WO2013170365A1 - Compositions of sparc polypeptide and grp78, and methods and uses thereof for sensitizing cancer cells - Google Patents

Abstract

The present invention relates to compositions and methods of use thereof for cancer therapy sensitization. Such compositions may comprise full length Secreted Protein Acidic and Rich in Cysteine (SPARC) polypeptide; and functional fragments thereof and a GRP78 inhibitor; or the nucleotide sequences encoding them. The compositions may be used in combination with existing chemotherapeutic agents for contacting a cancer cell. There are also provided uses, pharmaceutical compositions, and commercial packages associated therewith.

Description

COMPOSITIONS OF SPARC POLYPEPTIDE AND GRP78, AND METHODS AND USES THEREOF FOR SENSITIZING CANCER CELLS

RELATED APPLICATIONS

This application claims priority to United States provisional patent application 61/648,837 titled "SPARCS INTERACTION WITH GRP78 IMPROVES CHEMOSENSITIVITY IN THERAPY- RESISTANT CANCER CELLS" and filed 18 May 2012.

TECHNICAL FIELD

This invention relates to the field of cancer therapies. In particular, the invention relates to the sensitization of cancer cells through the administration of SPARC polypeptide and GRP78.

BACKGROUND

Cancer is one of the leading causes of death in humans and while standard chemotherapy, radiotherapy and surgical intervention successfully reduce tumor load in many cases, resistance to chemotherapeutic intervention is not uncommon, especially in solid tumors. Resistance may develop following exposure to a chemotherapeutic agent and can further impede tumor regression. It is this chemotherapy resistance, which leads to treatment failure and subsequently accounts for the high mortality rates in cancer.

The molecular basis of chemotherapy resistance is largely genetic, and can take many forms. Many mutations responsible for the initial development of tumors may also contribute to drug resistance. For example, loss of DNA mismatch repair (MMR) gene function has been associated with a more rapid emergence of clinical drug resistance in some cancers and mutations in the K- ras gene are associated with an increased relapse rate, mortality and a poor chemotherapeutic response. Aberrant expression and dysregulation of proteins involved in the normally tightly regulated cell replication cycle may also be protective of tumors. These proteins may be loosely referred to as Oncogenes'. Gene products p21 and p27, for example, have been shown to protect tumors from undergoing apoptosis elicited by various anticancer agents. Adhesion molecules, such as E-cadherin, may also confer resistance to cells exposed to chemotherapeutic agents. The mechanisms involved in therapeutic resistance are varied and may be very complex. Chemosensitizers may act in concert with the chemotherapeutic agent, or may serve to counteract resistance mechanisms in the cell. Existing chemosensitizers include small molecule drugs such as photosensitizers or drug efflux pump inhibitors, and more recently, antisense oligonucleotides. New compounds with chemosensitizing activity include US 5,776,925 and WO 02/00164, which provide examples of novel chemical compounds that enhance cytotoxicity of therapeutic agents.

Antisense sequences with chemosensitizing activity - often specifically targeting oncogenes - are varied and may be found for almost any target. For example, survivin is a protein that modulates apoptosis and is frequently overexpressed in cancer cells. Antisense survivin oligonucleotides have been demonstrated to downregulate expression of Survivin, and sensitize cells to chemotherapeutic agents such as docetaxel and etopotide.

Similarly, cancer therapy sensitizers may act in concert with cancer therapeutic agents, (for example, radiotherapy or chemotherapy), or may serve to counteract resistance mechanisms in the cell to the cancer therapeutic agent.

Secreted protein acidic and rich in cysteine (SPARC) is one example of a gene with significantly decreased expression in multidrug resistant cell lines in vitro, with a possible tumor suppressor role (Tai, LT. et al. 2005. J. Clin Invest. 1 15:1492-1502). SPARC, also known as osteonectin, belongs to a family of matricellular proteins having counter-adhesive properties, disruptive of cell-matrix interactions (Bornstein P. 1995. J. Cell Biol 130:503-6; Sage E. H. and Bornstein P. 1991. J Biol Chem; 266: 14831- 4).

