WO2012153139A1 - Compositions/substances for the treatment of renal cell carcinoma - Google Patents

Abstract

A composition comprising a slingshot phosphatase inhibitor for use in the treatment of renal cell carcinoma. The composition is also for use in the treatment of renal cell carcinoma associated with Birt-Hogg-Dubé syndrome. Also provided is a composition comprising an inhibitor of a slingshot phosphatase for use in the treatment of cancer associated with FLCNinactivation. The slingshot phosphatase inhibitor may be an RNAi molecule specific to a slingshot phosphatase gene.

Description

COMPOSITIONS/SUBSTANCES FOR THE TREATMENT OF RENAL CELL

CARCINOMA

This invention relates to treatment of diseases. More particularly, this invention relates to compositions for use in the treatment of cancers such as renal carcinoma, and in particular Birt-Hogg-Dube syndrome.

Renal cell carcinoma (RCC) accounts for 2-3% of all cancers and is the most common type of kidney cancer in adults. RCC is a heterogeneous disorder with a number of histopathological subtypes, although conventional clear cell RCC (ccRCC) accounts for more than 75% of cases of RCC. Non-clear-cell forms of RCC comprise papillary (or chromophil) RCC, chromophobe tumours, oncocytoma, collecting duct carcinoma and the rare medullary carcinoma. Surgical resection is currently the preferred treatment for locally confined RCC and can often achieve a cure in the earlier stages of RCC. RCC has traditionally been considered to be largely resistant to radiotherapy and in vitro studies have shown that renal cancer cells are among the least radiosensitive of human cell types. Furthermore, the majority of advanced RCC tumours have proved to be resistant to cytotoxic agents and therefore chemotherapy has had a very limited role in the treatment of metastatic renal cancer.

Most cases of RCC are sporadic and only about 3% of all cases have a genetic cause. However, investigations into rare inherited forms of RCC have provided seminal insights into the molecular pathogenesis of both familial and sporadic RCC.

Von Hippel-Lindau (VHL) disease is a dominantly inherited multisystem familial cancer syndrome characterised by the development of clear cell renal cell carcinoma (ccRCC) as well as haemangioblastomas, pancreatic lesions and phaeochromocytoma. VHL is the most common cause of inherited RCC.

The identification of the gene for VHL disease has led to recognition that the most frequent genetic event in the evolution of sporadic ccRCC is somatic inactivation of the VHL tumour suppressor gene (TSG). Further work has led to an understanding that VHL TSG inactivation leads to dysregulation of the HIF-1 and HIF-2 transcription factors and activation of hypoxia-responsive gene pathways (Latif et al., Science 1994;260:1317-20; Foster et al., Cancer 1994;69:230-4; Gnarra et al., Nat Genet 1994;7:85-90; Clifford et al., Genes Chromosomes Cancer 1998;22:200- 9; Maxwell et al., Nature 399:271 -275, 1999; Banks et al., Cancer Res 2006;66:2000-7 ).

It is now known that under normoxic conditions, the VHL tumour suppressor gene product, pVHL, functions in a ubiquitin ligase complex that targets hypoxia-response transcription factor subunits (HIF-1 a and HIF-2a) for destruction in the proteasome. VHL inactivation results in elevated levels of HIF-1 and HIF-2, leading to overexpression of target genes involved in growth and angiogenesis, such as VEGF and PDGF.

These findings have provided a rationale for the use of drugs such as sorafenib and sunitinib (inhibitors of HIF target gene pathways) in the treatment of metastatic RCC (Patel et al., Br J Cancer. 2006; 94:614-9; Motzer et al., J Clin Oncol. 2009; 27:3584-90).

Birt-Hogg-Dube (BHD) syndrome is another dominantly inherited familial cancer syndrome associated with susceptibility to RCC. BHD is also associated with benign skin fibrofolliculomas (hamartomatous tumours of the hair follicle) and multiple lung cysts and spontaneous pneumothrorax (Toro et al., J. Med. Genet. 2008; 45: 321 - 331 ); BHD-associated renal tumours are of variable histopathology but are often chromophobe RCC/oncocytoma. BHD syndrome results from inactivating mutations in the folliculin (FLCN) gene (Nickerson et al., Cancer Cell 2002; 2: 157-164; Schmidt et al., Am J Hum Genet. 2005; 76: 1023-33; Lim et al., Hum Mutat. 2010 Jan 31 (1 ):E1043-51 ) and renal tumours from BHD patients demonstrate somatic FLCN loss. The precise function of the FLCN gene product is still being elucidated, but folliculin (and the folliculin interacting proteins FNIP1 and FNIP2) have been linked to the mTOR and AMPK signalling pathways (Baba et al., Proc. Natl. Acad. Sci U.S.A. 2006; 103: 15552- 15557; Hasumi et al., Gene 2008; 415: 60-7; Takagi et al., Oncogene 2008; 27: 5339-47). In mice with kidney-targeted homozygous inactivation of Flcn, renal tumours and cysts developed with activation of mTOR and the mTOR inhibitor rapamycin diminished kidney pathology and increased survival (Baba et al., J. Natl. Cancer Inst. 2008; 100: 140-154; Chen et al., PloS ONE 2008; 33: e3581 ). mTOR inhibitor drugs (e.g. Temsirolimus, Everolimus, etc) have shown promise as treatments for metastatic RCC (Molina and Motzer Clin Genitourin Cancer 2008 Dec; 6 Suppl 1 :S7-13 ). Patients with BHD syndrome are typically offered renal imaging to facilitate early detection of RCC. However, some patients may only be diagnosed after presentation with advanced RCC. Treatment of metastatic RCC is challenging for both familial and sporadic cases. Although occasional patients may respond to immunotherapy with the cytokines interferon and interleukin-2, recently treatment with targeted therapies to HIF downstream targets (e.g. Sunitinib, Sorafenib, Bevacizumab, etc) and the mTOR pathway (e.g. Temsirolimus, Everolimus) has emerged as the most frequent management strategy. However these agents, whilst prolonging life, are not cytotoxic and so the identification of targeted cytotoxic agents would be a significant advance.

Chromomycin A3 (ChA3) (an aureolic acid compound) has been identified as a HIF- dependent cytotoxin. ChA3 shows discriminate killing of VHL-deficient cells in ccRCC cell lines (Sutphin et al., Cancer Res 2007; 67 (12); 5896 - 5902). It has been shown that overexpression of HIF-2a in VHL-positive clear cell RCC cell lines phenocopies the effect of VHL inactivation on susceptibility to ChA3 toxicity. However, ChA3 does not show differential growth inhibitory activity in FLCN- deficient and FLCN-wild type cell lines suggesting it is not likely to be useful as drug treatment for BHD syndrome. There is a need for identification of alternative treatments for renal cell carcinoma. Furthermore, there is a need for a treatment that targets cells deficient in the FLCN gene and diseases associated with such defects.

According to a first aspect of the present invention there is provided a composition comprising a slingshot phosphatase inhibitor for use in the treatment of renal cell carcinoma.

Slingshot phosphatases are a family of protein phosphatases that play a role in regulating the activity of cofilin which regulates the assembly and disassembly of actin filaments. The slingshot family of phosphatases (SSH) includes in particular protein phosphatase Slingshot homolog 1 (SSH1 ), protein phosphatase Slingshot homolog 2 (SSH2), and protein phosphatase Slingshot homolog 3 (SSH3).

The inventors have surprisingly found that some renal cell carcinoma cells are sensitive to slingshot phosphatase inhibition. An agent comprising a slingshot phosphatase inhibitor can therefore be used in the treatment of renal cell carcinomas that are sensitive to slingshot phosphatase inhibition.

The invention provides use of a slingshot inhibitor in the treatment of renal cell carcinoma. The slingshot phosphatase inhibitor may inhibit one or more members of the slingshot phosphatase family.

