis of extracts from BJ cells showing protein levels of dually phosphorylated p44/42 MAPK (Erkl/2), total ERK, and YAP. BJ cells were treated with 10^"7M of 40HT (Ras^V12 ON) or without 40HT (Ras^V12 OFF) for 7 days. Cells extracts were then subjected to western blots using antibodies against Phospho-p44/42 MAPK (diphosphorylated ERK-1 and 2 (dpERK); positive control for Ras^V12 activation), total ERK (from mouse, Sigma-Aldrich), and YAP (Rabbit, Cell Signaling Technology). Antibody against tubulin (from mouse, Sigma- Aldrich) was used as a loading control.

[0020] Fig. 4 B shows a pair of bar graphs related to mRNA levels of amphiregulin (AREG) and survivin, two transcriptional targets of YAP. The graph were obtained by plotting the values obtained from quantitative real-time polymerase chain reaction (RT-PCR) performed on mRNA obtained from BJ cells treated as in Fig. 4A. The amount of mRNA was expressed mRNA fold-change using Glyceraldehyde 3- phosphate dehydrogenase (GAPDH) to normalize the Ct value. Delta Ct (ACt) value was normalized to values obtained in the cells that were not treated with 40HT, that is mRNA fold change is 1 in untreated cells.

[0021] Fig. 5 A and B show light microscopic images of colonies after MTT staining and a bar graph resulting from the quantification of colonies obtained using the images. Light microscopic images (IX) of colonies after staining with MTT for lhour at 37°C. 10,000 cells were seeded and treated with 40HT together with either empty vector control or SOCS6 shRNA-1 or shRNA-2. The lower panel shows cells without 40HT treatment as background control (Fig. 5A). Quantification of 40HT-treated colonies. Data were normalized to control vector and averages of three biological repeats are shown (Fig. 5B).

[0022] Figure 6 is a bar graph showing that Ras signaling downregulates SOCS6 expression. Quantitative real-time PCR was used to measure SOCS6 mRNA (SEQ ID No: 1) levels in the genetically engineered human BJ fibroblasts. Ct value was normalization to GAPDH and ACt value of SOCS6 from cells treated with 40HT for 7 days was normalized to cells without 40HT treatment. Average of two independent experiments.

[0023] Fig. 7 is a pair of western blots and a dot plot graph showing stability of YAP protein in BJ cells in the absence of SOCS6. Immunoblot quantification of YAP protein turnover upon cycloheximide (CHX) treatment in BJ cells transfected with either SOCS6 small interfering RNA (siR A; 10 nM) or mock transfected (control) using a protocol provided by the manufacturer (Santa Cruz, Biotechnology Inc.). In the control (left panel), YAP protein level decreased in a time-dependent manner from 0 hour to 5 hours of 10 μ^ηιΐ of CHX treatment; while in BJ cells transfected with SOCS6 siRNA, YAP protein was stabilized (right panel). The relative amount of YAP protein was quantified using image J software and normalized using the relative amount of tubulin (loading control). A resulting protein decay curve was plotted indicating stabilization of YAP protein in SOCS6 silenced cells.

[0024] Fig. 8 A and B is a pair of western blots showing that SOCS6 overexpression leads to decreased YAP protein levels (the cDNA of human YAP having a SEQ ID No: 5 and human YAP protein has a SEQ ID No: 6). H1299 lung cancer cells grown in 6-well dishes were transfected with ^g of a plasmid construct encoding for an human influenza hemagglutinin labelled SOCS6 (pcDNA3.1-HA-SOCS6; SEQ ID No: 3 encoding a protein having a SEQ ID No:4) or an empty vector (pcDNA3.1). 48 hours after transfection, the cells were harvested and lysed and the protein extracted. Western blot analysis was carried out to detect endogenous YAP protein level using an anti-YAP rabbit antibody (Santa Cruz Biotechnology, Inc.; Fig. 8 A) as well as the exogenous HA-tagged SOCS6 protein level using an anti-HA antibody (rat anti-HA from Roche Applied Science; Fig. 8B). Tubulin was used as loading control and detected using an anti-tubulin antibody (mouse anti-Tubulin, Sigma- Aldrich).

[0025] Fig. 9 is a Western Blot showing physical interaction between YAP and SOCS6 proteins. HEK293T cells were cotransfected with combinations of pcDNA3.1-HA-SOCS6 and pcDNA-c-myc-ubiquitin by calcium phosphate method. 48 hours later cells were treated with the proteasome inhibitor N- (benzyloxycarbonyl)leucinyleucinylleucinal (MG132; 20μ /ιη1) for five (5) hours. The cells were harvested and lysed, and the total protein extract isolated for immunoprecipitation experiments. Anti-YAP antibody (rabbit polyclonal, Santa Cruz) was used for the co-immunoprecipitation. 1% of input and 30% of the immunoprecipitated fraction was loaded for immunoblot analysis using either anti- HA (upper panels) or anti-YAP antibodies (lower panels).

[0026] Fig. 10 A and B is a Western Blot analysis assessing ubiquitination levels of immunopurified YAP proteins using HEK293T cells (Fig. 10A and B). HE 293T cells transfected with SOCS5 siRNA, SOCS6 siRNA or mock control (Fig. 10A) or with pcDNA3.1-SOCS5, pcDNA3.1-SOCS6 or mock (Fig. 10B) were pre-treated with MG132 for 5 hours before immunoprecipitation using rabbit polyclonal anti- YAP. 30% of immunoprecipitated beads or 1% of input lysates was subjected to immunoblot analysis using antibodies against ubiquitin or against YAP. Cells overexpressing YAP not subjected to MG132 treatment (i.e. an equal amount of the dimethyl sulfoxyde (DMSO) that was used to prepare MG132 was added to the cell culture medium) were used as control for ubiquitin detection. _.

[0027] Fig. 11 shows a pair of Western Blots analysis assessing RAS activity in SOCS6 depleted BJ cells. Genetically engineered BJ cells were transfected with either a control vector (EV) or a shRNA against SOCS6. Proteins extracted from the cells that were harvested 48 hours after transfection were subjected to immunoblot analysis using antibody against dp-ERK. An antibody against tubulin (Mouse, Sigma- Aldrich) was used as a loading control, to demonstrate that equal amounts of protein were loaded into the gel.

[0028] Fig. 12 A is a cartoon showing pairing between mir-17/mir20a and SOCS6 3'- untranslated region. Fig.12 B is a histogram plot showing the effectiveness of mir- 17/20a-mediated repression of SOCS6 expression. The SOCS6 3'UTR (untranstlated genomic sequence; SEQ ID No.: 8) was fused to the 3' end of a firefly luciferase coding sequence (the sequence of the tubulin-luciferase plasmid is given in SEQ ID No.: 7). The construct was cloned in a plasmid vector and transcription was driven by a tubulin promoter (pCasper4 with tubulin promoter; SEQ ID No.: 9). The plasmid encoding the above construct or a control construct (that is a plasmid encoding a firefly luciferase reporter gene without the SOCS6 3'-UTR) was transfected in Drosophila S2 cells (that do not express either endogenous mir-17 (SEQ ID No: 10) or mir-20 (SEQ ID No: 11) sequences) that were either expressing a control vector (empty vector) or overexpressing mir-17 or mir-20a. The luciferase activity was assessed by techniques known in the art.

[0029] Fig. 13 A and B is a pair of histogram plots showing quantitative Q-PCR analysis (A) of SOCS6 mRNA level upon mir-17 depletion in human fibroblast cells and (B) the quantitative microRNA Q-PCR data demonstrating the effectiveness of depleting mir- 17/20 expression by anti-sense oligonucleotides.

[0030] Fig 14 is a histogram plot showing miRNA expression in Ras^V12 transformed B J cells. Transformed colonies either not induced or induced by activated Ras^V12 were harvested upon 10-12 days of growing and total RNA was extracted. Exemplary miRNA levels were measured by quantitative reverse transcription PCR (qRT-PCR). Values were averaged from two independent experiments. Data were normalized to a control small RNA, U6.

[0031] Fig. 15 A is a series of light microscope images showing colonies of BJ cells after staining with MTT for 1 hour at 37°C that demonstrate that mir-17 overexpression enhances colony formation. Fig. 15 B is a histogram plot summarizing the colony formation obtained from Fig. 15 A. (A) 10,000 BJ cells cells were seeded and treated with either EGF (4(^g/ml), or 40HT (107M) in cells transduced with a retroviral vector encoding either no miRNA (that is, no miRNA is expressed when the virus transduces the cells) or mir-17 under control of a Cytomegalovirus (CMV) promoter. The left panels showing cells without any treatment as background control. (B) Quantification of EGF or 40HT-treated colonies shown in (A)

[0032] Fig 16 is a pair of boxplot graphs showing expression of SOCS6 mRNA from liver and lung cancer array sets. Expression of SOCS6 mRNA from liver and lung cancer array sets from GENT: https://medical-genome.kribb.re.kr/GENT/index.php. Y-axis, SOCS6 expression level in log2 scale. Bottom and top of the box represent 25^th & 75^th percentiles. The middle band is the median. X-axis, tissue types (Cancer vs Normal) for Affymetrix platforms U133A. Number in brackets indicates total No. of individual cancer or normal tissues analyzed. Student's T tests were performed to assess the statistical significance.

[0033] Fig. 17 is a cartoon summarizing the findings disclosed herein and depicting a regulatory positive feedback loop for cellular transformation according to those findings.

DETAILED DESCRIPTION OF THE INVENTION

[0034] Before the present compounds and methods are described, it is to be understood that this invention is not limited to particular compounds, methods and experimental conditions described herein, as such compounds, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0035] The invention is predicated, at least in part, on the surprising discovery that YAP overexpression can promote transformation of normal human cells and that the YAP oncogene and the Ras/MAP oncogenic signaling pathway are linked. The inventors made the surprising and unexpected discovery that Ras/MAPK signaling leads to elevated YAP protein levels. This contributes to RAS-induced cellular transformation. YAP was not previously known to be regulated by the RAS/MAPK pathway.

[0036] The inventors have identified the mechanism by which Ras/MAPK signaling leads to increased YAP levels. As disclosed herein, Ras/MAPK signaling reduces the levels of the SOCS6. protein. Subsequently, reduction of SOCS6 levels leads to elevated YAP protein levels. As described herein, reduced levels of SOCS6 gene expression correlates with Hepatocellular carcinoma (HCC), cirrhosis and Hepatitis C status.

[0037] The subsequent identification of specific factors involved in the Ras/YAP mediated cellular transformation and tumourigenesis, combined with bioinformatic analysis, enables the creation of a detailed molecular signature of potential candidate to increase the therapeutic effect of chemotherapeutic agent and to decrease side effects of said agent by allowing the use of lower dosage. This signature can guide the development of combination therapies that are optimally effective for a specific patient suffering from cancer. Such a molecular map has certain advantages over the more common genetic signature approach because most anti-cancer agents are small molecules that target proteins and not genes, and many small molecules targeting specific molecular alterations are currently in pharmaceutical development.