SPARC has been demonstrated to play a role in bone mineralization, tissue remodeling, endothelial cell migration, morphogenesis and angiogenesis.

Additional studies suggest that cleavage of SPARC by MMP-3 results in peptides that affect angiogenesis (Sage et al. 2003 J. Biol Chem 287: 37849-37857). SPARC also has a role in malignancy, as variable gene and protein expression of SPARC have been linked to cancer progression in a number of tumors.

The SPARC protein is under expressed in several types of cancer, including colorectal cancer (CRC), and has been shown to enhance chemosensitivity and inhibit tumor growth in vivo. SPARC is also known to bind collagen-IV, a component of the extracellular matrix (ECM), in a calcium-dependent manner in vitro.

The use of the full length isolated SPARC protein as a chemo sensitizer is described by WO 2004/064785. Furthermore, SPARC polypeptides are described as chemotherapeutic sensitizers by WO 2008/000079.

SUMMARY

The present application is based in part on the discovery that an inhibitor of GRP78 in the presence of SPARC polypeptide sensitizes cancer cells to chemotherapeutic treatments.

Furthermore, it was also found that there is a synergism between SPARC and a GRP78 inhibitor in promoting cell death and/or growth inhibition of cancer, whereby the collective effect of SPARC polypeptide sensitization and sensitization associated with GRP78 inhibition is predicted to be greater than the sensitization effect of each of the SPARC polypeptide and the GRP78 inhibitor when added together.

In accordance with a first aspect of the invention, there is provided a method of sensitizing a cancer cell, the method including: contacting the cancer cell with a SPARC polypeptide and a GRP78 inhibitor. The method may further include contacting the cancer cell with a

chemotherapeutic agent.

In accordance with another aspect of the invention, there is provided a composition including a SPARC polypeptide and a GRP78 inhibitor, wherein the composition sensitizes a cancer cell to a chemotherapeutic agent. The composition may further include a chemotherapeutic agent. The composition may be formulated as a gel or paste. In accordance with another aspect of the invention, there is provided a pharmaceutical composition for treating colorectal cancer, including a SPARC polypeptide and a GRP78 inhibitor. The pharmaceutical composition may further include a chemotherapeutic agent. The pharmaceutical composition may be formulated as a gel or paste.

In accordance with another aspect of the invention, there is provided a use of a composition including a SPARC polypeptide and a GRP78 inhibitor for sensitizing a cancer cell.

In accordance with another aspect of the invention, there is provided a use of a composition comprising a SPARC polypeptide and a GRP78 inhibitor and a pharmaceutically acceptable carrier for sensitizing a cancer cell.

In accordance with another aspect of the invention, there is provided a use of a SPARC polypeptide and a GRP78 inhibitor for sensitizing a cancer cell.

In accordance with another aspect of the invention, there is provided a use of a SPARC polypeptide and a GRP78 inhibitor in the manufacture of a medicament for sensitizing a cancer cell.

In accordance with another aspect of the invention, there is provided a commercial package including (a) a SPARC polypeptide and a GRP78 inhibitor; and (b) instructions for the use thereof for sensitizing a cancer cell.

In accordance with another aspect of the invention, there is provided a commercial package including (a) a pharmaceutical composition comprising a SPARC polypeptide, a GRP78 inhibitor and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for sensitizing a cancer cell.

In accordance with another aspect of the invention, there is provided a vector, the vector including a SPARC polypeptide and a GRP78 inhibitor encoding polynucleotides. In accordance with another aspect of the invention, there is provided a method of sensitizing a CRC cell including contacting the cancer cell with a SPARC polypeptide and an agent that inhibits GRP78 in said cancer cells.