The terms composition and agent can be used interchangeably herein. The composition is particularly useful for use in the treatment of renal cell carcinoma associated with Birt-Hogg-Dube syndrome.

Also provided is a composition comprising a slingshot phosphatase inhibitor for use in the treatment of cancer associated with FLCN inactivation. In particular, there is provided a composition for use in the treatment of renal cell carcinoma associated with FLCN inactivation.

The inventors have also surprisingly found that cells in which FLCN has been inactivated are sensitive to slingshot inhibition as compared to wild type cells.

Gene inactivation may come about, for example, due to a mutation in the gene, the absence of the gene, or defective expression of the gene product. Genes may be inactivated by genetic mutation. Mutations may include point mutations (transitions or transversions), insertions or deletions. Point mutations may be silent (code for the same amino acid), missense (code for a different amino acid) or nonsense (code for a stop). Insertions may alter splicing of the mRNA or cause a frameshift altering the gene product. Deletions may also alter the reading frame thereby affecting the gene product. On a larger scale, mutations in chromosomal structure can include amplifications or gene duplications, deletions of chromosomal regions, chromosomal translocations, interstitial deletions and chromosomal inversions. The composition may be used as a cytotoxic agent against renal cell carcinoma cells, such as FLCN-null renal cell carcinoma cells. Cytotoxic agents are agents that are toxic to cells and can lead to a variety of outcomes for cells. Cells may stop actively growing and dividing, or may undergo necrosis, or the cells may undergo programmed cell death (apoptosis). Cells undergoing necrosis lose membrane integrity, exhibit rapid swelling, shut down metabolism and release the cell contents into their surroundings. The process of apoptosis is an ordered sequence of events characterised by a change in refractive index, cytoplasmic shrinkage, nuclear condensation and DNA cleavage. Apoptotic cells shut down metabolism, lose membrane integrity and lyse.

The composition may be used in the inhibition of growth of renal cell carcinoma cells and/or cancer cells associated with FLCN inactivation. According to a further aspect of the present invention there is provided a composition comprising a slingshot phosphatase inhibitor for use in the differential growth inhibition of FLCN-null cells over FLCN-wild type cells. The term "inhibiting" means decreasing, slowing or stopping. Thus, a composition of this invention can decrease, slow or stop the growth of a tumour cell. As used herein, "growth" means increase in size or proliferation or both. Thus, a composition of this invention can inhibit a tumour cell from becoming larger and/or can prevent the tumour cell from dividing and replicating and increasing the number of tumour cells. A cell can be in vitro. Alternatively, a cell can be in vivo and can be found in a subject. A "cell" can be a cell from any organism including, but not limited to, a bacterium. Preferably the slingshot phosphatase inhibitor inhibits Protein Phosphatase Slingshot Homolog 2 (SSH2). Suitably, the slingshot phosphatase inhibitor is an inhibitor of the activity of SSH2. The inventors have found that renal cell carcinoma cells are particularly sensitive to SSH2 inhibition. References herein to the inhibition of a particular slingshot phosphatase encompass inhibition of any isoform thereof.

The slingshot phosphatase inhibitor may inhibit the activity of one or more slingshot phosphatases selected from the group consisting of SSH2, SSH 1 and SSH3, or any isoform thereof. Preferably the composition is an inhibitor of more than one slingshot phosphatase enzyme. Preferably the composition comprises first and second slingshot phosphatase inhibitors. Preferably the composition comprises more than one slingshot phosphatase inhibitor, each for inhibiting a different member of the slingshot phosphatase family. The slingshot phosphatase inhibitors may inhibit Protein Phosphatase Slingshot Homolog 2 (SSH2) and Protein Phosphatase Slingshot Homolog 1 (SSH1 ). Alternatively the slingshot phosphatase inhibitors may inhibit Protein Phosphatase Slingshot Homolog 2 (SSH2) and Protein Phosphatase Slingshot Homolog 3 (SSH3). The inventors have found that multiple SSH inhibition can potentiate SSH2 knockdown effect. Suitably, use of more than one different inhibitor of a slingshot phosphatase is provided.

Preferably the slingshot phosphatase inhibitors inhibit Protein Phosphatase Slingshot Homolog 2 (SSH2), Protein Phosphatase Slingshot Homolog 1 (SSH1 ), and Protein Phosphatase Slingshot Homolog 3 (SSH3).

Preferably the slingshot phosphatase inhibitor is an RNAi molecule specific to a slingshot phosphatase gene. The RNAi molecule is suitably directed against slingshot phosphatase mRNA. Suitably, the slingshot phosphatase inhibitor may be a short interfering nucleic acid molecule that down-regulates expression of a slingshot phosphatase gene by RNA interference. The inventors have found that selective inhibition of a slingshot phosphatase enzyme by using the mechanism of RNA Interference (RNAi) targeted against its mRNA can be used to treat renal cell carcinoma, including renal cell carcinoma associated with FLCN inactivation.

The slingshot phosphatase inhibitor may comprise more than one type of RNAi molecule, each being specific to a particular slingshot phosphatase gene.

Preferably the slingshot phosphatase inhibitor is an RNAi molecule specific to SSH2. Suitably, the RNAi molecule inhibits the expression of protein encoded by SSH2 or an isoform thereof. Reference herein to inhibition of the expression of a protein encoded by a particular gene should be taken to include inhibition of the expression of any isoform thereof. Preferably the slingshot phosphatase inhibitor comprises an RNAi molecule specific to a first slingshot phosphatase gene and an RNAi molecule specific to a second slingshot phosphatase gene. The slingshot phosphatase inhibitor may comprise RNAi molecules specific to SSH2 and SSH1 . Alternatively the slingshot phosphatase inhibitor may comprises RNAi molecules specific to SSH2 and SSH3.

Preferably the slingshot phosphatase inhibitor comprises an RNAi molecule specific to a first slingshot phosphatase gene, an RNAi molecule specific to a second slingshot phosphatase gene, and an RNAi molecule specific to a third slingshot phosphatase gene.

Preferably the slingshot phosphatase inhibitor comprises RNAi molecules specific to SSH2, SSH1 and SSH3.

Preferably the RNAi molecule comprises siRNA.

Preferably the siRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding a slingshot phosphatase. Suitably, the siRNA may have at least one modified nucleotide. Preferably the siRNA comprises at least one nucleotide sequence from the group comprising SEQ ID NOs: 1 to 18. Suitably the siRNA comprises a sense strand and its corresponding antisense strand from the group comprising SEQ ID NOs: 1 to 18.

Preferably the RNAi molecule is a short hairpin RNA (shRNA).

Alternatively the RNAi molecule may be miRNA, DNAzyme, ribozyme, morpholino or other RNAi molecule targeted against mRNA associated with slingshot phosphatase. Preferably the composition further comprises a delivery vehicle, such as but not limited to a viral vector, an antibody, an aptamer or a nanoparticle, for delivering the RNAi molecule to a target cell. According to a further aspect of the invention there is provided a cell comprising an RNAi molecule specific to a slingshot phosphatase gene.

There is also provided a vector comprising an RNAi molecule specific to a slingshot phosphatase gene and a cell comprising such the vector.

Said cell or vector can be used in the treatment of renal cell carcinoma, Birt-Hogg- Dube syndrome, and/or cancer associated with FLCN inactivation.

According to a further aspect of the invention there is provided a method of identifying a candidate compound for treating renal cell carcinoma, the method comprising identifying an inhibitor of a slingshot phosphatase by contacting a cell expressing a slingshot phosphatase with a candidate compound and determining whether activity of slingshot phosphatase is inhibited, wherein inhibition of activity of slingshot phosphatase indicates that the candidate compound is a candidate compound for treating renal cell carcinoma.