[0038] It was previously reported that EGFR/Ras are among the most frequently mutated oncogenes in a variety of human cancer types. YAP/TAZ are recently discovered oncogenic effectors of the hippo pathway involved in organ size control and tumorigenesis. Though both Ras and YAP were shown to play important roles in many solid tumors, like liver and lung cancer, there was no evidence that there is direct cross-talk between the two factors. However, as described herein, overexpression of YAP acts synergistically with Ras^V12 to promote cellular transformation. In addition, depletion of SOCS6 enhances Ras^vl2-mediated cellular transformation. The inventors surprisingly discovered YAP over accumulation as a potential effector of solid tumors, such as liver and lung cancers.

[0039] Based on the above results, the present invention provides a method for treating cancer in a patient or a patient suspected to suffer from cancer. The method may comprises increasing expression of SOCS6 protein in a cancer cell by administering to the patient an effective amount of at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, as it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure.

[0041] Units, prefixes, and symbols are denoted in their Systeme International d'Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0042] As used herein "inhibiting" or "treating" a disease refers to the following. Inhibiting the full development of a disease, disorder or condition, for example, in a subject who is at risk for a disease such as a cancer. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term "ameliorating," with reference to a disease, pathological condition or symptom, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease or condition.

[0043] As used herein the term "inhibitor" or grammatical variation thereof refers to a substance or a compound or an agent capable of delaying, slowing or preventing the activity of a gene product. For example, the present invention provides a substance capable of inhibiting gene expression of at least one gene belonging to Raf/MAPK pathway and/or the PI3K pathway to reduce the level of gene expression or capable of binding to the expression product of at least one gene belonging to Raf/MAPK pathway and/or the PI3K pathway to reduce or prevent the activity of the at least one gene product.

[0044] In the present invention, there is no special limitation on the type of the inhibitors capable of inhibiting gene expression of at least one _> gene belonging to Raf/MAPK pathway and/or the PI3K pathway or binding to the expression product of gene at least one gene belonging to Raf/MAPK pathway and/or the PI3K pathway, as long as it can silence the at least one gene expression or inhibit the function of the gene product. It is understood that the inhibitor may be a reversible, quasi-irreversible or irreversible inhibitor. The reversibility of the inhibitor may be determined by method known in the art.

[0045] In one example, the Raf/MAPK pathway inhibitor as disclosed herein includes but is not limited to a Raf kinase inhibitor, a MEK inhibitor, an Erk inhibitor and combinations of the aforementioned inhibitors and other inhibitors capable of inhibiting gene expression or binding to the expression product of gene belonging to the Raf/MAPK pathway.

[0046] In one example, the inhibitor as disclosed herein includes but is not limited to an active organic compound, a silencing oligonucleotide, a ribozyme, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), an antibody, and other inhibitors capable of inhibiting gene expression or binding to the expression product of gene belonging to aforementioned pathways.

[0047] The silencing oligonucleotide as disclosed herein include but is not limited to a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a morpholino oligomer, and a micro-RNA (miRNA) mimic. The silencing oligonucleotide of the invention is capable of inhibiting expression of a gene of interest by interfering with the expression mechanism. For example, inhibition can occur through direct or indirect binding to the genomic region of the gene of interest, or interfering with the splicing mechanism of the pre-mRNA of the gene of interest, or binding to the mRNA of the gene of interest thereby inhibiting translation to a polypeptide encoded by the gene of interest. Other contemplated mechanisms of action of silencing oligonucleotide are well known in the art.

[0048] As used herein, the term "siRNA" is meant to refer to a small inhibitory RNA duplex that induces gene silencing by operating within the RNA interference ("RNAi") pathway. These molecules can vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5 Or 3' end of the sense strand and/or the antisense strand. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.

[0049] Each siRNA can include between 17 and 31 base pairs, more preferably between 18 and 26 base pairs, and most preferably 19 and 21 base pairs. Some, but not all, siR A have unpaired overhanging nucleotides on the 5' and/or 3' end of the sense strand and/or the antisense strand. Additionally, the term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region, which may be referred to as short hairpin RN A ("shRNA").

[0050] siRNA may be divided into five (5) groups (non-functional, semi-functional, functional, highly functional, and hyper-functional) based on the level or degree of silencing that they induce in cultured cell lines. As used herein, these definitions are based on a set of conditions where the siRNA is transfected into said cell line at a concentration of ΙΟΟηΜ and the level of silencing is tested at a time of roughly 24 hours after transfection, and not exceeding 72 hours after transfection. In this context, "non-functional siRNA" are defined as those siRNA that induce less than 50% (<50%) target silencing. "Semi-functional siRNA" induce 50-79% target silencing. "Functional siRNA" are molecules that induce 80-95% gene silencing. "Highly- functional siRNA" are molecules that induce greater than 95% gene silencing. "Hyperfunctional siRNA" are a special class of molecules. For purposes of this document, hyperfunctional siRNA are defined as those molecules that: (1) induce greater than 95% silencing of a specific target when they are transfected at subnanomolar concentrations (i.e., less than one nanomolar); and/or (2) induce functional (or better) levels of silencing for greater than 96 hours. These relative functionalities (though not intended to be absolutes) may be used to compare siRNAs to a particular target for applications such as functional genomics, target identification and therapeutics.

[0051] As used herein, the terms "shRNA" or "hairpins" are meant to refer to unimolecular siRNA comprised by a sense region coupled to an antisense region through a linker region. A shRNA may have a loop as long as, for example, 4 to 30 or more nucleotides. In some embodiments it may be preferable not to include any non- nucleotides moieties. The shRNA may also comprise RNAs with stem-loop structures that contain mismatches and/or bulges, micro-RNAs, and short temporal RNAs. RNAs that comprise any of the above structures can include structures where the loops comprise nucleotides, non-nucleotides, or combinations of nucleotides and non- nucleotides. The sense strand and antisense strand of an shRNA are part of one longer molecule or, in the case of fractured hairpins, two (or more) molecules that form a fractured hairpin structure. [0052] As used herein the term "morpholino oligomer" refers to a polymeric molecule having a backbone which supports bases capable of hydrogen bonding to typical polynucleotides, wherein the polymer lacks a pentose sugar backbone moiety, and more specifically a ribose backbone linked by phosphodiester bonds which is typical of nucleotides and nucleosides, but instead contains a ring nitrogen with coupling through the ring nitrogen. A morpholino oligomer is composed of "morpholino subunit" structures, such as shown below, which in the oligomer are preferably linked together by phosphoramidate or phosphorodiamidate linkages, or their thio analogs, joining the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit. Each subunit includes a purine or pyrimidine base- pairing moiety Pi which is effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.

[0053] The term "phosphorodiamidate" group as used herein comprises phosphorus having two attached oxygen atoms and two attached nitrogen atoms, and herein may also refer to phosphorus having one attached oxygen atom and three attached nitrogen atoms. In the intersubunit linkages of the oligomers described herein, one nitrogen is typically pendant to the backbone chain, and the second nitrogen is the ring nitrogen in a morpholino ring structure. Alternatively or in addition, a nitrogen may be present at the 5 '-exocyclic carbon.

[0054] As used herein, the term "oligonucleotide" or "nucleotide" refers to a short, single-stranded nucleic acid molecule, in the context of the present invention, to an oligomer or nucleic acid polymer (e.g. ribonucleic acid ( NA) or deoxyribonucleic acid (DNA)) or nucleic acid analogue of those known in the art, for example Locked Nucleic Acid (LNA), or a mixture thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly or with specific improved functions. A fully or partly modified or substituted oligonucleotide is often preferred over native forms because of several desirable properties of such oligonucleotides such as for instance, the ability to penetrate a cell membrane, good resistance to extra- and intracellular nucleases, high affinity and specificity for the nucleic acid target. Methods of modifying oligonucleotides in this manner are known in the art.

[0055] An oligonucleotide is a plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules.

[0056] Nucleotide analogs include nucleotides having modifications in the chemical structure of the base, sugar and/or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2 '-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2 -OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, or CN, wherein R is an alkyl moiety. Nucleotide analogs are also meant to include nucleotides with bases such as inosine, queuosine, xanthine, sugars such as 2 '-methyl ribose, non-natural phosphodiester linkages such as methylphosphonates, phosphorothioates and peptides.

[0057] In some oligonucleotides sometimes called oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitroge atoms of the amide portion of the backbone.

[0058] Examples include, but are not limited to oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular-CH₂-NH-0-CH₂-, -CH₂-N(CH₃)-0-CH₂- [known as a methylene (methylimino) or MMI backbone], -CH₂-0-N(CH₃)-CH₂-, -CH₂-N(CH₃)-N(CH₃)- CH₂- and -0-N(CH₃)-CH₂-CH₂- [wherein the native phosphodiester backbone is represented as -0-P-0-CH₂-]. Also usable are oligonucleotides having morpholino backbone structures. [0059] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-0- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Q to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particular examples include, but are not limited to 0[(CH₂)nO]mCH₃, 0(CH₂)nOCH₃, 0(CH₂)nNH₂, 0(CH₂)nCH₃, 0(CH₂)nONH₂, and 0(CH₂)nON[(CH₂)nCH₃)]₂, where n and m are from 1 to about 10. Other examplary oligonucleotides comprise one of the following at the 2' position: Ci to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O- alkaryl or O-aralkyl, SH, SCH₃, OCN, CI, Br, CN, CF₃, OCF₃, SOCH₃, S0₂CH₃, ON0₂, N0₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. One examplary modification includes 2'-methoxyethoxy (2'-0-CH₂CH₂OCH₃, also known as 2'-0-(2- methoxyethyl) or 2 -MOE) i.e., an alkoxyalkoxy group. A further modification includes 2'-dimethylaminooxyethoxy, i.e., a 0(CH₂)20N(CH₃)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-0- dimethylaminoethoxyethyl or 2*-DMAEOE), i.e., 2*-0-CH₂-0-CH₂-N(CH₂)₂, also described in examples hereinbelow.

[0060] A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (- CH2-)n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.

[0061] Other modifications include 2'-methoxy (2'-0-CH₃), 2'-aminopropoxy (2'- OCH₂CH₂CH₂NH₂), 2'-allyl (2'-CH₂-CH-CH₂), 2*-0-allyl (2'-0-CH₂-CH-CH₂) and 2'- fluoro (2'-F). The 2 '-modification may be in the arabino (up) position or ribo (down) position. An exemplary 2'-arabino modification is 2 -F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. [0062] Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C[identical to]C-CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7- methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases can include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H- pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin- 2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.

[0063] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention can include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenan-thridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence- specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion.

[0064] Oligonucleotides composed of 2'-deoxyribonucleotides

(oligodeoxyribonucleotides) are fragments of DNA and are often used in the polymerase chain reaction, a procedure that can greatly amplify almost any small amount of DNA. There, the oligonucleotide is referred to as a primer, allowing DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

[0065] The term "nucleotide" refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof. Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs.

[0066] Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups. Some examples of types of modifications that can comprise nucleotides that are modified with respect to the base moieties include but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination. More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, Ν,Ν,-dimethyladenine, 2-propyladenine, 2- propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2- amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1- methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2- methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5- methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza- adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N- alkylated purines and pyrimidines such as N6-methyladenosine, 5- methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2- one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'- thioribose, and other sugars, heterocycles, or carbocycles.