In accordance with another aspect of the invention, there is provided a method of sensitizing a tumor in an animal, the method including: (a) measuring the level of SPARC polypeptide and GRP78 polypeptide in said tumor or the level of SPARC niRNA and GRP78 mRNA in said tumor, to determine the ratio of GRP78 to SPARC polypeptide or mRNA in said tumor; and (b) administering either a SPARC polypeptide; or a GRP78 inhibitor; or both a SPARC polypeptide and a GRP78 inhibitor, to establish a low GRP78 to SPARC ratio in the animal. Steps (a) and (b) may be repeated until a low GRP78 to SPARC ratio is established. The low GRP78 to SPARC ratio may be <1.5. The SPARC polypeptide may be measured by immune- histochemistry, western-blot or mass spectroscopy. The method may further include contacting the cancer cell with a chemotherapeutic agent. The animal may be a human.

The contacting of the cancer cell may be ex vivo. The contacting of the cancer cell may be in vivo. The SPARC polypeptide may be provided via gene therapy. The GRP78 inhibitor may be provided via gene therapy. The SPARC polypeptide may be provided in a pharmaceutical composition. The GRP78 inhibitor may be provided in a pharmaceutical composition. The SPARC polypeptide may include the full length protein. The SPARC polypeptide may include a sensitizing fragment or variant. The SPARC polypeptide and the GRP78 inhibitor may form a pharmaceutical composition. The SPARC polypeptide, the GRP78 inhibitor, and the chemotherapeutic agent may be combined in a pharmaceutical composition.

The GRP78 inhibitor may be selected from one or more of the following: (a) a GRP78 inhibitory nucleic acid molecule; (b) an antibody or antibody fragment thereof that specifically binds to a GRP78 polypeptide; (c) an immunogenic composition or vaccine which comprises a GRP78 polypeptide; (d) a vector encoding an inhibitory polypeptide or an inhibitory nucleic acid molecule; (e) a polypeptide; and (f) a small molecule. The GRP78 inhibitory nucleic acid molecule may be selected from an antisense oligonucleotide, a ribozyme, and an RNA interference (RNAi) molecule. The RNAi may be selected from one or more of microRNA (miRNA); small interfering

(siRNA); short-hairpin RNA (shRNA); primary-microRNA (pri -miRNA); asymmetric interfering RNA (aiRNA); small internally segmented RNS (sisiRNA); RNA-DNA chimeric duplex; trans-kingdom RNA (tkRNA); tRNA-shRNA; tandem siRNA (tsiRNA); tandem hairpin RNA (thRNA); pri-miRNA mimic cluster; and transcriptional gene silencing (TGS).

The small molecule may be selected from versipelostatin (VST), a glycosylated derivative of versipelostatin, a versipelostatin analogue, geldanamycin, and epigallocatechin Gallate (EGCG).

The cancer cell may be a colorectal cancer cell. The colorectal cancer cell may be chemotherapy resistant. The cancer cell may be a human cancer cell.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate embodiments of the invention:

FIGURE 1 shows SPARC and GRP78 expression in colorectal cancer cells, where (A) shows mRNA levels in colorectal cancer cell lines and (B) is an immune-blot of GRP78 as compared to beta-actin;

FIGURE 2 shows a series of Western blots where the activation of signaling events in ER-stress pathway in SPARC- overexpressing CRC cells (MIP/SP) are demonstrated, (A) shows co-immuno-precipitation studies with an interaction between GRP78 and SPARC in MIP/SP cells, (B) shows the treatment with Tm (0.5 mg/ml) for 30 min, increased GRP78, IREl and SPARC expression in the membrane fraction (with calnexin (a membrane bound protein) and actin as controls), and (C) shows a Western blot analysis of proteins involved in ER stress signaling: MIP/SP (high SPARC expression) and HCTl 16 (moderate SPARC expression) showed early activation of PERK, p-PERK, and GRP78 following treatment with Tm, compared to MlP/Zeo cells (low SPARC expression), wherein the increasing CHOP expression was most pronounced in MIP/SP cells;