According to a further aspect of the invention there is provided a method of identifying a composition for treating cancer, wherein the cancer is characterised by FLCN inactivation, the method comprising identifying an inhibitor of a slingshot phosphatase by contacting a cell expressing a slingshot phosphatase with a candidate compound and determining whether activity of slingshot phosphatase is inhibited, wherein inhibition of activity of slingshot phosphatase indicates that the candidate compound is a candidate compound for treating cancer characterised by FLCN inactivation.

The invention provides methods of identifying compositions comprising inhibitors of enzymes of slingshot phosphatase. An inhibitor of such an enzyme is a substance which reduces, attenuates, decreases or eliminates the expression and/or activity of such an enzyme. Expression in this context is used to refer to any of the steps of transcription and translation. Activity of the enzyme in this context is used to refer to enzymatic activity of a polypeptide encoded by a slingshot phosphatase gene. An inhibitor may exert inhibition via any mechanism.

The slingshot phosphatase inhibitor may be a competitive inhibitor of slingshot phosphatase. A competitive inhibitor can be bound by an enzyme in place of the substrate. The enzyme cannot bind the inhibitor and the substrate at the same time. A competitive inhibitor may bind in the active site of the enzyme and therefore prevent the binding of the substrate. A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate.

For example, such a method may comprise the steps of providing a cell expressing a slingshot phosphatase inhibitor, contacting the cell with at least one substrate and at least one candidate compound, and determining whether the substrate is cleaved. Preferably the composition further comprises mithramycin or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective derivative or mithramycin.

The inventors have previously found that mithramycin could be used in the treatment of renal cell carcinoma (see previously filed application number PCT/GB1 1/050313). The inventors have surprisingly found that inhibition of one or more slingshot phosphatases potentiates the activity of mithramycin in treating renal cell carcinoma and/or cancer associated with FLCN inactivation. Mithramycin (also known as aurelic acid, plicamycin or mitramycin) is an aureolic- acid type polyketide antibiotic produced by various soil bacteria of the genus Streptomyces. Mithramycin has formula:

Mithramycin has been used as a targeted therapy to treat hypercalcaemia in patients with bone metastases, Paget's disease, testicular carcinoma and leukaemia (Yuan et al, Cancer 2007; 1 10: 2682-2690). It has also been shown that mithramycin has potential as a neuroprotective drug for the alleviation of symptoms associated with β-thalassemia and sickle cell anaemia.

Mithramcyin binds to GC-rich regions in the minor groove of DNA and inhibits the transcription of genes with GC-rich promoters. Mithramycin therefore inhibits transcription of genes regulated by transcription factors that bind to such sequences, such as the Sp1 family. Sp1 has been shown to be involved in the regulation of the angiogenesis stimulator vascular endothelial growth factor (VEGF), and the use of mithramycin for the inhibition of angiogenesis in mammals has been reported.

Also provided according to the invention is a composition comprising a slingshot phosphatase inhibitor for use in therapy.

Also provided according to the invention is use of a composition comprising a slingshot phosphatase inhibitor for the manufacture of a medicament for the treatment of renal cell carcinoma.

The invention also provides a method of treating renal cell carcinoma comprising administering to a subject a composition comprising a slingshot phosphatase inhibitor. The "subject" can include domesticated animals, such as cats, dogs etc., livestock (e.g. cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g. mouse, rabbit, rat, guinea pig etc.) and birds. Preferably, the subject is a mammal such as a primate, and more preferably a human. The composition may further comprise a pharmaceutically acceptable carrier. Suitably the composition is administered in amount that is effective to treat renal cell carcinoma in a subject. In general an "effective amount" of a compound or composition is that amount needed to achieve the desired result or results.

A composition of the instant invention may be administered to a subject by any of number of routes of administration including, for example, orally (for exampl drenches as in aqueous or non-aqueous solutions or suspension, tablets, boluses, powders, granules, pastes for application to the tongue); sublingually, anally, rectally, or vaginally (for example as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecal^ as for example a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); or topically (for example as a cream, ointment or spray applied to the skin). The composition may also be formulated for inhalation. As used herein, the term "RNA" refers to a molecule comprising at least one ribonucleotide residue. By "ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2' position of a [beta]-D-ribofuranose moiety. The terms encompass double stranded RNA, single stranded RNA, RNAs with both double stranded and single stranded regions, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA, or analog RNA, that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material. Nucleotides in the RNA molecules of the presently disclosed subject matter can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of a naturally occurring RNA.

RNA interference (RNAi) is a natural process by which living cells can control which genes are expressed or suppressed. As used herein, the term "RNAi" refers to a mechanism that silences specific genes by inhibiting an RNA molecule and stopping or at least substantially reducing the expression of the protein encoded by this RNA molecule. If the target protein has a function in the cell, RNAi approaches can result in loss of that function. As such, RNAi technology is an attractive therapeutic tool to modulate the expression of genes in a way to suppress disease. RNAi can be mediated by several natural and synthetic constructs, including double stranded RNA (dsRNA), or smaller dsRNA known as small interfering RNAs (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or synthetic hammerhead ribozymes. These can be referred to as examples of RNAi molecules. The terms "small interfering RNA", "short interfering RNA" and "siRNA" are used interchangeably and refer to any nucleic acid molecule capable of mediating RNA interference (RNAi) or gene silencing. See e.g., Bass, Nature 41 1 :428-429, 2001 ; Elbashir et al., Nature 41 1 :494-498, 2001. Non-limiting examples of suitable siRNA molecules are shown in Table 1 having SEQ ID Nos: 1 -18, wherein the siRNA comprises the sense and antisense regions (19 nucleotide sequences in upper case).

The siRNA may comprise a double stranded polynucleotide molecule comprising complementary sense and antisense regions, wherein the antisense region comprises a sequence complementary to a region of a target nucleic acid molecule (for example, an mRNA encoding an SSH, such as SSH 1 , SSH2 or SSH3). Suitably, the antisense strand comprises a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an SSH gene, and wherein the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein said siRNA, upon contact with a cell expressing said SSH gene, inhibits the expression of said SSH gene by at least 25%, or preferably by least 50%. The siRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the siRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of an SSH gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30, preferably between 18 and 25, yet more preferably between 19 and 24 base pairs in length, and yet more preferably 19 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more preferably between 18 and 25, yet more preferably between 19 and 24 nucleotides in length. The siRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s). The siRNA can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer. In specific embodiments, the antisense strand of the siRNA is selected from the group comprising the antisense sequences of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 16, 18 and the sense sequence is selected from the group consisting of the corresponding sense sequences of SEQ ID Nos: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17. In a preferred embodiment, the SSH gene is the SSH2 gene. In other preferred embodiments, RNAi molecules targeted against multiple SSH genes can be used, including RNAi molecules targeted against two or more of SSH1 , SSH2 and SSH3. In one preferred embodiment, an RNAi molecule targeted against SSH2 in combination with an RNAi molecule targeted against one of SSH1 or SSH3 can be used.

It will be understood that siRNAs other than the examples in Table 1 may be used for RNAi. Furthermore, siRNAs based on one or more of the sequences of Table 1 may be used, but comprising shorter or longer sequences.

At least one end of the siRNA may have a single -stranded nucleotide overhang of 1 to 4, generally 2 nucleotides. siRNAs having at least one nucleotide overhang are known to those skilled in the art to have superior inhibitory properties than their blunt-ended counterparts. Generally, the single-stranded overhang is located at the 3'-terminal end of the antisense strand and/or, at the 3'-terminal end of the sense strand.

Alternatively, the siRNA may comprise a single stranded polynucleotide having self- complementary sense and antisense regions, wherein the antisense region comprises a sequence complementary to a region of a target nucleic acid molecule. As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompass chemically modified nucleotides and non- nucleotides.

The siRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the siRNA of the invention contains no more than 3 mismatches. The terms "short hairpin RNA" and "shRNA" are used interchangeably and refer to any nucleic acid molecule capable of generating siRNA. Suitably, the shRNA may comprise a polynucleotide having one or more loop structures and a stem comprising self complementary sense and antisense regions, wherein the antisense region comprises a sequence complementary to a region of a target nucleic acid molecule, and wherein the polynucleotide can be processed either in vivo or in vitro to generate an active siRNA capable of mediating RNAi. Alternatively retroviral vectors encode shRNA, which are processed intracellularly, to generate siRNA that silence the expression of a target gene, such as a gene encoding a slingshot phosphatase. Another RNAi molecule that could be used as the inhibitor of a slingshot phosphatase is a morpholino. Morpholinos are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages. Morpholino nucleic acids typically comprise heterocyclic bases attached to the morpholino ring. A number of linking groups may link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups has been selected to give a non-ionic oligomeric compound. Morpholino-based oligomeric compounds are non- ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins. The invention will now be described by way of example with reference to the figures, in which:

Figure 1 shows graphs relating to the results of exponential growing cells UOK257- FLCN- and UOK257-FLCN+, FTC-12 and FTC-22-FLCN that were exposed to mithramycin, or transfected with SSH2 siRNA (A), or in combination of SSH2 siRNA and mithramycin (B) at indicated concentrations for 72 hours and caspase3/7 activity were then determined as described in "Materials and Methods";

Figure 2 relates to exponential growing cells FTC-12 and FTC-22-FLCN were transfected with siRNAs at indicated concentrations for 72 hours and caspase3/7 activity and cell viability were then determined as described in "Materials and Methods";

Figure 3 relates to exponential growing cells UOK257-FLCN- and UOK257-FLCN+ were transfected with siRNAs at indicated concentrations for 72 hours and caspase3/7 activity and cell viability were then determined as described in "Materials and Methods";

Figure 4 relates to validation of SSH2 siRNA knockdown by Real time PCR. Exponential growing FTC-12 and FTC-22-FLCN cells were transfected with either luciferase or SSH2 siRNAs at indicated concentrations for 72 hours and RNA was then extracted by RNAeasy(RTM) as described in "Materials and Methods". SSH1 , SSH2 and SSH3 transcript levels after SSH2 siRNA treatment were examined by Taqman(RTM) gene assay and normalized by GAPDH expression before compared with Luciferase siRNA control;

Figure 5 is a schematic diagram showing actin reorganization involving the slingshot family;

Figure 6 relates to cellular protein expression in UOK cells with/without FLCN Expression after siRNA transfection for 48 hours. Exponential growing cells were mixed with diluted siRNA INTERFERin(RTM) mixture and transfected continuously for 48 hours and then western blotting was carried out by using antibodies as described in "Materials and Methods"; Figure 7 relates to exponential growing cells FTC12-FLCN- and FTC22-FLCN+ were transfected with single siRNAs, or combination of multiple siRNAs at indicated concentrations for 72 hours and caspase3/7 activity were then measured as described in "Materials and Methods"; Figure 8 relates to cellular protein expression in FTC cells with/without FLCN Expression after single siRNA or multiple siRNAs transfection (10nM, 48 hours). Exponential growing cells were mixed with diluted siRNA INTERFERin(RTM) mixture and transfected continuously for 48 hours and western blotting was then carried out by using antibodies as described in "Materials and Methods";

Figure 9 relates to: (A) relates to exponential growing cells were transfected with SSH 1 , SSH2, or SSH3 siRNAs at indicated concentrations for 72 hours and caspase3/7 activity were then measured as described in "Materials and Methods". (B) Cellular transcript FLCN and SSH2 levels measured by Real time PCR. RNA was extracted from exponential growing cells by RNAeasy(RTM) as described in "Materials and Methods". FLCN, and SSH2 transcript levels were examined by Taqman(RTM) gene assay and normalized by GAPDH expression before compared with those from UOK-257; Figures 10 to 14 relate to experiments, some of which are similar to those of previous Figures, but wherein UOK-257-2 cells (also referred to herein as "UOK257- 2-FLCN+ ", "UOK-2", or "U2") were used in place of UOK-FLCN; Figure 10 shows data similar to that for Figure 1 but including data for UOK-257-2 - FLCN+ cells instead of UOK257-FLCN+;

Figure 11 shows data similar to that for Figure 3 but including data for UOK-257-2- FLCN+ cells instead of UOK257-FLCN+;

Figure 12 shows Western Blots similar to that of Figure 6, but for UOK and UOK- 257-2 cells ("U" and "U2" respectively);

Figure 13 shows data similar to that of Figure 7, but including data for UOK-257-2 cells;

Figure 14 relates to cell cycle population changes in relation to single or multiple SSH siRNA treatment in UOK and UOK-2 cells. Exponential growth cells were transfected with siRNA at indicated concentrations for 72 hours. At the end of incubation, cells were fixed and stained with PI as indicated in Materials and Methods before being scanned with the Acumen eX3 cytometer.

Materials and Methods Cell Lines and Cell Culture.

Human renal carcinoma of BHD origin cells UOK-257 (UOK-FLCN^") and FLCN transfected UOK-257 cells UOK-FLCN⁺ (Yang et al., 2008) were characterized and kindly provided by Dr Marston Linehan and Dr. Laura S Schmidt (Urologic Oncology Branch, Centre for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA). The previously reported FLCN mutation was confirmed in both cell lines and the expected expression of folliculin in the two cell lines was demonstrated (see Examples section). For FLCN transfected UOK-257 cells, UOK-FLCN was used for the results shown in Figures 1 to 7 and UOK-257-2 was used for the results shown in Figures 10 to 14. UOK-257-2 is a cell line in which FLCN expression has been restored by lentivirus infection. FTC-133 cells, originally derived from a lymph node metastasis of a follicular thyroid carcinoma from a 42-year-old male, were purchased from ECACC (Salisbury, United Kingdom) and a FLCN containing construct and an empty vector were introduced into parental FTC-133 cells respectively and stably transfected cells with/without FLCN expression were selected with neomycin (Lu et al 2010). Renal carcinoma cell lines, 786-0 and KTCL26 were obtained. All cell lines were cultured in DMEM with supplement of 10% foetal bovine serum except for FTC-133 cells which were incubated in medium containing DMEM and Ham's F12(1 :1 ). Phosphatase siRNA library Screening.

The differential sensitivities of the isogenic UOK cell lines to siRNA-reduced cell viability was determined using CellTiter-Blue Cell Viability Assay (Promega, Southampton, the United Kingdom). Briefly, diluted siRNA (Silencer^® Select human Phosphatase siRNA Library, Ambion, Warrington, the United Kingdom) was mixed with transfection reagent (INTERFERin(RTM), Polyplus Transfection, 10 μΙ) in 96- well plate before adding exponentially growing cells (6x 10³ cells/90 μΙ/well). The final siRNA library screening concentration was at 20nM. After siRNA transfected for 72 hours, the cells were incubated with 20 μΙ of CellTiter-Blue Cell Viability Assay reagent for an additional 4 hours. The cell viability was measured by fluorescence with a 570nm excitation and 590nm emission set in a Victor X3 Multilabel Plate Reader (PerkinElmer, Beaconsfield, United Kingdom).

Caspase -Glo 3/7 Assay

The ability to induce caspase 3/7 activation after exposure to siRNA was measured by Caspase-Glo 3/7 Assay (Promega, Southampton, United Kingdom) according to the manufacturer's instruction. The cells were added to diluted siRNA/INTERFERin(RTM) mixture as described in Phosphatase siRNA library screen. At the end of incubation, 50 μΙ of medium was removed and 50 μΙ of assay reagent was added to the remaining medium. Additional one hour incubation with shaking was carried out at room temperature. The resultant luminescent light was measured in a Victor X3 Multilabel Plate Reader (PerkinElmer, Beaconsfield, United Kingdom).