[0067] The term nucleotide is also meant to include what are known in the art as universal bases. By way of example, universal bases include but are not limited to 3- nitropyrrole, 5-nitroindole, or nebularine. The term "nucleotide" is also meant to include the N3' to P5' phosphoramidate, resulting from the substitution of a ribosyl 3' oxygen with an amine group.

[0068] Further, the term nucleotide also includes those species that have a detectable label, such as for example a radioactive or fluorescent moiety, or mass label attached to the nucleotide.

[0069] As used herein, the term "nucleic acid" refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules") in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit to any particular tertiary forms. Thus, this term includes double- stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

[0070] [0071] In another example, the Raf kinase inhibitor includes but is not limited4-[4- [[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine- 2-carboxamide (Sorafenib), 5-(2-(4-(2-(dimethylamino)ethoxy)phenyl)-5-(pyridin-4- yl)-lH-imidazol-4-yl)-2,3-dihydroinden-l-one oxime (SB590885), N-(3-(5-chloro- lH-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-l-sulfonamide (PLX4720), XL281 , 1 -methyl-5-(2-(4-(trifluoromethyl)-l H-imidazol-2-yl)pyridin-4- yloxy)-N-(4-(trifluoromethyl)phenyl)-lH-benzo[d]imidazol-2-amine (RAF265), (E)- 5-( 1 -(2-hydroxyethyl)-3 -(pyridin-4-yl)- 1 H-pyrazol-4-yl)-2,3-dihydro- 1 H-inden- 1 -one oxime (GDC-0879), 4-[4-({[4-Chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)- 3-fluorophenoxy]-iV-methylpyridine-2-carboxamide (Regorafenib), 4-Methyl-3-[[l- methyl-6-(3 -pyridinyl)- 1 H-pyrazolo[3 ,4-d]pyrimidin-4-yl] amino] -N-[3- (trifluoromethyl)phenyl]benzamide trifluoroacetate (NVP-BHG712), (E)-5-[l-(2- Hydroxy-ethyl)-3-pyridin-4-yl-lH-pyrazol-4-yl]-indan-l-one oxime (AZ628), -(5- (3-dimethylaminobenzamido)-2-methylphenyl)-4-hydroxybenzamide (ZM336372) or N-[3-[5-(4-chlorophenyl)-lH-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4- difluorophenyl]propane- 1 -sulfonamide (Vemurafenib).

[0072] In another example, the MEK inhibitor includes but is not limited(S)-(3,4- difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydroxy-3-(piperidin-2- yl)cyclobutyl)methanone (XL518), 2-(2-Chloro-4-iodophenylamino)-N- (cyclopropylmethoxy)-3,4-difluorobenzamide (CI-1040), PD035901, 6-(4-bromo-2- chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (selumetinib), l,4-diamino-2,3-dicyano-l,4-bis(2-aminophenylthio)butadiene (U0126) or N-(3- {3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl- 2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin- 1 (2H)-yl}phenyl)acetamide (GSK112021).

[0073] In yet another example, the Erk inhibitor includes but is not limited to PD98059, 5-(2-phenylpyrazolo[l,5-a]pyridin-3-yl)-lH-pyrazolo[3,4-c]pyridazin-3- amine (FR180204) or N-((2R)-2,3-dihyroxypropoxy)-3,4-difluoro-2-((2-fluoro-4- iodophenyl)amino)-benzamide (PKI-ERK-005).

[0074] As disclosed herein, the at least one PI3K pathway inhibitor includes but is not limited to (lS,6bi?,9aS,l 1^,1 lbi?)l l-(Acetyloxy)-l,6b,7,8,9a,10,l l,l lb-octahydro-1- (methoxymethyl)-9a,l lb-dimethyl-3H-iuro[4,3,2- e]indeno[4,5,-A]-2-/2]-2- benzopyran-3,6,9-trione (Wortmannin), 18-Norandrosta-5,8,l l,13-tetraeno[6,5,4- bc]furan-3,7,17-trione, l-hydroxy-,(lb)-(demethoxyviridin), or 2-(4-Morpholinyl)-8- phenyl-4H- 1 -benzopyran-4-one (LY-294002).

[0075] As disclosed herein, the cancer is a solid tumour type of cancer. As disclosed herein solid tumour is an abnormal mass of tissue that usually does not contain cysts or liquids areas. Examples of solid tumours include but are not limited to sarcomas, carcinomas, and lymphomas. More specifically, cancer as disclosed herein includes but is not limited to colon cancer, lung cancer, ovarian carcinoma, hepatocellular carcinoma, breast cancer, throat cancer, esophageal cancer, prostate cancer, testicular cancer, stomach cancer, bowel cancer, anal cancer, or kidney cancer. In yet another example, the method of the present invention may be used in the treatment of hepatocellular carcinoma (HCC) that is liver cancer.

[0076] Owing to the surprising discovery that the Ras signalling pathway and YAP protein are linked in the promotion of tumourigenesis, the present invention provides a method for determining the susceptibility of a patient suffering or suspected to suffer from cancer to a treatment with at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor. As disclosed herein, the method may comprise comparing mRNA level and/or protein expression level and/or miRNA levels of genomic sequences or any combination of transcriptional targets of YAP activator. If levels of mRNA level and/or protein expression and/or miRNA of or any combination of transcriptional targets of YAP activator differ in a patient suffering or suspected to suffer from cancer from a control group then the patient is susceptible to a treatment comprising the at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor.

[0077] As disclosed herein, the term "control group" refers to a group of subjects who do not suffer or are not suspected to suffer from cancer. The control group includes but is not limited to at least one individual not having cancer (healthy individual) and/or having previously treated against cancer. The control group is preferably a group of individuals having or sharing some characteristics with the patient. Some characteristics include but are not limited to the sex, the age, the race, or the socio- economic background. In general for the purpose of the invention, the control group may comprise healthy individuals i.e. individuals without any known disorders, diseases or conditions or without having had any recent disorders, diseases or conditions that may affect the levels of the mRNA and/or protein expression levels and/or miRNA levels to be compared. [0078] The control group may comprise individuals that do not undergo any therapeutic or prophylactic treatments. For example, preferably the individuals may not have taken any medicines or drugs or may not have had any surgery.

[0079] In another example, the control group may include but is not limited to tissue sample obtained from the patient suffering or suspected to suffer from cancer, wherein the tissue sample obtained from said patient is not affected by the cancer (healthy tissue (non-tumour tissue) of the patient to be treated).

[0080] As such, examples of mRNA levels and/or protein expression levels and/or miRNA levels to be determined include but are not limited to mRNA levels and/or protein expression levels and/or miRNA levels for SOCS6 and/or YAP and/or AREG (Locus: NM_001657) and/or Survivin (BRIC5; Locus: NM_001012271) and/or mir- 17 and/or connective tissue growth factor (CTGF; Locus: NM 001901) and/or cysteine-rich, angiogenic inducer, 61 (CYR61; Locus: NM_001554) or any combination of transcriptional targets of YAP obtained from a patient suffering or suspected to suffer from cancer with the mRNA level and/or protein expression level and/or miRNA results of a control group.

[0081] For example, increased level of mRNA and/or protein expression for YAP protein in a patient suffering or suspected to suffer from cancer compared to the level of mRNA and/or protein expression for YAP protein indicates that the patient is susceptible to a treatment comprising the at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor as disclosed herein.

[0082] In another example, increased level of mRNA and/or protein expression for AREG protein in a patient suffering or suspected to suffer from cancer compared to the level of mRNA and/or protein expression for AREG protein indicates that the patient is susceptible to a treatment comprising the at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor as disclosed herein.

[0083] In yet another example, increased level of mRNA and/or protein expression for SOCS6 protein in a patient suffering or suspected to suffer from cancer compared to the level of mRNA and/or protein expression for SOCS6 protein indicates that the patient is susceptible to a treatment comprising the at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor as disclosed herein.

[0084] It will be appreciated that the genes of interest can be detected in a variety of ways. In one example, the method comprises obtaining nucleic acid sequence data from the cellular sample. Suitable methods of directly analyzing a nucleic acid molecule include, for instance, denaturing high pressure liquid chromatography (DHPLC), DNA hybridization, computational analysis, automated fluorescent sequencing, clamped denaturing gel electrophoresis (CDGE), denaturing gradient gel electrophoresis (DGGE), mobility shift analysis, restriction enzyme analysis, heteroduplex analysis, chemical mismatch cleavage (CMC), RNase protection assays, use of polypeptides that recognize nucleotide mismatches, and direct manual sequencing. These and other methods are described in the art.

[0085] In one example, diagnosis of (or identification of a predisposition to) cancer can be accomplished using a hybridization method. A biological sample of genomic DNA, RNA, or cDNA is obtained from a subject suspected of having, being susceptible to, or experiencing symptoms associated with cancer. Optionally, the nucleic acid encoding the gene of interest is amplified by polymerase chain reaction (PCR). The DNA, RNA, or cDNA sample is then examined. The presence of the gene of interest can be determined by sequence-specific hybridization of a nucleic acid probe specific for particular mutation within the gene of interest coding sequence. As discussed above, a nucleic acid probe is a DNA molecule or an RNA molecule that hybridizes to a complementary sequence in genomic DNA, RNA, or cDNA. In some aspects, the presence of more than one gene of interest mutation is determined by using multiple nucleic acid probes, each being specific for a particular mutation.

[0086] One of skill in the art has the requisite knowledge and skill to design a probe so that sequence-specific hybridization will occur only if a particular mutation is present in a gene of interest coding sequence. By "sequence-specific hybridization" is meant that the probe(s) preferentially bind to a nucleic acid sequence encoding Sun 1. In some embodiments, specific hybridization is achieved using "stringent conditions," which are conditions for hybridization and washing under which nucleotide sequences at least 60% identical to each other typically remain hybridized. It is appreciated in the art that stringent conditions can differ depending on sequence content, probe length, and the like. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since target sequences are generally present at excess, 50% of the probes are occupied at equilibrium at Tm. Stringent conditions also may include a salt concentration less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes, primers, or oligonucleotides (e.g., 10 nucleotides to 50 nucleotides) and at least about 60° C. for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6xSSC, 50 mM Tr-is-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65° C, followed by one or more washes in 0.2xSSC, 0.01% BSA at 50° C

[0087] Specific hybridization, if present, is detected using standard methods. For example, the probe can comprise a fluorescent moiety at its 3' terminus, a quencher at its 5' terminus, and an enhancer oligonucleotide to facilitate detection. In this detection method, an enzyme cleaves the fluorescent moeity from a fully complementary detection probe, but does not cleave the fluorescent moeity if the probe contains a mismatch. The presence of a particular target sequence is signalled by the fluorescence of the released fluorescent moiety. Alternatively, nucleic acids encoding the gene of interest are dot-blotted using standard methods, and the blot is contacted with one or more oligonucleotide probes specific for a gene of interest mutation. Similarly, arrays of oligonucleotide probes complementary to target nucleic acid sequence(s) can be employed in the inventive diagnostic method. Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes coupled to a surface of a substrate (e.g., plastic, complex carbohydrate, or acrylic resin) in different known locations. Such arrays are generally produced using mechanical synthesis methods or light-directed synthesis methods, although other methods are known to the ordinary skilled practitioner.