FIGURE 3 shows ER-stress induced activity in SPARC-overexpressing CRC cells, (A) shows three bar graphs of the caspase 3/7 activity of MlP/Zeo, MIP/SP and HCTl 16 cells treated with Tm (0.5 g/ml) for 30 min., where all three cell lines showed a significant increased in caspase 3/7 activity (*P O.05), (B) shows a Western blot of ER-stress induced proteins, GRP78, p-PERK, p-eIF2 in MIP/Zeo and MIP/SP after treatment with 5Fu (5 μΜ) at various time points, (C) shows a bar graph comparing caspase 3/7 activity in MIP/SP and MIP/Zeo cell lines following treatment with 5FU (5 μΜ) (*P value < 0.05), and (D) shows a Western blot comparing activation of ER-stress proteins in MIP/SP cells and MIP/Zeo cells, where the cells are exposed to 5FU (6 h) treatment and optionally exposed to knockdown of CHOP by transient siRNA transfection;

FIGURE 4 is a series of bar graphs which show that SPARC inhibits GRP78-mediated cell survival in CRC cells, (A) shows an analysis of cell viability at 48 hours following treatment with 5FU and Tm by MTT assay where there was no significant change in GRP78- overexpressing MIP101 cells (MIP/78) as compared to control (MIP or MIP/Zeo empty vector), but overexpressing GRP78 in SPARC-overexpressing MIP/SP (MIP/SP/78) cells showed that the reduction in cell viability in Tm or 5FU-treated MIP/SP cells could be abolished in MIP/SP/78 cells (*P<0.05, **P<0.005), (B) shows a knockdown of GRP78 in MIP/SP cells reduced cell viability following treatment with 5FU (10 μΜ), CPT (10 μΜ), and Cetuximab (10 μ /ηι1) after 72 h (*P< 0.05), and (C) shows that the detection of apoptosis by TUNEL assay in MIP, MIP/78, MIP/SP, MIP/SP/78 treated with 5FU (10 μΜ) and Tm (0.5 mg/ml) demonstrated a significant reduction in TUNEL-positive cells in GRP78- overexpressing cells (*P < 0.05, **P < 0.0057);

FIGURE 5 shows a bar graph comparing the relative gene expression levels of GRP78 to SPARC in MIP101, MIP-ZEO, MIP-SP, and MIP-5FU cell lines, where the ratio of GRP78 to SPARC expression was highest in the MIP-5FU chemoresistant cell line and the lowest in the MIP-SP chomo sensitive SPARC-overexpressing cell line; and

FIGURE 6 shows a survival plot comparing patients having colorectal cancer, where the individuals are grouped as having tumors with either a high ratio of GRP78 to SPARC expression or with a low ratio of GRP78 to SPARC expression, wherein the individuals having a high ratio of GRP78 to SPARC expression have a significantly shorter overall survival than individuals that have a lower ratio of GRP78 to SPARC expression. DETAILED DESCRIPTION

Various alternative embodiments and examples are described herein. These embodiments and examples are illustrative and should not be construed as limiting the scope of the invention.

SPARC protein is encoded by a 912 bp mRNA (for example, see GenBank Accession:

CR456799.1). The Homo sapiens SPARC, is on chromosome 5 (NCBI Reference Sequence: NC 000005.9). Human SPARC encodes a 303 amino acid protein (see for example, see GenBank: CAG33080.1).

Human SPARC protein

MRAWIFFLLC LAGRALAAPQ QEALPDETEV VEETVAEVTE VSVGANPVQV EVGEFDDGAE ETEEEVVAEN PCQNHHCKHG KVCELDENNT PMCVCQDPTS CPAPIGEFEK VCSNDNKTFD SSCHFFATKC TLEGTKKGHK LHLDYIGPCK YIPPCLDSEL TEFPLRMRDW LKNVLVTLYE RDEDNNLLTE KQKLRVKKIH ENEKRLEAGD HPVELLARDF EKNYNMYIFP VHWQFGQLDQ HPIDGYLSHT ELAPLRAPLI PMEHCTTRFF ETCDLDNDKY IALDEWAGCF GIKQKDIDKD LVI