Measurement of Gene Transcript Levels.

Total RNA was isolated from FTC 12 and FTC22 cell lines after siRNAs of Luciferase or SSH2 treatment or from exponential growing cells in five cell lines including UOK- 257, FTC12, 786-0, FTC22 and KTCL26 using the RNAeasy(RTM) kit according to the manufacturer's instructions (Qiagen, Crawley, United Kingdom). The cDNAs were synthesized from RNA (1 μg) by Superscriptll reverse-transcriptase (lnvitrogen,RTM). Real time PCR was carried out in a total volume of 20 μΙ containing 1 x TaqMan(RTM) Universal PCR Master Mix, ~200ng of cDNA and TaqMan(RTM) Gene Expression Assay Mix (1 μΙ) for GAPDH, SSH1 , SSH2, SSH3 and FLCN (Applied Biosystems, Cheshire, United Kingdom) in a Bio-Rad(RTM) Q5 Real-Time PCR machine. PCR conditions were as follows: (a) 95°C for 10min; (b) 40 cycles of 95°C for 15 s and 60°C for 1 min. At least three separate PCR reactions for each gene were performed. Luciferase siRNA treatment in both FTC12 and FTC22 cells was used as untreated control and the real-time amplification data were normalized relative to the control GAPDH gene. Relative gene expression levels after SSH2 siRNA treatment was analysed using REST software (Qiagen). Western Blot Analysis

Cellular protein expression in UOK and FTC Cells in response to siRNA treatment was determined using total cell extracts at 48 h after treatment, according to standard procedures. Protein concentration was determined using DC protein assay kit according to the manufacturer's instructions (Bio-Rad Laboratories Ltd, Hertfordshire, United Kingdom). 20μg of protein from each sample was electrophoresed on 15% (w/v) SDS-PAGE gels and electroblotted on to nitrocellulose membrane (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, United Kingdom). Antibodies against FLCN (a gift from Prof. Arnim Pause, Rosalind and Morris Goodman Cancer Centre Montreal, Canada), caspase3, Cofilin and phosphor-Cofilin (Cell Signalling Technology, Hertfordshire, United Kingdom), and actin (Sigma-Aldrich Company Ltd., Dorset, United Kingdom) were used. The signal was detected using the enhanced chemiluminescence (ECL+Plus, Amersham) system after addition of anti-mouse IgG-HRP conjugate (DAKO, Ely, United Kingdom).

Cell Cycle Analysis

UOK-257 cells with/without FLCN expression were transfected with siRNAs at a range of concentrations (0, 1 , 2, 5, 10 and 20 nM) at 2000 cells per well in replicates of eight in 96-well plates and incubated at 37°C/5% C02 for 72 hours. At the end of the time course, media was carefully removed and cells were fixed in 85% ice-cold ethanol. After removal of ethanol, cells were incubated in the dark at 37°C for 20 mins in PBS buffers containing 0.1 % Triton X-100, 10C^g/ml RNase A and 10 μg ml Propidium iodide (PI). The 96-well plates were subsequently scanned using the Acumen eX3 cytometer (TTP LabTech, Melbourn, Royston, UK). Examples

Identification of Candidate Targets by Screening of a Phosphatase siRNA Library in Folliculin Deficient Renal Cancer Cell Lines

Screen of a siRNA Library targeting of 298 human phosphatase genes was undertaken in a matched pair of folliculin deficient (UOK257-FLCN^") and folliculin replete (UOK257-FLCN⁺) isogenic renal cell carcinoma cell lines. UOK-257 is the only RCC cell line available that has been derived from a patient with BHD and harbours a germline FLCN frameshift mutation (c.1285dupC) (predicted, in the absence of nonsense mediated mRNA decay, to lead to premature protein truncation (p.His429ProfsX27)). Both UOK257-FLCN^" and UOK257-FLCN⁺ cells were examined for their cellular viability after being transfected by phosphatase siRNAs for 72 hours. Each phosphatase gene contains three siRNAs. The ratio of cell viability from UOK257-FLCN⁺ and UOK257-FLCN^" were calculated and used as indicators for the sensitivity to mutant FLCN cells. The potential positive hits identified by the higher ratio of cell viability were selected from at least two of the three siRNAs for each gene and used in the subsequent screen. In an alternative screening method UOK257-FLCN^" cells were examined for their cellular viability after being transfected by phosphatase siRNAs or luciferase siRNA control for 72 hours. The ratio of cell viability from phosphatase siRNA vs control siRNA in UOK257-FLCN^" were calculated and used as indicators for the sensitivity to mutant FLCN cells (<1 ). The potential positive hits identified by the lower ratio of cell viability were selected from at least two of the three siRNAs for each gene.

The initial screen identified siRNA against the slingshot phosphatases (SSH 1 , SSH2 and SSH3) as a potential hit and further investigation were undertaken. These demonstrated that knockdown of SSH2 reduced cellular viability of UOK257-FLCN^" cells but not UOK257-FLCN⁺ cells. Furthermore, when investigated for their ability to induce Caspase3/7 activity in the pair of UOK cell lines, SSH2 knockdown induced —5-fold caspase3/7 activity (detected with the Caspase-Glo 3/7 Assay) in UOK257- FLCN^" cells when compared with the luciferase siRNA control but not in UOK257- FLCN⁺ cells (see Figurel A at 20nM). Previously we found that Mithramycin (a cytotoxic chemotherapy drug) selectively induces caspases 3/7 activity in UOK257- FLCN^" cells but not UOK257-FLCN⁺ cells; however, in a non-RCC cell line model (FTC-133 a human thyroid carcinoma) with/without FLCN expression there were no apparent differential mithramycin sensitivity in FTC-133 cells with different levels of FLCN expression (Figure 1A and Lu et al 201 1 ). Therefore we proceeded to investigate whether SSH2 knockdown in FTC-133 cells had a similar effect to as in UOK cells. We found that siRNA-induced SSH2 knockdown increased caspase3/7 activity in FTC133-FLCN^" cells but not FTC133-FLCN⁺ cells (see Figure 1A), indicating that reduced cell viability and induced caspase3 activity triggered by knockdown of SSH2 expression might be specifically to FLCN-null cell lines.

Next we examined whether SSH2 knockdown might potentiate mithramycin activity in activation of caspase 3/7 activity (Figure 1 B) in FLCN-null UOK cells. Low concentration of either SSH2 or luciferase siRNA (5nM) was incubated with increased mithramycin concentrations for 72 hours and caspase 3/7 activities were then measured. As indicated in UOK cells in Figure 1 B, there was 1 -4 fold increase in caspase 3/7 activity in the luciferase control plus mithramycin treatment (25 to 100nM; grey bars), however, more caspase3/7 induction was found in the combination of SSH2 knockdown and mithramycin treatment (2-6 fold increase, black bars) at the same concentration range of mithramycin, suggesting that SSH2 knockdown might increase mithramycin activity in triggering of caspase 3/7 activation in the UOK cells. However, there was little change in caspase 3/7 activity in either mithramycin alone, or in combination with SSH2 knockdown by siRNA in UOK-FLCN cells (Figurel B).