[0088] In another hybridization method, Northern analysis is used to identify the presence of a gene of interest encoded by mRNA in a subject's sample. Specific hybridization between the nucleic acid probe and the nucleic acid in the subject sample indicates that the gene of interest is present, and the subject is suffering from or is at risk of developing a cancer.

[0089] Sequence analysis can also be used to detect specific gene mutations associated with cancer. Therefore, in one embodiment, determination of the presence or absence of mutant gene entails directly sequencing DNA or RNA obtained from a subject. If desired, PCR is used to amplify a portion of a nucleic acid encoding the gene of interest, and the presence of a specific mutation is detected directly by sequencing the relevant site(s) of the DNA or RNA in the sample.

[0090] Mutations in the gene of interest coding sequence may lead to altered expression levels, e.g., a decrease in the expression level of an mRNA or protein, which leads to an abnormal phenotype. Such mutations are detected via, e.g., ELISA, radioimmunoassays, immunofluorescence, Northern blotting, and Western blotting to compare the protein expression levels in a subject compared to a biologically-matched control or reference. These processes are described in the art.

[0091] Methods to determine the mRNA levels of the mRNA as disclosed herein are known to the person skilled in the art. For example, mRNA levels can be measured using reverse transcriptase polymerase chain reaction for quantitating the mRNA levels of the mRNA encoding the proteins disclosed herein. The proteins levels are measured using antibodies against said proteins via, e.g. Western Blotting or Immunofluorescence as described above. The present invention also provides for a combination of the aforementioned methods. For example, total cell lysis may be carried out. Total RNA including but not limited to mRNA, miRNA, piRNA, rRNA, tRNA, hnRNA and pre-mRNA may be isolated from the cell lysate and total protein extract may be isolated from the same lysate. Method of detecting mRNA levels and protein expression levels may then be carried out on the respective extracts.

[0092] As used herein, the term "miRNA" refers to microRNA. MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 21-23 nucleotides. As used herein, the term miRNA mimic refers to a single-stranded RNA, chemically synthetized or isolated, capable of reproducing the function, structure and activity of a naturally occurring miRNA. miRNAs have been shown to be important in post-transcriptional control of gene expression. miRNAs are misregulated in human diseases including cancer. As disclosed herein, by using available publicly available databases, it is possible to predict the target sites for microRNAS in the SOCS6 3'UTR. Predicted pairing between miRNAs and SOCS6 3'UTR allows the design of a method for treating cancer in a patient. The method as disclosed herein includes but is not limited to sequestering a microRNA capable of decreasing SOCS6 protein levels. The sequestration of microRNA capable of decreasing SOCS6 protein levels will result in an increase in the SOCS6 protein levels.

[0093] Sequestration of miRNA capable of decreasing SOCS6 protein levels may be achieved by administering to a patient in need thereof an antisense oligonucleotide suitable to sequester said miRNA. The antisense oligonucleotide may be an antisense oligonucleotide as defined above.

[0094] As used herein, the term "antisense strand" is meant to refer to a polynucleotide or region of a polynucleotide that is at least substantially (e.g., about 80% or more) or 100% complementary to a target nucleic acid of interest. Also, the antisense strand of a dsRNA is at least substantially complementary to its sense strand. An antisense strand may be comprised of a polynucleotide region that is RNA, DNA, or chimeric RNA/DNA. Additionally, any nucleotide within an antisense strand can be modified by including substituents coupled thereto, such as in a 2' modification. The antisense strand can be modified with a diverse group of small molecules and/or conjugates. For example, an antisense strand may be complementary, in whole or in part, to a molecule of messenger RNA ("mRNA"), an RNA sequence that is not mRNA including non-coding RNA (e.g., tRNA and rRNA), or a sequence of DNA that is either coding or non-coding. The terms "antisense strand" and "antisense region" are intended to be equivalent and are used interchangeably.

[0095] The antisense region or antisense strand may be part of a larger strand that comprises nucleotides other than antisense nucleotides. For example, in the case of a unimolecular structure the larger strand would contain an antisense region, a sense region and a loop region, and might also contain overhang nucleotides and additional stem nucleotides that are complementary to other stem nucleotides, but not complementary to the target. In the case of a fractured hairpin, the antisense region may be part of a strand that also comprises overhang nucleotides and/or a loop region and two other regions that are self-complementary. The present invention provides antisense oligonucleotide suitable to sequester miRNA capable of decreasing SOCS6 protein levels. The antisense oligonucleotide includes but is not limited to

Anti-mir-17: 5' - CUACCUGCACUGUAAGCACUUUG - 3 ';

Anti-mir-20a: 5 '- CUACCUGCACUAUAAGCACUUUA -3

Anti-mir-183: 5^'- AGUGAAUUCUACCAGUGCCAUA-3 ';

Anti-mir-155: 5 ^'-ACCCCUAUCACGAUUAGCAUUAA-3 ';

Anti-mir-21 : 5 '-UCAACAUCAGUCUGAUAAGCUA-3

Anti-mir-30a: 5 ' -CUUCCAGUCGAGGAUGUUUACA-3 ' ;

Anti-mir-25 : 5 ' -UC AG ACCGAGAC AAGUGC AAUG-3 ' ;

Anti-mir-92a: 5 ' - AC AGGCCGGG AC AAGUGC AAUA-3 ' ; Anti-mir-92b: 5 ' -GGAGGCCGGG ACG AGUGC AAU A-3 ' ;

Anti-mir-32: 5'- UGCAACUUAGUAAUGUGCAAUA-3';

Anti-mir-19a: 5'-UCAGUUUUGCAUAGAUUUGCACA-3';

Anti-mir-27a: 5'-GCGGAACUUAGCCACUGUGAA-3'; and

Anti-mir-27b: 5'-GCAGAACUUAGCCACUGUGAA-3'; and functional equivalents thereof (SEQ ID No. 26 to 38, respectively).

[0096] By "functional equivalents" thereof, it is understood that the functional equivalents may achieve a similar or better inhibitory sequestration of the target miRNA. Examples of equivalents are given in the definition section above that may include but is not limited to chemically modified antisense oligonucleotides. For example the functional equivalents include but are not limited to antisense oligonucleotide disclosed herein with at least one modified or substituted nucleotide. Examples of modified of substituted nucleotides are provided above.

[0097] Functional equivalents include but are not limited to analog, derivative or mimetic. An analog is a molecule that differs in chemical structure from a parent or reference compound, for example a homolog (differing by an incremental change in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques known in the art. A derivative is a substance related to a base structure, and theoretically derivable from the base structure. A mimetic is a biomolecule that mimics the activity of another biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound, for instance a native siRNA.

[0098] In an example, the modified nucleotides comprise modified bases that include but are not limited to phosphorothioate, methyl phosphonate, peptide nucleic acids, 2'-0-methyl, fluoro- and carbon, methylene and other locked nucleic acid molecules. Further examples of modified bases are provided in the definition section.

[0099] Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as DNA or RNA) that is at least 6 bases (or nucleotide), for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 8 to 30 bases, or about 8 to 25 bases or about 20 to 25 bases or about 10 to about 2.0 bases or about 12 bases to about 16 bases such as 12, 14, 16, 18 or 20 bases.

[00100] Based on the findings as disclosed herein, it has been found that some miRNAs are capable of binding to the 3' UTR region of SOCS6, thereby inhibiting expression of the SOCS6 protein. In one example, microRNA as disclosed herein include but is not limited to the miRNA as disclosed in Table 1 below:

Table 1 :

[00101] In an example, the miRNA capable of decreasing" SOCS6 protein levels is a member of the miR-17 family of miRNA. For example the inventors have found that miR-17 and miR-20 are capable of decreasing SOCS6 protein levels. In another example, miRNA that may be capable of decreasing SOCS6 levels includes but is not limited to miR-203; miR-499/499-5p; miR-183; miR-23ab; miR-216/216b; miR-128; miR-204/211 ; miR-192/215; miR-15/16/195/424/497; miR-144; miR-218; miR-17- 5p/20/93.mr/106/519.d; miR-30a/30a-5p/30b/30b-5p/30cde/384-5p; miR-216/216a; miR-182; miR-208/208ab; miR-2 l/590-5p; miR-26ab/1297; miR- 25/32/92/92ab/363/367; miR-141/200a; miR-19; miR-190; miR-200bc/429; miR- 33/33ab; miR-130/301 ; miR-137; miR-27ab; miR-34a/34b-5p/34c/34c- 5p/449/449abc/699; miR-142-3p; miR-138; miR-96/1271 ; miR-155; miR-193ab; miR-101; miR-139-5p; miR-338/338-3p; miR-24; miR-503; miR-494; miR-329/362- 3p; miR-186; miR-590/590-3p; miR-340/340-5p; miR-421 ; miR-296/296-3p; miR- 377; miR-300; miR-376c; miR-431; miR-544; miR-382; miR-149; miR-592/599; miR-495/1192; miR-326/330/330-5p; miR-539; miR-378/422a; miR-361/361-5p; miR-758; miR-542/542-3p; miR-410; miR-342/342-3p; miR-339-5p; miR-504; miR- 154; miR-374/374ab; miR-224; or miR-384/384-3p. The sequences of these microRNAs can be obtained in publicly available databases such as "mirbase" (www.mirbase.org; SEQ ID No. 52 to 263).

[00102] The present invention also provides for a kit for use in the method as disclosed herein. The kit may include means to detect SOCS6 proteins levels and/or mRNA levels and/or miRNA levels. For example, the kit may include PCR primers to detect SOCS6 mRNA by reverse transcription polymerase chain reaction, antibodies to detect SOCS6 protein. The antibodies include but are not limited to monoclonal antibodies, polyclonal antibodies, humanized antibodies or fragments thereof. The antibodies may be used to quantify the relative amount of SOCS6 protein by methods known in the art. The method may include for example ELISA, radioimmunoassays, immunofluorescence, Northern blotting, and Western blotting to compare the protein expression levels in a subject compared to a biologically-matched control or reference.

[00103] The inventive method is preferably performed as soon as possible after it has been determined that a subject is at risk for developing a cancer (e.g., diagnosis of close family member) or as soon as possible after onset of the cancer is detected. To this end, the compound to increase SOCS6 protein level is administered before symptoms appear to protect, in whole or in part, against the onset of cancer. The compound as disclosed herein also can be administered after symptoms are detected to prevent, in whole or in part, additional symptoms or an increase in symptom severity.