Full length human SPARC cDNA

gttgcctgtc tctaaacccc tccacattcc cgcggtcctt cagactgccc ggagagcgcg ctctgcctgc cgcctgcctg cctgccactg agggttccca gcaccatgag ggcctggatc ttctttctcc tttgcctggc cgggagggcc ttggcagccc ctcagcaaga agccctgcct gatgagacag aggtggtgga agaaactgtg gcagaggtga ctgaggtatc tgtgggagct aatcctgtcc aggtggaagt aggagaattt gatgatggtg cagaggaaac cgaagaggag gtggtggcgg aaaatccctg ccagaaccac cactgcaaac acggcaaggt gtgcgagctg gatgagaaca acacccccat gtgcgtgtgc caggacccca ccagctgccc agcccccatt ggcgagtttg agaaggtgtg cagcaatgac aacaagacct tcgactcttc ctgccacttc tttgccacaa agtgcaccct ggagggcacc aagaagggcc acaagctcca cctggactac atcgggcctt gcaaatacat ccccccttgc ctggactctg agctgaccga attccccctg cgcatgcggg actggctcaa gaacgtcctg gtcaccctgt atgagaggga tgaggacaac aaccttctga ctgagaagca gaagctgcgg gtgaagaaga tccatgagaa tgagaagcgc ctggaggcag gagaccaccc cgtggagctg ctggcccggg acttcgagaa gaactataac atgtacatct tccctgtaca ctggcagttc ggccagctgg accagcaccc cattgacggg tacctctccc acaccgagct ggctccactg cgtgctcccc tcatccccat ggagcattgc accacccgct ttttcgagac ctgtgacctg gacaatgaca agtacatcgc cctggatgag tgggccggct gcttcggcat caagcagaag gatatcgaca aggatcttgt gatctaaatc cactccttcc acagtaccgg attctctctt taaccctccc cttcgtgttt cccccaatgt ttaaaatgtt tggatggttt gttgttctgc ctggagacaa ggtgctaaca tagatttaag tgaatacatt aacggtgcta aaaatgaaaa ttctaaccca agacatgaca ttcttagctg taacttaact attaaggcct tttccacacg cattaatagt cccatttttc tcttgccatt tgtagctttg cccattgtct tattggcaca tgggtggaca cggatctgct gggctctgcc ttaaacacac attgcagctt caacttttct ctttagtgtt ctgtttgaaa ctaatactta ccgagtcaga ctttgtgttc atttcatttc agggtcttgg ctgcctgtgg gcttccccag gtggcctgga ggtgggcaaa gggaagtaac agacacacga tgttgtcaag gatggttttg ggactagagg ctcagtggtg ggagagatcc ctgcagaacc caccaaccag aacgtggttt gcctgaggct gtaactgaga gaaagattct ggggctgtgt tatgaaaata tagacattct cacataagcc cagttcatca ccatttcctc ctttaccttt cagtgcagtt tcttttcaca ttaggctgtt ggttcaaact tttgggagca cggactgtca gttctctggg aagtggtcag cgcatcctgc agggcttctc ctcctctgtc ttttggagaa ccagggctct tctcaggggc tctagggact gccaggctgt ttcagccagg aaggccaaaa tcaagagtga gatgtagaaa gttgtaaaat agaaaaagtg gagttggtga atcggttgtt ctttcctcac atttggatga ttgtcataag gtttttagca tgttcctcct tttcttcacc ctcccctttt ttcttctatt aatcaagaga aacttcaaag ttaatgggat ggtcggatct cacaggctga gaactcgttc acctccaagc atttcatgaa aaagctgctt cttattaatc atacaaactc tcaccatgat gtgaagagtt tcacaaatcc ttcaaaataa aaagtaatga cttagaaact gccttcctgg gtgatttgca tgtgtcttag tcttagtcac cttattatcc tgacacaaaa acacatgagc atacatgtct acacatgact acacaaatgc aaacctttgc aaacacatta tgcttttgca cacacacacc tgtacacaca caccggcatg tttatacaca gggagtgtat ggttcctgta agcactaagt tagctgtttt catttaatga cctgtggttt aacccttttg atcactacca ccattatcag caccagactg agcagctata tccttttatt aatcatggtc attcattcat tcattcattc acaaaatatt tatgatgtat ttactctgca ccaggtccca tgccaagcac tggggacaca gttatggcaa agtagacaaa gcatttgttc atttggagct tagagtccag gaggaataca ttagataatg acacaatcaa atataaattg caagatgtca caggtgtgat gaagggagag taggagagac catgagtatg tgtaacagga ggacacagca ttattctagt gctgtactgt tccgtacggc agccactacc cacatgtaac tttttaagat ttaaatttaa attagttaac attcaaaacg cagctcccca atcacactag caacatttca agtgcttgag agccatgcat gattagtggt taccctattg aataggtcag aagtagaatc ttttcatcat cacagaaagt tctattggac agtgctcttc tagatcatca taagactaca gagcactttt caaagctcat gcatgttcat catgttagtg tcgtattttg agctggggtt ttgagactcc ccttagagat agagaaacag acccaagaaa tgtgctcaat tgcaatgggc cacataccta gatctccaga tgtcatttcc cctctcttat tttaagttat gttaagatta ctaaaacaat aaaagctcct aaaaaatcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa

A 'SPARC polypeptide' as used herein refers to the full length 303 amino acid SPARC protein sequence and to any fragment or variant thereof, known in the art, that retains chemo-sensitzing activity. For example, Rahman M. et al. (PLOS ONE 10.1371/journal.pone.0026390 Published: 1 November 201 1) describe a number of SPARC polypeptides that have sensitizing activity. Furthermore, TABLE 1, below shows fragments that were tested in WO/2008/000079.

TABLE 1. Peptide sequences (from earlier filed WO/2008/000079)

9 B 14 CQNHHCKHGKVCELDENNTPMCVCQDPTSCP Y

13 A ESALCLPPACLPLRVPSTMRAWIFFLLCLAGRALA N

14 A l MRA IFFLLCLAGRALA N

15 A2 ACL P LR VP S TMR AW I F F L L C L AGRAL A N

16 A3 CLPPACLPLRVPSTMRAWIFFLLCLAGRALA N

17 B8- QPLEAVQPTAVEEDAEVETTEEV N

SCR

18 B 14- VPCKTGSKCHNNDPPTCNCEVDMCLQHQCEH N

SCR

SCR = scrambled peptide

Endoplasmic reticulum transmembrane glucose regulated protein 78 (GRP78) in a human is encoded by the 78 kdalton GRP78 gene (for example, the 5470 bp cDNA sequence represented by GenBank: M19645.1 or the 3973 bp mRNA sequence represented by NM_005347.4). Human G P78 encodes a 654 amino acid protein (see for example, NCBI Reference Sequence: NP_005338.1). GRP78 is a heat shock protein 70 (HSP70) family member of molecular chaperones important for endoplasmic reticulum integrity. GRP78 is also involved in regulating the unfolded protein response (UPR) and stress-induced autophagy.

A 'GRP78 inhibitor', as used herein, is meant to include any molecule capable of inhibiting GRP78 function. For example, a GRP78 inhibitor may be selected from one or more of the following: an RNA interference (RNAi) molecule for GRP78, an antibody or antibody fragment thereof that specifically binds to a GRP78 polypeptide, an immunogenic composition or vaccine which comprises a GRP78 polypeptide or an antigenic fragment thereof, or any other inhibitor of GRP78 function.