Identification of SSH2 as a Synthetic Lethal Candidate Target for Folliculin Deficient Cancer Cell Lines

The human slingshot family is encoded by three genes, however only SSH2 siRNA treatment induced upto 5-fold increase in caspase3/7 activity and reduced cell viability in folliculin deficient FTC and UOK cells (see Figures 2 and 3). Although SSH2 siRNA treatment also increased up to 2-fold caspase3/7 activity in FLCN- expressing FTC22 cells, the viability of the cells were not apparently changed (Figure 2). It was interesting to note that reduction of cell viability (25%) can be observed in FTC-12 cells after SSH3 siRNA treatment, irrespective of no remarkable induction of Caspase3/7 activity in FLCN-null cells. Similar results seen in FTC cells were found in UOK cells with/without FLCN expression as shown in Figure 3. Next we expanded siRNAs treatment to a range of relative genes, such as PDXP, LIMK1 , LIMK2 and Cofilin to examine whether any relative gene siRNA treatment induced caspase 3/7 activity and reduced cell viability in UOK cells. As shown in Figure 3, although LIMK1 and Cofilin b siRNA treatment induced upto 3-fold caspase activity in UOK cells, cell viability remained unchanged. There was neither induction of Caspase3/7 activity nor reduction of cell viability found in UOK-FLCN cells (Figure 3). Validation of SSH2 Knockdown by Real-Time PCR

To validate the specificity of SSH2 knockdown in FTC133 cells, the transcript levels of SSH 1 , SSH2 and SSH3 were analysed by real time PCR after SSH2 siRNA treatment for 72 hours. SSH2 transcripts levels were reduced -60% at the concentrations of 5 and 10nM of SSH2 siRNA treatment when compared with luciferase siRNA control in both FTC 12 and FTC22 cells. In addition, SSH1 transcript levels increased up to 200% and SSH3 transcript levels increased up to 130% in FTC 12 and FTC22 cells following SSH2 transcript knockdown (Figure 4), indicating that there was a compensatory regulation mechanism in transcripts among members of SSH family.

Examination of Cofilin Expression after SSH siRNA Treatment

The slingshot family of phosphatases has a crucial role in regulating the activity of cofilin which regulates the assembly and disassembly of actin filaments (see Figure 5). Thus phosphorylated cofilin is inactive. LIM-kinases (LIMK) and testis-specific protein kinases (TESKS) phosphorylate cofilin on a single serine residue (Ser 3). Conversely the Slingshot family phosphatases and PDXP reactivate cofilin.

We investigated whether SSH2 knockdown in UOK cell lines might have differential effects on cofilin phosphorylation according to FLCN status and whether SSH2 knockdown in the presence of compensatory increases in SSH1 and SSH3 transcript levels would be associated with a detectable change in cofilin protein levels and phosphorylation status. However no detectable changes in cofilin were observed after siRNA knockdown of SSH2 in UOK cells (Figure 6). Interestingly, none of Slingshot family member siRNAs (including PDXP) reduced expression of Cofilin protein but a slight reduction of Cofilin expression was detected after LIMK1 siRNA treatment in UOK cells without FLCN expression (this was not seen with LIMK2 siRNA treatment). The only siRNA treatment to reduce both Cofilin and phosphor-Cofilin expression was Cofilin b in UOK-FLCN^" cells, whereas only reduction of Cofilin not phosphor-Cofilin expression was found in UOK-FLCN⁺ cells. Similar experiments were also carried out for UOK compared to UOK-257-2 cell lines (Figure 12). As shown in Figure 12 in FLCN-expressing UOK-2 cells, FLCN expression was reduced (up to 0.3 fold) in response to SSH 1 , SSH2 and PDXP knockdown whereas increased phosphorylation of cofilin (2-3 fold), as expected, can be found in knockdown of SSH phophatases (SSH 1/2/3 but not PDXP). In addition treatment of LI M-kinase1 and LI M-kinase2 siRNAs decreased phosphorylated cofilin expression (0.8-1 fold) in UOK-2 cells. In contrast, in FLCN-null UOK cells only slight increase (up to 1 .5 fold) was found in cofilin phosphorylation in response to SSH siRNA treatment. Furthermore, more than 2 fold increase in phosphorylated cofilin (in opposite of reduction in UOK-2 cells) was found in treatment of LI M-Kinases1/2 siRNAs, indicating an impaired function in cofilin de/phosphorylation by SSH phosphatases and LI M-Kinases in UOK cells. The most striking difference was that in UOK cells strong activated caspase 3 bands (17 and 19 bands) were found only in the treatment of SSH2 siRNA, whereas caspase3 was activated in a lesser degree in UOK-2 cells in response to knockdown of SSH 1 , SSH2, LI MK1 and LI MK2, suggesting that caspase 3 activation by knockdown of SSH2 is a specific event in FLCN-null UOK cells. Cofilin siRNA treatment was used as a positive control and decreased expression of phosphorylated cofilin but no cofilin was found in both UOK cells with/without FLCN expression, implying abundance of cofilin expression and/or rate-limiting controlled expression of phosphor-cofilin in the UOK cells. From the above results, we speculate that (a) in the absence of FLCN, de/phosphorylation of cofilin by SSH phosphatases and LI M-Kinases might be impaired in UOK cells; (b) FLCN may be directly or indirectly involved in the process of actin reorganization. - (c) apoptotic cell death in FLCN-nuW cells can be triggered by SSH2 knockdown through impaired cofilin de/phosphorylation.

Examination of Cofilin Expression after multiple SSH siRNA Treatment

Considering that there was a compensatory regulation mechanism among Slingshot transcripts in the FTC12 and FTC22 cells (Figure 5), we next examine whether multiple SSH siRNA treatment can potentiate SSH2 knockdown effect in the activation of caspase 3/7 activity. Cells were incubated with a range of SSH2 siRNA, either alone, or in combination with SSH 1 , or/and SSH3, at 5 and 10nM concentrations for 72 hours. Luciferase siRNA was used as control in replacement of SSH2. As shown in Figure 7, more induction of caspase 3/7 activities were observed in FTC12 cells in the combination of SSH2 and SSH1 , or all three slingshot siRNAs, when compared with SSH2 knockdown alone. Similar results were found in the UOK cells with additional Caspase 3/7 activity when SSH1 siRNA was included in the treatment of SSH2 siRNA, but not seen with SSH3 siRNAs (Figure 5). In contrast, in the FLCN-expressing FTC22 and UOK-FLCN cells similar pattern can be seen at smaller degree (upto 2-fold Caspase3/7 induction in all three SSH siRNAs treatment), suggesting that when multiple SSH siRNAs used, the increased Caspase3/7 activity might be independent to the FLCN status.

As also shown in Figure 13, in FLCN-null UOK and FTC12 cells more induction of caspase 3/7 activities were observed in knockdown of multiple SSHs (up to 5-fold in FTC 12 and 7-fold in UOK cells) than SSH2 knockdown alone (2.5-fold in FTC12 and 4.5-fold in UOK cells). In FLCN-expressing FTC22 and UOK-2 cells similar pattern can be seen at a much smaller degree (upto 2-fold caspase3/7 induction in SSH2 knockdown alone and upto 3-fold in multiple SSH siRNAs treatment), suggesting that FLCN protein might interfere with caspase activation. Western blot was then carried out to investigate whether reduction of cofilin expression, or induction of cofilin phosphorylation expression can be observed following knockdown of all three SSHs in FTC12 and FTC22 cells (Figures 8 and 12). As shown in Figure 8, there was neither apparent reduction of cofilin expression, nor clear increased phosphorylation of cofilin expression in both cell lines throughout SSH siRNAs treatment when compared with Luciferase siRNA control. The possible explanation of it is due to the abundant expression of cofilin in these cell lines. However, phosphorylated cofilin was reduced, in opposite of increased, when combination of SSH1 and SSH3 were knocked down in FTC22 cells.

As shown in Figure 12, only triple SSH knockdown in FTC22 cells increased upto 2- fold expression in phosphor-cofilin when compared with Luci+SSH1 +SSH3 siRNA treatment. In contrast in FLCN-null FTC12 cells, phosphor-cofilin expression was reduced in knockdown of either SSH2 alone or multiple SSHs. There was no apparent reduction of FLCN in FTC22 cells in all treatments, suggesting abundance of exogenous FLCN over-expression.