[00104] A particular administration regimen for a subject will depend, in part, upon the form of the compound administered (e.g., polypeptide or nucleic acid molecule or organic compound), the amount administered, the route of administration, and the cause and extent of any side effects. The amount of compound administered to a subject (e.g., a mammal, such as a human) in accordance with the invention should be sufficient to effect the desired response over a reasonable time frame. Dosage typically depends upon a variety of factors, including the particular agent employed, the age and body weight of the subject, as well as the existence of any disease or disorder in the subject. The clinician may titer the dosage and may modify the route of administration to obtain the optimal therapeutic effect, and conventional range-finding techniques are known to those of ordinary skill in the art. Purely by way of illustration, the inventive method can comprise administering, e.g., from about 0.1 μg/kg to up to about 100 mg/kg of compound or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 1 μg/kg up to about 100 mg/kg; or 5 g/kg up to about 100 mg kg; or 10 μg/kg up to about 100 mg/kg. Some conditions or disease states require prolonged treatment, which may or may not entail administering lower doses of agent over multiple administrations. In addition, when appropriate, the compound is administered in combination with other substances (e.g., therapeutics) and/or other therapeutic modalities to achieve an additional (or augmented) biological effect.

[00105] To deliver the a compound capable of increasing expression of SOCS6 protein in cancer cell to a subject having or suspected to have a cancer, the present invention provides for a delivery vehicle to be formulated with said compound. The delivery vehicle when formulated with the a compound capable of increasing expression of SOCS6 protein in cancer cell may allow delivery of the compound to the target site in a patient having or suspected to have a cancer. The delivery vehicle may be such that the compound capable of increasing expression of SOCS6 protein in cancer cell is protected from degradation, has an increased half-life, and is capable of delivering the compound to the mRNA and/or protein and/or miRNA target thereby inhibiting the SOCS6 decreased expression level. As used in the present disclosure, the term "subject" or "patient" refers to a mammal such as a rodent, cat, dog, primate or human, preferably said subject or patient is a human.

[00106] In one example, the delivery vehicle may be a nanoparticle. The nanoparticle of the invention includes but is not limited to a liposome, a peptide, an aptamer, an antibody, a polyconjugate, a microencapsulation, a virus like particle (VLP), a nucleic acid complex and a mixture thereof. For example, the liposome as disclosed herein includes but is not limited to a stable nucleic acid-lipid particle (SNALP), a l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) based delivery system, and a lipoplex. As described herein, the term "liposome" refers to an artificial vesicle composed of one or more concentric phospholipid bilayers and used especially to deliver microscopic substances (as drugs or nucleic acid) to body cells.

[00107] The term "aptamer" refers to oligonucleic acid or peptide molecules that bind to a specific molecular target such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. The term "lipoplex" as used herein refers to non- viral vehicles, such as cationic liposomes and the complexes they form with nucleic acid molecules. Lipoplexes are often presented as the most promising alternative to the use of viral vectors for gene therapy.

[00108] Suitable methods of administering a physiologically acceptable composition, such as a pharmaceutical composition comprising a compound capable of increasing expression of SOCS6 protein in cancer cell, are well known in the art. Although more than one route can be used to administer an agent, a particular route can provide a more immediate and more effective reaction than another route. Depending on the circumstances, a pharmaceutical composition comprising a compound capable of increasing expression of SOCS6 protein in cancer cell is applied or instilled into body cavities, absorbed through the skin or mucous membranes, ingested, inhaled, and/or introduced into circulation.

[00109] In the present invention, the compound capable of increasing expression of SOCS6 protein in cancer cell may be administered by the same or different routes. For example, the silencing oligonucleotide is administered systemically. The present disclosure also envisages administering the compound capable of increasing expression of SOCS6 protein in cancer cell locally.

[00110] In some instances, the silencing oligonucleotide may be administered orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularally, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, orally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, via localized perfusion, bathing target cells directly, or any combination thereof. For example, in some variations, the compound capable of increasing expression of SOCS6 protein in cancer cell is administered intravenously, intra-arterially or orally. For example, in some variations, the compound is administered intravenously. In one example, the compound as disclosed herein may be formulated for systemic administration. To facilitate administration, a protein or nucleic acid molecule can be formulated into a physiologically-acceptable composition comprising a carrier (i.e., vehicle, adjuvant, or. diluent). The particular carrier employed is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the therapeutic, and by the route of administration. Physiologically-acceptable carriers are well known in the art. Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Injectable formulations are further described in the art. A pharmaceutical composition comprising a compound capable of increasing expression of SOCS6 protein in cancer cell may be placed within containers, along with packaging material that provides instructions regarding the use of such pharmaceutical compositions. Generally, such instructions include a tangible expression describing the reagent concentration, as well as, in certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline or PBS) that may be necessary to reconstitute the pharmaceutical composition.

[00111] The pharmaceutically effective amount of the compound to be used for treatment of cancer can be a daily dose is 0.01 - 50 mg of composition per kg of body weight. In some variations, the daily dose is 5-50 mg of composition per kg of body weight. In some variations, the daily dose is 0.05 - 20 mg of composition per kg of body weight. In some variations, the daily dose is 0.1 - 10 mg of composition per kg of body weight, or 1 - 10 mg of composition per kg of body weight. In some variations, the daily dose is 0.1 - 5 mg of composition per kg of body weight. In some variations, the daily dose is 0.1 - 2.5 mg of composition per kg of body weight.

[00112] The amount of compound capable of increasing expression of SOCS6 protein in cancer cell in the formulation can be from about 0.1 mg to about 500 mg. In some variations, the daily dose can be from about 1 mg to about 300 mg. In some variations, the daily dose can be from about 10 mg to about 200 mg of the formulation. In some variations, the daily dose can be about 25 mg of the formulation. In other variations, the daily dose can be about 75 mg of the formulation. In still other variations, the daily dose can be about 150 mg of the formulation. In further variations, the daily dose can be from about 0.1 mg to about 30 mg of the formulation. In some variations, the daily dose can be from about 0.5 mg to about 20 mg of the formulation. In some variations, the daily dose can be from about 1 mg to about 15 mg of the formulation. In some variations, the daily dose can be from about 1 mg to about 10 mg of the formulation. In some variations, the daily dose can be from about 1 mg to about 5 mg of the formulation.

[00113] For example, for oral administration of Raf/MAPK/P 13 pathway inhibitor in mice, the daily dose can be from about 5mg to about 50 mg of inhibitor per kg of body weight or about 10 mg to about 25 mg of inhibitor per kg of body weight, or about 15 mg to about 20 mg of inhibitor per kg of body weight, or 12 mg to about 18 mg of inhibitor per kg of body weight. The variations may depend on the potency and toxicity index of individual drugs and the individual (or subject, or patient). In another example, for intravenous injection of Raf/MAPK/P 13 K pathway inhibitor in mice, the dose may be about 20 mg to 200 mg of inhibitor per kg of body weight every three days, or about 40 mg to 100 mg of inhibitor per kg of body weight every three days, or about 50 mg to 80 mg of inhibitor per kg of body weight every three days, or about 60 mg to 70 mg of inhibitor per kg body weight every days depending on the potency and toxicity index of individual drugs. In another example, for intraperitoneal injection of Raf/MAPK/PI3K pathway inhibitor in mice, the dose may be about 20 mg to 200 mg of inhibitor per kg of body weight every three days, or about 40 mg to 100 mg of inhibitor per kg of body weight every three days, or about 50 mg to 80 mg of inhibitor per kg of body weight every three days, or about 60 mg to 70 mg of inhibitor per kg body weight every days depending on the potency and toxicity index of individual drugs.

[00114] In another example, for oral administration of antisense oligonucleotides, the dosage may be about 0.5 mg to about 10 mg per kg of body weight every 2 days, or about 1 mg to about 5 mg per kg of body weight every 2 days, or about 2 mg to about 4 mg per kg of body weight every 2 days depending on the potency and toxicity index of individual drugs. In another example, for intravenous injection of antisense oligonucleotides, the dosage may be about 2 mg to about 20 mg per kg of body weight every 2 days, or about 5 mg to about 10 mg per kg of body weight every 2 days, or about 6 mg to about 8 mg per kg of body weight every 2 days, or about 7 mg to about 9 mg per kg of body weight every 2 days depending on the potency and toxicity index of individual drugs. In yet another example, for intraperitoneal injection of antisense oligonucleotides, the dosage may be about 10 mg to about 100 mg per kg of body weight every 2 days, or about 20 mg to about 80 mg per kg of body weight every 2 days, or about 30 mg to about 70 mg per kg of body weight every 2 days, or about 40 mg to about.60 mg per kg of body weight every 2 days depending on the potency and toxicity index of individual drugs.

[00115] In the present disclosure, the two components may be administered by the same or different routes. For example, the composition is administered locally. The present disclosure also envisages administering the compounds systemically. In some instances, the compounds may be administered orally, intraadiposally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularally, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, intrarectally, intrathecally, intratracheally, intratumorally, intraumbilically, intravaginally, intravenously, intravesicularlly, intravitreally, liposomally, locally, mucosally, orally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, transbuccally, transdermally, vaginally, in cremes, in lipid compositions, via a catheter, via a lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, via localized perfusion, bathing target cells directly, or any combination thereof. For example, in some variations, the compounds are administered intravenously, intra-arterially or orally. For example, in some variations, the compounds are administered intravenously.

EXAMPLES

[00116] Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[00117] Example 1:

[00118] To test whether there is a functional interaction between Ras^V12 and YAP, an in vitro model of cellular transformation based on genetically engineered human fibroblast cells was used. The cells were transduced with retroviral constructs to direct expression of (1) hTERT, which encodes the catalytic subunit of the telomerase to allow replicative immortality; (2) Inducible oncogenic H-RasV fused to the ligand binding domain of the Estrogen Receptor (ER-H-Ras^V12), to activate Ras signaling upon addition of 4 hydroxy-tamoxifen (40HT); (3) shRNAs to deplete p53 and pl6, to overcome Ras -induced growth arrest and confer colonigenic outgrowth; (4) SV40 small T antigen, which allows anchorage-independent growth in soft agar.

[00119] The transgenic cells were either transduced to express YAP cDNA or empty vector control (EV) to compare the effect of YAP overexpression on soft agar colony formation. Fully confluent cells were trypsinized, mixed with 0.8% agar in DMEM medium containing 15% Fetal Calf Serum (FCS) and pooled into 6-well plates with 2ml pre-solidified 1% agar/DMEM at the bottom (10,000 cells/well). The agar plates were either treated with human recombinant Epidermal Growth Factor (EGF; O^g/ml) or 40HT (10^"7M, to activate ER-H-Ras^V12). Colonies were stained and quantified after incubation for 12 days.

[00120] As shown in Fig. 1A, in control cells with normal YAP expression, addition of either EGF or 40HT give rise to colonies ranging from 50μηι to ΙΟΟμιη in size (cells labeled by GFP, incubated for 12 days). In comparison, cells overexpressing YAP (YAP) alone (i.e. without Ras activation) could give rise to colonies of similar size than those obtained from cells treated with EGF alone (EV+EGF). Surprisingly, overexpression of YAP together with EGF/40HT gives rise to bigger colonies (YAP+EGF; YAP+40HT).

[00121] These results suggest that YAP and EGFR/RAS function synergistically in promoting colonigenic growth in soft agar.