The pharmaceutical compositions described herein for the inhibition of GRP78 activity may be selected from any number of molecules known in the art. For example, GRP78 may be inhibited by an inhibitory nucleic acid molecule, for example, an antisense oligonucleotide, ribozymes, or an RNA interference (RNAi) molecule; an antibody or antibody fragment thereof that

specifically binds to a GRP78 polypeptide and inhibits GRP78 activity; an immunogenic composition or vaccine, which comprises a GRP78 polypeptide or an antigenic fragment thereof; a vector encoding an inhibitory polypeptide or an inhibitory nucleic acid molecule, for example, an siRNAs that include sequences sufficiently complementary to a portion of the GRP78 nucleic acid for inhibiting GRP78 transcription or translation levels; a polypeptide; or a small molecule. 'RNAi' as used herein is meant to include any of the gene silencing methods known in the art, including post-transcriptional gene silencing (PTGS) methods. These may include, but are not limited to any one or more of the following: microRNA (miRNA); small interfering (siRNA); short-hairpin RNA (shRNA); primary-microRNA (pri-miRNA); asymmetric interfering RNA (aiRNA); small internally segmented RNS (sisiRNA); RNA-DNA chimeric duplex; trans- kingdom RNA (tkRNA); tRNA-shRNA; tandem siRNA (tsiRNA); tandem haiφin RNA

(thRNA); pri-miRNA mimic cluster; and transcriptional gene silencing (TGS).

For example, an inhibitory GRP78 siRNA is described insi-GRP78, 5'- GGAGCGCAUUGAUACUAGATT-3' (sense) and 5'-UCUAGUAUCAAUGCGCUCCTT-3' (antisense) are described in 7,981,917. Similarly, Mhaidat, N. et al. (INT J BIOL AND BIOMED ENG (201 1) 2(5) :41-48) report the use of siRNA constructs (M-008198- 01 siGENOME SMARTpool™ Dharmacon (Lafayette, CO) to inhibit GRP78 in colorectal cancer cell lines. Also, US

201 100591 1 1 teaches a siRNA having the nucleotide sequence 5'-CTTGTTGGTGGCTCGACTCGA- 3' for inhibiting GRP78 transcription or translation levels in endothelial cells. An alternative GRP78 siRNA sequence 5'-AAGGTTACCCATGCAGTTGTT-3' (sense) and 3'- TTCCAATGGGTACGTCAACAA-5 ' (antisense) is described in US20120251543. US201 10008882 also described RNAi sequences 5'-AAGGATGGTTAATGATGCTGAGAA-3'; 5'- AAGGATGGTTAATGATGCTGAGAAgaagcttgTTCTCAGCATCATTAACCATCCTT-3'; and 5'- GGTTAATGATGCTGAGAActtcgaacTTCTCAGCATCATTAACC-3'. US20100135904 describes the following shRNAs 5'-CTGTCTAGACAAAAACAATGACTCTGAATTAAAGTCTCTTGAACTTTAATTCAG AGTCATTGCGGGGATCTGTGGTCTCATACA-3' and 5 ' -CTGTCTAGAC AAAAAACC ATAC ATTC AAGTTGA TTCTCTTGAAATCAACTTGAATGTATGGTCGGGGATCTGTGGTCTCATACA-3'.

For example, antibodies that bind to GRP78 are described in WO2013019730 (i.e. anti-GRP78 antibody 2D6F9) and WO/2008/105560.

A small molecule that inhibits GRP78 may be selected from one or more of the following:

versipelostatin (VST) is useful to inhibit transcription from GRP78 (Park et al, J. Nat. Cane. Inst., 96(17): 1300-1310, 2004; glycosylated derivatives of versipelostatin (Zhao P. et al. Org Biomol Chem. (2009) 7(7): 1454-60); versipelostatin analogues (Ueda J. et al. The Journal of Antibiotics (2008) 61 :752-755); Geldanamycin (Lawson B. et al. J of Cellular Physiol (1998) 174: 170-178); dATP; and Epigallocatechin Gallate (EGCG) as described in US20100135904.

Additional strategies for inhibiting GRP78 are described in the art. For example,

US20120251543; EP2393836; US20100009435; US20090181913; WO2007083101 ; and

WO/2008/105560.

For gene therapy strategies, endoplasmic reticulum stress elements (ERSE) associated with GRP78 are described in US20110268701.