Cofilin siRNA was used as positive control to knockdown cellular cofilin levels and only phosphorylated form was reduced in the cells examined, which was similar to those seen in UOK cells (Figures 6 and 12). It is interesting to note that in FTC12 cells, cleaved active caspase 3 (lowest 17kDa band) was found in all SSH2 siRNA treated lysate in comparison with those from luciferase control, no matter whether it was transfected with either single or in combination of additional member of SSH siRNAs, indicating that knockdown SSH2 alone is sufficient to activate caspase3 in FLCN-nuW FTC12 cells. In contrast in FTC22 cells, there was no similar active caspase 3 pattern as seen in FTC12 (Figure 8). It provided further evidence based on the multiple SSH siRNA results that triggering cell death by SSH2 knockdown might be cofilin-unrelated (as yet uncharacterized) functions of SSH2 in FLCN-null cells, which are distinctive from SSH 1 and SSH3.

Analysis of Cell Cycle Changes in UOK-257 Cells in Response to Single or Multiple SSH siRNAs Treatment To study the underlining mechanism of differential induction of Caspase3/7 activity after SSH2 knockdown, cell cycle analysis was carried out in UOK cells with/without FLCN expression using cytometry (Acumen Explorer). Cells were transfected with single siRNAs (Luciferase as control, SSH 1 , SSH2 and SSH3) or multiple siRNAs (SSH2+SSH 1 , SSH2+SSH3 and SSH2+SSH 1 +SSH3) at a range of concentrations (as indicated in Figure 14) for 72 hours. The cell cycle populations at the same treatment but without siRNA were used as control. In FLCN-null UOK cells, single SSH2 siRNA treatment elongated G1 cell cycle phase (up to 20% increase) and shortened S and G2M cell cycle phases (30%, 40% decrease respectively). This cell cycle change pattern was easily observed after SSH2 knockdown even in the lowest siRNA concentration (1 nM) examined. Similar pattern can be found in SSH3 knockdown but at a small extent (10% increase in G1 , 20% decrease in S and 10% decrease in G2M) in UOK cells. In comparison, consistent cell cycle change pattern was found in none of the single SSH siRNA treatment in FLCN-expressing UOK-2 cells (Figure 14). In multiple SSH siRNA treatment, similar cell cycle change pattern to those seen in single SSH2 knockdown can be observed in all three treatments with more prominent in combination treatment of SSH2+SSH3, or SSH2+SSH1 +SSH3 siRNAs (20% increase in G1 , 40% decrease and 40% decrease in G2M) in UOK cells, indicating that SSH3 knockdown may potentiate SSH2 activity in this cell line. In contrast, in UOK-2 cells similar cell cycle pattern can be found only in all three SSH siRNAs treatment (SSH2+SS1 +SSH3, upto 20% increase in G1 phase, 20%, 20% decrease in S and G2M phases, respectively), suggesting that in the presence of FLCN, it is required to knockdown all three SSH in order to have similar effect seen in single SSH2 knockdown in UOK cells.

Analysis of Caspase3/7 Induction and Cell Viability Reduction in Other Cell Lines after SSH siRNA Treatment

Next we investigated whether SSH2 siRNA treatment might induce caspase3/7 activity and reduce cell viability (as seen in FLCN-null UOK and FTC cell lines) in non-BHD RCC cells lines. FTC 12 cells and two RCC cell lines (786-0, and KTCL- 26) cells were transfected with siRNAs of luciferase, SSH 1 , SSH2 and SSH3 for 72 hours and caspase3/7 activity and cell viability assay were examined and normalized by luciferase siRNA control as before. 786-0 is a kidney cancer cell line that harbours an inactivating VHL gene mutation. As shown in Figure 9A, caspase3/7 activity were remained unchanged or slightly increased (20-50%) with the largest increase in KTCL-26 cells (100%) after SSH 1 siRNA treatment, whereas around 20% reduction in cell viability in KTCL-26 at highest 20nM siRNA treatment. Although reductions in cell viability were detected after SSH3 siRNA transfection this was not associated with caspase 3/7 induction. For SSH2 siRNA treatment similar levels of caspase3/7 induction to those in FTC12 cells were observed in 786-0 and KTCL-26 (upto 3-fold, 20nM), although the degree of reduction in cell viability in these two cell lines were less than those in FTC12 which reached 50% reduction at 5nM. The induction of caspase3/7 and reduction in cell viability in RCC cell lines (786-0 and KTCL-26) without a detectable FLCN mutation might suggest that the pathway targeted by SSH2 siRNA may also be druggable in some RCC cancers without FLCN inactivation.

In order to examine whether there is a correlation between expression levels of FLCN and SSH2, and induction of caspase3/7 activity, cellular transcript levels of FLCN and SSH2 were determined by Real-Time PCR in UOK-257, FTC 12 and FTC22 cells, and in two other cells (786-0 and KTCL-26). Each gene was measured at least three times and normalized by GAPDH before compared with those expressed in UOK-257 cells. As shown in Figure 9B, differential expression of FLCN transcripts ranged from the lowest for UOK-257 and FTC 12 to the highest KTCL26 (7 fold). However, UOK-257 cells expressed the highest SSH2 transcripts and its level was 3.2 fold as those in FTC12 cells. Statistical analysis showed that there was no correlation between expression from FLCN and SSH2 (p=0.8397), and between induction of caspase3/7 activity and either FLCN or SSH2 expression in these cells (data not shown). It is interesting to note that FLCN transcript level was 4-fold higher in FTC22 cells than its counterpart, whereas SSH2 transcript level in FTC22 was also elevated as 2-fold as those in FTC12. siRNAs and siRNA design

siRNAs targeting the SSH genes were identified and the selected siRNAs are provided in Table 1 . Table 1 shows sense and anti-sense strand of nine different SSH siRNAs. Combination of one sense strand (eg. SEQ ID No: 1 ) with its complementary antisense strand (eg., SEQ ID No: 2) results in formation of a base- paired 19 nucleotide duplex with 2 base pair overhangs. The human slingshot family is encoded by three genes: SSH1 , SSH2, SSH3. More than one siRNA to a given gene target may be used. Table three includes three individual siRNAs per gene. For each gene, one or more of each of the siRNAs in Table 1 targeted against that gene may be used.

Table 1

SSH1

SiRNA no. Sense siRNA Sequence Antisense siRNA Sequence

CCUCCACAGUCAUAGCCUAtt UAGGCUAUGACUGUGGAGGcc

1 SEQ ID NO: 1 SEQ ID NO: 2

GGCUUAUUUGCAUAUCAUAtt UAUGAUAUGCAAAUAAGCCag

2 SEQ ID NO: 3 SEQ ID NO: 4

GCAUGGAGGAUGAUGCUAUtt AUAGCAUCAUCCUCCAUGCcg

3 SEQ ID NO: 5 SEQ ID NO: 6 SSH2

SiRNA no. Sense siRNA Sequence Antisense siRNA Sequence

G G AC U U G AAU U G AC U AG U U tt AAC U AG U CAAU U CAAG U CCtg

1 SEQ ID NO: 7 SEQ ID NO: 8

G U AU CAU AACAU U C GG G U Att UACCCGAAUGUUAUGAUACtc

2 SEQ ID NO: 9 SEQ ID NO: 10

GGAGCGACACGCUAAUUCAtt UGAAUUAGCGUGUCGCUCCag

3 SEQ ID NO: 1 1 SEQ ID NO: 12

SSH3

SiRNA no. Sense siRNA Sequence Antisense siRNA Sequence

AGACUGAACUCCGAACAGAtt UCUGUUCGGAGUUCAGUCUct

1 SEQ ID NO: 13 SEQ ID NO: 14

CCACGAG U CU U CACAU GAAtt UUCAUGUGAAGACUCGUGGga

2 SEQ ID NO: 15 SEQ ID NO: 16

G C AC C U C AG AG AC C AG U G Att UCACUGGUCUCUGAGGUGCtc

3 SEQ ID NO: 17 SEQ ID NO: 18

It will be understood that siRNAs specific to SSH1 , SSH2, or SSH3 other than those listed in Table 1 can be used. Other suitable siRNAs can be designed using techniques that are known to those skilled in the art. As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompass chemically modified nucleotides and non-nucleotides.