[00122] Example 2:

[00123] If EGFR/Ras and YAP promote colony formation by independent mechanisms, they would be expected to have an additive effect. The observed synergy suggests that they act in a common pathway. To test this, YAP was depleted by RNA interference in genetically engineered fibroblast cells (that is by transduction of a retroviral shRNA vector, encoding a short hairpin RNA interfering with YAP mRNA). Colony formation in soft agar was measured for cells treated with EGF or 40HT. Total colony number was reduced by 50% in EGF-treated cells and by 58% in 40HT-treated cells, when the cells were pretreated with the YAP silencing RNA (Fig. 2). This suggested that EGFR/Ras-mediated cellular transformation is dependent on endogenous YAP activity.

[00124] Example 3:

[00125] It was next sought to understand whether YAP-mediated cellular transformation might reciprocally depend on Ras pathway activity. As indicated above, Ras-mediated transformation is dependent on endogenous YAP activity. Ras acts via three distinct kinase cascades: MAPK, PI3K and RalGEF. To address this, the inventors made use of U0126 to inhibit MEK1/2 activity, and LY294002 to inhibit PI3K activity in a soft agar assay. Cells were transfected either with an empty vector (i.e. no expressing any gene; EV (control cells)) or with a vector comprising the cDNA of the gene YAP protein. The control cells (EV) or YAP overexpressing cells were treated with 10^"7M of 40HT together with either U0126 or LY294002 at ΙΟμΜ or 30μΜ for 12 days.

[00126] Colony formation was reduced in control cells and Ras-activated (40HT treated) cells by treatment with U0126 (Fig. 3A) or LY294002 (Fig. 3B). Interestingly, colony formation by YAP overexpressing cells was more sensitive to U0126 or LY294002 than Ras-activated cells. This result suggests that YAP- dependent cellular transformation is dependent on both MAPK and P3IK activity.

[00127] Colony formation by synergistic activation of the Ras pathway kinase cascade and YAP was sensitive to both inhibitors. The synergistic action of Ras and YAP resulted in stronger signaling amplification of EGFR/Ras and more vigorous colony formation, shown by size and total number of colonies (Example 1).

[00128] Example 4:

[00129] To define the molecular mechanism of interaction between Ras and YAP, YAP protein levels were measured by Western blot. BJ cells treated with 40HT for 7 days were harvested and lysed and total protein extract were isolated. The extracts were separated by SDS-PAGE and a Western Blot was carried out. As a way to monitor activation of Ras , ERK phosphorylation state was measured by Western Blotting using a monoclonal anti-MAP Kinase, Activated antibody (Diphosphorylated ERK-1 and 2; dpERK). The antibody is specific for the active, dually-phosphorylated form of MAP kinase (ERK-1 and ERK-2, 44 kDa and 42 kDa, respectively). The epitope recognized by the antibody contains the phosphorylated threonine and tyrosine residues within the regulatory site of active MAP kinase (Thrl83 and Tyrl85 in ERK-2). It does not recognize the non-phosphorylated or the monophosphorylated forms of the MAP kinase molecule or the diphosphorylated form of Jun-kinase (INK) and p38 MAP kinase. Dp-ERK levels increased (Fig. 4 A) in 40HT treated cells. Interestingly, we also found a substantial increase in YAP protein upon Ras activation (Fig. 4A, right panel). Surprisingly, there was an increased expression of YAP transcriptional targets, AREG and survivin (Fig. 4B; as demonstrated using antibodies against the indicated proteins) when the cells were treated with 40HT, thereby suggesting increased YAP levels and activity.

[00130] This example demonstrates that the RAS pathway regulates YAP protein levels in the cells. This provides an explanation at a molecular level for the synergy between the two pathways in cellular transformation. [00131] Example s: .

[00132] The previous example suggested that RAS activation increases the level of YAP protein. Members of the SOCS protein family have been identified unexpectedly by the inventors as molecular mediators of this interaction. Therefore, to confirm the role of SOCS protein family as mediator of the interaction, the inventors depleted SOCS6 in BJ cells by using shRNAs vectors encoding shRNA capable of inhibiting SOCS6 expression. Two vectors were constructed encoding for two independent shRNA against SOCS6. 10,000 engineered fibroblast BJ cells were seeded and 24 hours later were transfected with either an empty vector control (same backbone as the vector used to construct the shRNA, without the shRNA sequence inserted) or SOCS6 shRNA- 1 or shRNA-2. Four days after transfection and selection, soft agar assay was performed and 2 weeks later, soft agar colonies were stained with MTT for 1 hour at 37°C. Pictures of the colonies were captures with a light microscope fitted with a camera (Fig. 5). The lower panel shows cells without 40HT treatment as background control. The colonies were then quantified using Matlab software and plotted. Data were normalized to control vector and averages of three biological repeats are shown in Fig. 5B.

[00133] The result indicates that depletion of human SOCS6 by two independent shRNAs enhanced the Ras^V12 cellular transformation induced by 40HT by ~2 fold compared with vector control.

[00134] Example 6:

[00135] The observation that SOCS6 depletion leads to increased cellular transformation, prompted the inventors to ask whether Ras^VI2 activation regulates SOCS6 expression. Genetically engineered fibroblast BJ cells were treated with 10^A- 7M 40HT for 7 days. The cells were then harvested and the total RNA isolated. Quantitative real time PCR was then used to measure SOCS6 mRNA levels in the cells treated with 40HT. The mRNA levels were normalized to GAPDH and compared to the SOCS6 mRNA levels in cells grown for 7 days without any 40HT treatment. SOCS6 mRNA levels were reduced by -60% in cells treated with 40HT. Hence, this result suggests that Ras signaling triggers downregulation of SOCS6 mRNA levels.

[00136] Example 7:

[00137] The human SOCS6 protein contains a highly conserved SOCS box domain, which acts as a substrate adaptor for E3 ubiquitin ligase to promote protein degradation via the ubiquitin-proteasome degradation pathway. To verify whether YAP protein is targeted by the ubiquitin-proteasome degradation pathway, protein translation was inhibited in BJ cells using cycloheximide (20 μ§/ι 1 amount of CHX). Cells were harvested at various times after CHX treatment and protein extract isolated. YAP protein levels were monitored by western blot using a YAP antibody. 48 hours prior to CHX treatment, the cells were mock transfected or transfected with siRNA against SOCS6 (i.e. a siRNA capable of silencing SOCS6 expression). In the control (Fig. 7; left panel), YAP protein level decreased in a time-dependent manner from 0 hour to 5 hours of CHX treatment; while in SOCS6 siRNA treated cells, YAP protein levels were stable (Fig. 7; right panel). The relative intensity of YAP and tubulin (loading control) was quantified using image J software and a protein decay curve was plotted.

[00138] Upon depletion of SOCS6 by siRNA treatment, YAP proteins were more stable than in control cells (mock). Thus, depletion of SOCS6 increased YAP stability, indicating that the function of SOCS6 is to promote YAP protein turnover.

[00139] Example 8:

[00140] Elevated or at least stable YAP protein levels and activity were unexpectedly found in SOCS6 depleted cells, thus this result prompted the inventors to ask whether the reverse is true, that is whether overexpression of SOCS6 could influence YAP expression levels. HI 299 lung cancer cells grown in 6-well dishes were transfected with ^g of pcDNA3.1-HA-SOCS6 vector (i.e. a vector comprising the cDNA of SOCS6 fused to the influenza Hemagglutin gene) or of an empty vector (that is the plasmid pcDNA3.1 not comprising any coding region. 48 hours after transfection, proteins were harvested and western blot analysis was carried out to detect endogenous YAP protein level (Fig. 8A) as well as HA-tagged SOCS6 (Fig. 8B) using an anti-HA antibody. Tubulin was used as loading control. YAP protein level was dramatically reduced upon SOCS6 overexpression as shown in (Fig. 8A). By overexpressing HA-tagged SOCS6 (HA-SOCS6) in H1299 lung cancer cell lines, effective depletion of YAP protein in SOCS6 overexpressing cells was observed. This validated SOCS6 as a potent regulator of YAP protein level.

[00141] Example 9:

[00142] The SOCS protein family is reported to act as a core component of the E3 ubiquitin ligase system. To ask whether YAP is a direct substrate of SOCS6, co- immunoprecipitation experiment were performed between SOCS6 and YAP. HEK293T cells were, cotransfected with combinations of pcDNA-HA-SOCS6 and pcDNA-c-myc-ubiquitin by calcium phosphate method. 48 hours later, cells were treated with the proteasome inhibitor MG132 (2(^g/ml) for 5 hours and the cells were harvested for immunoprecipitation experiments. Anti-YAP antibody (rabbit polyclonal, Santa Cruz) was used for the Co-IP. 1% of input and 30% of Immunoprecipitated fraction was loaded for immunoblot analysis using either anti- HA (Fig. 9; upper panels) or anti-YAP antibodies (Fig. 9; lower panels).

[00143] Using anti-YAP antibody as bait, we are able to pull down transfected HA- SOCS6. This suggests that the endogenous YAP is capable of binding to the exogenous HA-SOCS6 to form an interacting molecular complex in the 293T cells.

[00144] Example 10:

[00145] YAP physically interacts with SOCS6 as demonstrated above and SOCS6 is part of the ubiquitin-mediated proteasome degradation system, thus the next question that the inventors sought to answer was whether YAP ubiquitylation is regulated by SOCS6. In this example, the highly homologous SOCS5 protein was used as comparison

[00146] 293T cells were cotransfected with myc-ubiquitin and siRNAs directed against either SOCS5 or SOCS6. 48 hours later the cells were treated with MG132 for 5 hours. The cells were then harvested and lysed, and the proteins extracts isolated. The extracts were immunoprecipitated using anti-YAP antibody. YAP ubiquitylation was significantly reduced in the immunoprecipitated portion in SOCS6 depleted cells. YAP ubiquitylation was also mildly reduced in SOCS5 depleted cells (as shown in Fig. 10A). Conversely, we also observed a strong enhancement of SOCS5/6 overexpression in YAP ubiquitylation (Fig. 10B). Taken together, SOCS5/6 appears to be potent regulator of YAP ubiquitylation. The same experiment was performed in SOCS6 depleted BJ cells and similar results were observed (Fig. IOC).

[00147] Example 11:

[00148] Previous work reported that amphiregulin (AREG), an EGFR ligand, is an important transcriptional target of YAP in mediating cellular transformation in mammary epithelia cells. For example, fig. 4B shows upregulation of AREG in cells with activated Ras^V12. Given the evidence that depletion of SOCS6 increased YAP levels (Fig. 7), the inventors asked whether depletion of SOCS6 would lead to an increase in MAPK pathway activity. Using western blot analysis on cells treated with SOCS6 shRNA or a negative control, an increase in dp-ERK staining was observed, indicating elevated MAPK activity in SOCS6 depleted fibroblast BJ cells.

[00149] Example 12:

[00150] miRNAs have been shown to be important in post-transcriptional control of gene expression. miRNAs are misregulated in human diseases including cancer. Predicted target sites for the oncogenic microRNAS miR-17 and miR-20a in the SOCS6 3'UTR were identified by the inventors. By screening all members of the mir- 17-92 cluster, which contains multiple SOCS6 binding site on the 3'UTR, the inventors were able to validate one member of the cluster, the mir-17 family (including mir-17 and mir-20, which have the same seed sequences and hence the same target genes), that could repress SOCS6 expression. The pairing between mir-17 and SOCS6 was shown in (Fig. 12A). Luciferase assay was used to validate the ability .≤¾ of mir- 17/20 overexpression to suppress the activity of luciferase fused with SOCS6 3'UTR. As shown in (Fig. 12B), SOCS6 3'UTR luciferase was robustly suppressed to 50% of the wild type luciferase level (empty; i.e a luciferase vector without the SOCS6 3'UTR) upon mir-17 or mir-20 overexpression. Therefore, mir-17 could be a potent regulator SOCS6 expression.

[00151] Cancer cells expressing elevated levels of mir-17/20a can be predicted to have reduced levels of SOCS6 mRNA. Lower levels of SOCS6 mRNA will lead, as shown in examples 7 and 10, to lower ubiquitination of YAP and thereby to enhanced YAP stability.

[00152] Example 13:

[00153] Since a functional mir-17 miRNA binding site was identified at the 3'UTR of SOCS6, the ability of mir-17 to regulate endogenous SOCS6 mRNA expression was further investigated. An antisense oligonucleotide, which was fully complementary to the endogenous mir-17 mature sequence, was designed to inhibit mir-17 expression in human fibroblast cells. A control random scrambled oligonucleotide, which does not bind to any human miRNA sequences, was used as a control (ctrl). Upon delivery of this antisense oligonucleotide, both mir-17 and mir-20 were effectively blocked as shown in (Fig. 13B) by the robust reduction of mature miRNA expression as determined by Q-PCR. As shown in (Fig. 13 A), depletion of mir-17 and mir-20 caused upregulation of SOCS6 mRNA but not the house-keeping gene, HPRT1, suggesting mir-17/20 could modulate SOCS mRNA expression, and presumably the SOCS6 protein activity. [00154] Example 14:.

[00155] Transformed colonies induced by activated Ras^V12 were harvest upon 10-12 days of growing and RNAs extracted. The indicated miRNA levels were measured by real-time quantitative PCR. mir-17 and mir-184 were upregulated in colonies while mir-7 was downregulated. Surprisngly, cellular transformation driven by activated Ras leads to increased expression of miR-17. As shown in example 6, activated RAS leads to reduced levels of SOCS6 mRNA, whereas, mir-17 can reduce SOCS6 reporter expression via its 3' UTR (example 12).

[00156] Example 15:

[00157] The preceding examples demonstrate a mechanistic link between mir-17, SOCS6 and YAP, which could play an important role in cancer. The oncogenic miRNA mir-17 was upregulated in Ras^V12 transformed soft agar colonies. Upregulation of mir-17/20a may contribute to the downregulation of SOCS6 mRNA observed in example 6. Lower SOCS6 activity can reduce YAP ubiquitination and protein turnover, and thereby promote cellular transformation and tumor growth.

[00158] Consistent with this model, it is shown that mir-17 expression in BJ cells is able to potentiate cellular transformation by Ras^V12 or EGF expression as indicated in Fig. 15.

[00159] Example 16.

[00160] YAP activity has been shown to be involved in a variety of human solid tumors, including liver and lung cancer. The inventors observed significant reduction of SOCS6 levels in liver cancer samples (left panel, p = 0.0004) and lung cancer samples (right panel, p = 2.28E^"12) compared to normal liver and lung tissues (data from GENT database; Fig. 16).

[00161] It confirms that lower SOCS6 protein levels contribute to elevated YAP activity and via the YAP target AREG to increase RAS/MAPK activity. This is an advantageous discovery that allow physicians to identify cancers with low SOCS6 protein levels or elevated YAP protein levels to be treated with drugs that inhibit the RAS/MAPK pathway, including (but not limited to) sorafenib, and in cases where AREG is elevated with drugs that inhibit EGFR, including (but not limited to) imatinib and Herceptin. These data indicate that SOCS6, YAP, AREG and miR-17, and functionally related RNAs and proteins are useful biomarkers for determining whether a patient having cancer or suspected to have cancer is susceptible to the treatment of cancer as described herein. Fig. 18 is a working model of a positive feedback loop for cellular transformation based on the evidence provided by the examples above.

[00162] Experimental procedures

[00163] Reverse Transcribed-Polymerase Chain Reaction (RT-PCR): Reverse transcription was performed to synthesize the first strand used oligo-dT primers and Superscript RT-III (Invitrogen). The reaction was performed in PCR cycler following the protocol from Supperscript III kit (Invitrogen). After cD A synthesis, 20 μΐ cDNA was diluted in 60-80 μΐ water and used as templates for real-time quantitative PCR (Q-PCR). For each Q-PCR reaction, 12.5 μΐ POWER SYBR GREEN Master Mix (Applied Biosystems), 1 μΐ diluted PCR primer mixture (stock in 6.25 μΜ) and 6 ul DNA template were used to make a 25 μΐ PCR solution with water. Real-time Q- PCR was performed in 96 well-plate using Applied Biosystems 7500 fast real-time PCR system.]

[00164] The primers that were used in the method of the present invention are referred to in the following:

[00165] Q-SOCS6-F1 (SEQ ID No: 12) : 5 '-GTTTAGGGGTGGGGAAGTGT-3 ' [00166] Q-socs6-Rl (SEQ ID No:13): 5'-GGGCATTGAGGAGAATTTGA-3 ' [00167] Q-socs6-F2(SEQ ID No:14): 5'-CTGGCTTTGTCATTCAAGCA-3 '

[00168] Q-socs6-R2(SEQ ID No: 15): 5 '-GCATTGTGCCTTTTCTTGGT-3 '

[00169] Q- YAP-F 1 (SEQ ID No : 16) : 5 '- ACGTTC ATCTGGG AC AGC AT-3 '

[00170] Q-YAP-Rl(SEQ ID No:17): 5'-GTTGGGAGATGGCAAAGACA-3 '

[00171] Q-YAP-F2 (SEQ ID No. 8): 5 '-GCCATGTTGTTGTCTGATCG-3 '

[00172] Q-YAP-R2 (SEQ ID No: 19): 5'-CACAGCTCAGCATCTTCGAC-3'

[00173] Q-AREG-F1(SEQ ID No:20): 5 '-ACGAACCACAAATACCTGGC-3 '

[00174] Q-AREG-R1 (SEQ ID No:21): 5 ^'-TTTCACTTTCCGTCTTGTTTTG-3 ' [00175] Q-AREG-F2 (SEQ ID No:22): 5 ' ACGAACCACAAATACCTGGC 3 ' [00176] Q-AREG-R2 (SEQ ID No:22): 5 ^'-CCATTTTTGCCTCCCTTTTT-3 '

[00177] Q-survivin-F (SEQ ID No:24): 5 '-TTGGTGAATTTTTGAAACTGGA-3 ' [00178] Q-survivin-R (SEQ ID No:25): 5'-CTTTCTCCGCAGTTTCCTCA-3'

[ 001791 Immunoprecipitation:

The following reagents were used for immunoprecipitation:

1. Lysis buffer: 20 mM HEPES at pH7.9, 200 mM KC1, 2% complete protease inhibitor cocktail [Roche], 100 U/mL RNasin [Promega], 20% glycerol, 0.5%NP-40 2. Antibodies of choice: Rabbit anti-YAP, (Santa Cruz Biotechnology Inc.) 3. Protein G-agarose beads (Roche)

4. 2x Protein loading buffer: 4% SDS, 20% Glycerol, 0.12M Tris pH 6.8, and 10% beta mercaptoethanol.

HEK29T cells grown in 10-cm culture dish were transfected with pcDNA3.1-HA- SOCS6 (5 μg) by calcium phosphate method. Cells were washed with chilled PBS, trypsinized and harvested 48 hours after transfection. After a second wash with chilled PBS, the cell pellet was resuspended in 0.8ml lysis buffer per dish. The cells were lysed using a 1ml syringe and 27G1/2 needle (BD PrecisionGlide™ needle) for 5-10 times. The lysed cells were incubated in ice for 10 minutes and nuclei were removed by centrifugation at 10,000g for 10 min at 4°C. The supernatant was pipetted out and filtered with 0.45 μπι syringe filter (Sigma). The lysates were pre-cleared using 50 μΐ protein-G agarose beads per sample for one hour by rotation at 4°C. A rabbit polyclonal anti-YAP at 1 :200 dilution was added to the pre-cleared lysate and rotated at 4C for 2 hours. ). 50 μΐ of protein-G agarose beads were prepared per sample and the antibody-incubated lysates were the beads, the mix was rotated at 4°C for 2 hours. The supernatant was removed after 2 hours incubation and the beads were washed 6 times, each time with 500 μΐ lysis buffer per sample. After the final wash, the wash buffer was removed, and 50μ1 protein of loading buffer was added directly to the beads. The samples was boiled and vortexed at 1400 rpm for 10 minutes. The sample was briefly spun down and loaded on a polyacrylamide gel. The proteins were separated by SDS-PAGE and protein interactions checked by western blot analysis.

Claims

Claims:

1. A method of treating cancer in a patient or a patient suspected to suffer from cancer, wherein the method comprises increasing expression of SOCS6 protein in a cancer cell by administering to the patient an effective amount of at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor.

2. The method of claim 1, wherein the at least one Raf/MAPK pathway inhibitor is selected from the group consisting of Raf kinase inhibitor, MEK inhibitor, Erk inhibitor and combinations of the aforementioned inhibitors.

3. The method of claim 2, wherein the Raf kinase inhibitor is selected from the group consisting of 4-[4-[[4-chloro-3- (trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2- carboxamide (Sorafenib), 5-(2-(4-(2-(dimethylamino)ethoxy)phenyl)-5-(pyridin-4- yl)-lH-imidazol-4-yl)-2,3-dihydroinden-l-one oxime (SB590885), N-(3-(5-chloro- lH-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-l-sulfonamide (PLX4720), XL281 , 1 -methyl-5-(2-(4-(trifluoromethyl)- 1 H-imidazol-2-yl)pyridin-4- yloxy)-N-(4-(trifluoromethyl)phenyl)-lH-benzo[d]imidazol-2-amine (RAF265), (E)- 5-(l -(2-hydroxyethyl)-3-(pyridin-4-yl)- 1 H-pyrazol-4-yl)-2,3-dihydro- 1 H-inden- 1 -one oxime (GDC-0879), 4-[4-({[4-Chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)- 3-fluorophenoxy]-N-methylpyridine-2-carboxamide (Regorafenib), 4-Methyl-3-[[l- methyl-6-(3-pyridinyl)-lH-pyrazolo[3,4-<i]pyrimidin-4-yl]amino]-N-[3- (trifluoromethyl)phenyl]benzamide trifluoroacetate (NVP-BHG712), (E)-5-[l-(2- Hydroxy-ethyl)-3-pyridin-4-yl-lH-pyrazol-4-yl]-indan-l-one oxime (AZ628), N-(5- (3 -dimethylaminobenzamido)-2-methylphenyl)-4-hydroxybenzamide (ZM336372) and N-[3-[5-(4-chlorophenyl)-lH-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4- difluorophenyl]propane-l -sulfonamide (Vemurafenib). 4. The method of claim 2, wherein the MEK inhibitor is selected from the group consisting of (S)-(3,4-difluoro-2-((2-fluoro-4-iodophenyl)amino)phenyl)(3-hydroxy- 3-(piperidin-2-yl)cyclobutyl)methanone (XL518), 2-(2-Chloro-4-iodophenylamino)- N-(cyclopropylmethoxy)-3,4-difluorobenzamide (CI- 1040), PD035901, 6-(4-bromo- 2-chloroanilino)-7-fiuoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5- carboxamide (selumetinib), l,4-diamino-2,3-dicyano-l,4-bis(2- aminophenylthio)butadiene (U0126) and N-(3-{3-cyclopropyl-5-[(2-fluoro-4- iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,

4,6,7-tetrahydropyrido[4,3- djpyrimidin- 1 (2H)-yl }phenyl)acetamide (GSK112021 ).