Many different tools, algorithms and programs are widely available for assisting in the design of siRNAs for RNAi. Ambion's siRNA Target Finder is just one example of the many different tools available. Many tools use the suggested guidelines for identifying targets based on the work of Tuschl et al (Elbashir,S.M., Lendeckel,W. and TuschlJ. (2001 b) RNA interference is mediated by 21 and 22 nt RNAs. Genes Dev., 15, 188-200). In order to design an siRNA, an mRNA or cDNA sequence of the gene of interest is required, which if available, can be retrieved from the NCBI (National Center for Biotechnology Information) database. Some programs provide homology searches for candidate siRNA targets against other mRNA sequences of the genome. SSH1 encodes three different isoforms. mRNA sequences for each isoform are available from the NCBI database.

The mRNA sequence for SSH 1 isoform 1 is provided at NCBI Reference Sequence No. NM_018984.3, which is hereby incorporated by reference in its entirety.

The mRNA sequence for SSH 1 isoform 2 is provided at NCBI Reference Sequence No. NM_001 161330.1 , which is hereby incorporated by reference in its entirety. The mRNA sequence for SSH1 isoform 2 is provided at NCBI Reference Sequence No. NM_001 161331.1 , which is hereby incorporated by reference in its entirety. The mRNA sequence for SSH2 is provided at NCBI Reference Sequence No. NM_033389.2, which is hereby incorporated by reference in its entirety.

The mRNA sequence for SSH3 is provided at NCBI Reference Sequence No. NM_017857.3, which is hereby incorporated by reference in its entirety.

One or more of the above mRNA sequences can be used to design a RNAi molecule(s) to be used in the present invention. shRNAs and shRNA design

Many different tools, algorithms and programs are widely available for assisting in the design of shRNAs for RNAi. BLOCK-iT™ RNAi Designer is just one example of the many different tools available and known to the skilled person. As with siRNA design, potential sequences for use as shRNAs targeted against the mRNA of interest can be generated by providing the target mRNA sequence. Many of the tools implement a multi-step procedure to identify potential shRNAs including: analysis of GC content, presence or absence of internal hairpins, differential thermal stability of ends, sequence complexity, and various position-specific criteria. shRNA molecules need not be limited to those molecules containing only RNA, but further encompass chemically modified nucleotides and non-nucleotides.

Claims

1 . A composition comprising a slingshot phosphatase inhibitor for use in the treatment of renal cell carcinoma.

2. A composition according to claim 1 for use in the treatment of renal cell carcinoma associated with Birt-Hogg-Dube syndrome.

3. A composition comprising a slingshot phosphatase inhibitor for use in the treatment of cancer associated with FLCN inactivation.

4. A composition according to claim 3 for use in the treatment of renal cell carcinoma associated with FLCN inactivation.

5. A composition according to any preceding claim wherein the slingshot phosphatase inhibitor inhibits Protein Phosphatase Slingshot Homolog 2 (SSH2).

6. A composition according to any preceding claim comprising a second slingshot phosphatase inhibitor.

7. A composition according to any preceding claim comprising more than one slingshot phosphatase inhibitor, each for inhibiting a different member of the slingshot phosphatase family.

8. A composition according to claim 6 or 7 wherein the slingshot phosphatase inhibitors inhibit Protein Phosphatase Slingshot Homolog 2 (SSH2) and Protein Phosphatase Slingshot Homolog 1 (SSH1 ).

9. A composition according to claim 6 or 7, wherein the slingshot phosphatase inhibitors inhibit Protein Phosphatase Slingshot Homolog 2 (SSH2) and Protein

Phosphatase Slingshot Homolog 3 (SSH3).

10. A composition according to claim 7, wherein the wherein the slingshot phosphatase inhibitors inhibit Protein Phosphatase Slingshot Homolog 2 (SSH2), Protein Phosphatase Slingshot Homolog 1 (SSH1 ), and Protein Phosphatase Slingshot Homolog 3 (SSH3).

1 1 . A composition according to any preceding claim, wherein the slingshot phosphatase inhibitor is an RNAi molecule specific to a slingshot phosphatase gene.

12. A composition according to claim 1 1 , wherein the slingshot phosphatase inhibitor is an RNAi molecule specific to SSH2.

13. A composition according to any preceding claim, wherein the slingshot phosphatase inhibitor comprises an RNAi molecule specific to a first slingshot phosphatase gene and an RNAi molecule specific to a second slingshot phosphatase gene.

14. A composition according to claim 13, wherein the slingshot phosphatase inhibitor comprises RNAi molecules specific to SSH2 and SSH1 .

15. A composition according to claim 13, wherein the slingshot phosphatase inhibitor comprises RNAi molecules specific to SSH2 and SSH3.

16. A composition according to claim 13, wherein the slingshot phosphatase inhibitor comprises an RNAi molecule specific to a first slingshot phosphatase gene, an RNAi molecule specific to a second slingshot phosphatase gene, and an RNAi molecule specific to a third slingshot phosphatase gene.

17. A composition according to claim 16, wherein the slingshot phosphatase inhibitor comprises RNAi molecules specific to SSH2, SSH 1 and SSH3.

18. A composition according to any of claims 1 1 to 17, wherein the RNAi molecule comprises siRNA.

19. A composition according to claim 18, wherein the siRNA comprises at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprises a second sequence comprising a region of complementarity which is substantially complementary to at least a part of a mRNA encoding a slingshot phosphatase.

20. A composition according to claim 18 or 19, wherein the siRNA comprises at least one nucleotide sequence from the group comprising SEQ ID NOs: 1 to 18.

21 . A composition according to any of claims 1 1 to 17, wherein the RNAi molecule is a short hairpin RNA (shRNA).

22. A composition according to any of claims 1 1 to 21 , wherein the composition further comprises a delivery vehicle, such as but not limited to a viral vector, an antibody, an aptamer or a nanoparticle, for delivering the RNAi molecule to a target cell.

23. A cell comprising an RNAi molecule of any of claims 1 1 to 21 for use in the treatment of renal cell carcinoma, Birt-Hogg-Dube syndrome, and/or cancer associated with FLCN inactivation.

24. A vector comprising an RNAi molecule of any of claims 1 1 to 21 for use in the treatment of renal cell carcinoma, Birt-Hogg-Dube syndrome, and/or cancer associated with FLCN inactivation.

25. A cell comprising the vector of claim 24 for use in the treatment renal cell carcinoma, Birt-Hogg-Dube syndrome, and/or cancer associated with FLCN inactivation.

26. A method of identifying a candidate compound for treating renal cell carcinoma, the method comprising identifying an inhibitor of a slingshot phosphatase by contacting a cell expressing a slingshot phosphatase with a candidate compound and determining whether activity of slingshot phosphatase is inhibited, wherein inhibition of activity of slingshot phosphatase indicates that the candidate compound is a candidate compound for treating renal cell carcinoma.

27. A method of identifying a composition for treating cancer, wherein the cancer is characterised by FLCN inactivation, the method comprising identifying an inhibitor of a slingshot phosphatase by contacting a cell expressing a slingshot phosphatase with a candidate compound and determining whether activity of slingshot phosphatase is inhibited, wherein inhibition of activity of slingshot phosphatase indicates that the candidate compound is a candidate compound for treating cancer characterised by FLCN inactivation.

28. A composition according to any preceding claim, wherein the composition further comprises mithramycin or a pharmaceutically acceptable salt or solvate thereof.

29. A composition comprising a slingshot phosphatase inhibitor for use in therapy.

30. A composition, cell, vector and/or method substantially as herein described or illustrated.