5. The method of claim 2, wherein the Erk inhibitor is selected from the group consisting of PD98059, 5-(2-phenylpyrazolo[l,5-a]pyridin-3-yl)-lH-pyrazolo[3,4- c]pyridazin-3-amine (FRl 80204) and N-((2R)-2,3-dihyroxypropoxy)-3,4-difluoro-2- ((2-fluoro-4-iodophenyl)amino)-benzamide (PKI-ERK-005).

6. The method of claim any one of the preceding claims, wherein the at least one PI3K pathway inhibitor is selected from the group consisting of (\S,6bR,9aS,nR,\ lbi?)l l-(Acetyloxy)-l,6b,7,8,9a, 10,11,1 lb-octahydro-1- (methoxymethyl)-9a,l lb-dimethyl-3H-furo[4,3,2-6?e]indeno[4,5,-/i]-2- i]-2- benzopyran-3,6,9-trione (Wortmannin), 18-Norandrosta-5,8,l l,13-tetraeno[6,5,4- bc]furan-3,7,17-trione, l-hydroxy-,(lb)-(demethoxyviridin), and 2-(4-Morpholinyl)- 8-phenyl-4H-l-benzopyran-4-one (LY-294002).

7. The method of any one of the preceding claims, wherein cancer is a solid tumor type of cancer. -

8. The method of any one of the preceding claims, wherein cancer is selected from the group consisting of colon cancer, lung cancer, ovarian carcinoma and hepatocellular carcinoma (HCC).

9. The method of claim 8, wherein the cancer is hepatocellular carcinoma.

10. A method of determining the susceptibility of a patient suffering or suspected to suffer from cancer, to a treatment with at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor, wherein the method comprises:

comparing mR A level and/or protein expression level and/or miRNA level results for SOCS6 protein and/or YAP protein and/or AREG protein (Locus: NM_001657) and/or Survivin (BRIC5; Locus: NM_001012271) and/or mir-17 and/or connective tissue growth factor (CTGF; Locus: NM_001901) and/or cysteine-rich, angiogenic inducer, 61 (CYR61; Locus: NM_001554) or any combination of transcriptional targets of YAP obtained from a patient suffering or suspected to suffer from cancer with the mRNA level and/or protein expression level results of a control group;

wherein an mRNA level and/or protein expression level and/or miRNA level for SOCS6 protein and/or YAP protein and/or AREG protein (Locus: NM 001657) and/or Survivin (BRIC5; Locus: NM_001012271) and/or mir- 17 and/or connective tissue growth factor (CTGF; Locus: NM 001901) and/or cysteine-rich, angiogenic* inducer, 61 ,(CYR61; Locus: NM_001554) or any combination of transcriptional targets of YAP in a patient suffering or suspected to suffer from cancer that differs from the control expression level for SOCS6 protein and/or YAP protein and/or AREG protein (Locus: NM_001657) and/or Survivin (BRIC5; Locus: NM_001012271) and/or mir- 17 and/or connective tissue growth factor (CTGF; Locus: NM 001901) and/or cysteine-rich, angiogenic inducer, 61 (CYR61; Locus: NM_001554) or any combination of transcriptional targets of YAP indicates that the patient is susceptible to a treatment comprising the at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor.

11. The method of claim 10, wherein the control group comprises at least one individual not having cancer and/or having previously been treated against cancer.

12. The method of claim 10, wherein the control group comprises tissue sample obtained from the patient suffering or suspected to suffer from cancer wherein the tissue sample obtained from this patient is not affected by the cancer.

13. The method of any one of claims 10 to 12, wherein an increased mRNA level and/or protein expression level for YAP protein in a patient suffering or suspected to suffer from cancer compared to the control expression level for YAP protein indicates that the patient is susceptible to a treatment comprising the at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor.

14. The method of any one of claims 10 to 13, wherein an increased mRNA level and/or protein expression level for AREG protein in a patient suffering or suspected to suffer from cancer compared to the control mRNA level and/or protein expression level for AREG protein indicates that the patient is susceptible to a treatment comprising the at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor.

15. The method of any one of claims 10 to 14, wherein a decreased mRNA level and/or protein expression level for SOCS6 protein in a patient suffering or suspected to suffer from cancer compared to the control mRNA level and/or protein expression level for SOCS6 protein indicates that the patient is susceptible to a treatment comprising the at least one Raf/MAPK pathway inhibitor and/or at least one PI3K pathway inhibitor.

16. The method of any one of claims 10 to 15, wherein the mRNA levels are measured using reverse transcriptase polymerase chain reaction for quantitating the mRNA levels encoding the proteins or wherein the protein levels are measured using antibodies against the proteins, or a combination of any of the aforementioned methods.

17. A method of treating a disease selected from the group consisting of cancer in a patient, wherein the method comprises sequestering a micro-RNA capable of decreasing SOCS6 protein levels.

18. The method of claim 17, wherein the method comprises administering to the patient an antisense oligonucleotide suitable to sequester a micro-RNA capable of decreasing SOCS6 protein levels.

19. The method of claim 18, wherein the antisense oligonucleotide suitable to sequester miRNA is selected from the group consisting of

Anti-mir-17: 5' - CUACCUGCACUGUAAGCACUUUG - 3';

Anti-mir-20a: 5 '- CUACCUGCACUAUAAGCACUUUA -3 ';

Anti-mir-183 : 5 '- AGUGAAUUCUACCAGUGCCAUA-3 ';

Anti-mir- 155: 5 '- ACCCCU AUC ACGAUU AGC AUUAA-3 ' ;

Anti-mir-21 : 5'-UCAACAUCAGUCUGAUAAGCUA-3 ';

Anti-mir-30a: 5'-CUUCCAGUCGAGGAUGUUUACA-3';

Anti-mir-25: 5 ' -UC AG ACCG AG AC AAGUGC A AUG-3 ' ; Anti-mir-92a: 5 '-ACAGGCCGGGACAAGUGC AAUA-3 ';

Anti-mir-92b: 5 ' -GGAGGCCGGGACGAGUGC AAUA-3 ' ;

Anti-mir-32: 5'- UGCAACUUAGUAAUGUGC AAUA-3';

Anti-mir-19a: 5'-UCAGUUUUGCAUAGAUUUGCACA-3';

Anti-mir-27a: 5'-GCGGAACUUAGCCACUGUGAA-3'; and

Anti-mir-27b: 5'-GCAGAACUUAGCCACUGUGAA-3'; and functional equivalents thereof.

20. The method of claim 19, wherein the functional equivalents thereof comprise at least one modified or substituted nucleotide.

21. The method of claim 20, wherein the modified nucleotides comprise modified bases selected from the group consisting of phosphorothioate, methylphosphonate, peptide nucleic acids, 2'-0-methyl, fluoro- and carbon, methylene and other locked nucleic acid (LNA) molecules.

22. The method of any one of claims 19 to 20, wherein the functional equivalents comprise between about 8 to 30, or 8 to 25, or 20 to 25, or 10 to 20, or 12 to 16 nucleotides.

23. The method of any one of claims 17 to 22, wherein the micro-RNA is selected from the group consisting of

5 '-CAAAGUGCUUACAGUGCAGGUAG-3 ' (hsa-miR-17);

5 '-UAAAGUGCUUAUAGUGCAGGUAG-3 ' (hsa-miR-20a);

5 '-UAUGGCACUGGUAGAAUUCACU-3 ' (hsa-miR-183-5p);

5 ' -UUA AUGCU AAUCGUG AU AGGGGU-3 ' (hsa-miR- 155);

5 '-UAGCUUAUCAGACUGAUGUUGA-3 ' (hsa-miR-21 );

5'-UGUAAACAUCCUCGACUGGAAG-3' (hsa-miR-30a);

5'- CAUUGCACUUGUCUCGGUCUGA-3' (hsa-miR-25);

5'- UAUUGCACUUGUCCCGGCCUGU-3' (hsa-miR-92a);

5'- UAUUGCACUCGUCCCGGCCUCC-3' (hsa-miR-92b);

5'- UAUUGCACAUUACUAAGUUGCA-3' (hsa-miR-32);

5'- UGUGC AAAUCUAUGC AAAACUG A-3 ' (hsa-miR- 19a);

5'-UUCACAGUGGCUAAGUUCCGC-3' (hsa-miR-27a); and 5'- UUCACAGUGGCUAAGUUCUGC-3' (hsa-miR-mir-27b).

24. The method of any one of claims 17 to 22, wherein the micro-RNA is selected from the group consisting of miR-203; miR-499/499-5p; miR-183; miR-23ab; miR- 216/216b; miR-128; miR-204/211; miR-192/215; miR-15/16/195/424/497; miR-144; miR-218; miR-17-5p/20/93.mr/106/519.d; miR-30a/30a-5p/30b/30b-5p/30cde/384- 5p; miR-216/216a; miR-182; miR-208/208ab; miR-21/590-5p; miR-26ab/1297; miR- 25/32/92/92ab/363/367; miR-141/200a; miR-19; miR-190; miR-200bc/429; miR- 33/33ab; „ miR-130/301; miR-137; miR-27ab; miR-34a/34b-5p/34c/34c- 5p/449/449abc/699; miR-142-3p; miR-138; miR-96/1271; miR-155; miR-193ab; miR-101; miR-139-5p; miR-338/338-3p; miR-24; miR-503; miR-494; miR-329/362- 3p; miR-186; miR-590/590-3p; miR-340/340-5p; miR-421; miR-296/296-3p; miR- 377; miR-300; miR-376c; miR-431; miR-544; miR-382; miR-149; miR-592/599; miR-495/1192; miR-326/330/330-5p; miR-539; miR-378/422a; miR-361/361-5p; miR-758; miR-542/542-3p; miR-410; miR-342/342-3p; miR-339-5p; miR-504; miR- 154; miR-374/374ab; miR-224; and miR-384/384-3p. (Sequences of these microRNAs can be obtained at www.mirbase.org) 25. A kit for use in the method of any one of claims 10 to 16, wherein the kit comprises means to detect SOCS6 protein levels and or mRNA levels in a sample obtained from a patient suffering or suspected to suffer from cancer.