As used herein, a 'patient" or a 'subject' are used interchangeably. A subject may be human, or a non-human animal, such as a rodent or transgenic mouse.

As used herein, a 'composition' may include small organic or inorganic molecules with distinct molecular composition made synthetically, found in nature, or of partial synthetic origin. Included in this group are nucleotides, nucleic acids, amino acids, peptides, polypeptides, proteins, peptide nucleic acids or complexes comprising at least one of these entities. A composition may be comprised of the effective composition alone (i.e. a pharmacologically effective amount) or in combination with a pharmaceutically acceptable excipient.

As used herein, a 'pharmaceutically acceptable excipient' includes any and all solvents, dispersion media, coatings, antibacterial, antimicrobial or antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The excipient may be suitable for intravenous, intraperitoneal, intramuscular, intrathecal or oral administration. The excipient may include sterile aqueous solutions or dispersions for extemporaneous preparation of sterile injectable solutions or dispersion. Use of such media for preparation of medicaments is known in the art.

As used herein, a 'pharmacologically effective amount' of a medicament refers to using an amount of a medicament present in such a concentration to result in a therapeutic level of drug delivered over the term that the drug is used. This may be dependent on the mode of delivery, time period of the dosage, age, weight, general health, sex and diet of the subject receiving the medicament. The determination of what dose is a 'pharmacologically effective amount' requires routine optimization, which is within the capabilities of one of ordinary skill in the art.

As used herein, the term 'cancer' refers to a proliferative disorder caused or characterized by the proliferation of cells, which have lost susceptibility to normal growth control. The term cancer, as used in the present application, includes tumors and any other proliferative disorders. Cancers of the same tissue type usually originate in the same tissue, and may be divided into different subtypes based on their biological characteristics. Four general categories of cancers are carcinoma (epithelial tissue derived), sarcoma (connective tissue or mesodermal derived), leukemia (blood-forming tissue derived) and lymphoma (lymph tissue derived). Over 200 different types of cancers are known, and every organ and tissue of the body may be affected. Specific examples of cancers that do not limit the definition of cancer may include melanoma, leukemia, astrocytoma, glioblastoma, retinoblastoma, lymphoma, glioma, Hodgkins' lymphoma and chronic lymphocyte leukemia. Examples of organs and tissues that may be affected by various cancers include pancreas, breast, thyroid, ovary, uterus, testis, prostate, thyroid, pituitary gland, adrenal gland, kidney, stomach, esophagus, colon or rectum, head and neck, bone, nervous system, skin, blood, nasopharyngeal tissue, lung, urinary tract, cervix, vagina, exocrine glands and endocrine glands. Alternatively, a cancer may be multicentric or of unknown primary site (CUPS).

As used herein, a 'cancerous cell' refers to a cell that has undergone a transformation event and whose growth is no longer regulated to the same extent as before the transformation event. A tumor refers to a collection of cancerous cells, often found as a solid or semi-solid lump in or on the tissue or in a subject.

A cancer or cancerous cell may be described as 'sensitive to' or 'resistant to' a given therapeutic regimen or chemotherapeutic agent based on the ability of the regimen to kill cancer cells or decrease tumor size, reduce overall cancer growth (i.e. through reduction of angiogenesis), and/or inhibit metastasis. Cancer cells that are resistant to a therapeutic regimen may not respond to the regimen and may continue to proliferate. Cancer cells that are sensitive to a therapeutic regimen may respond to the regimen resulting in cell death, a reduction in tumor size, reduced overall growth (tumor burden), or inhibition of metastasis. For example, this may manifest itself in a reduction in tumor size, overall growth/tumor burden, or the incidence of metastasis of about 10% or more, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or more, to about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold or more. Monitoring of a response may be accomplished by numerous pathological, clinical and imaging methods as known to persons of skill in the art.

A common theme for a chemotherapeutic agent or combination of agents is to induce death of the cancerous cells. For example, DNA adducts such as nitrosoureas, busulfan, thiotepa, chlorambucil, cisplatin, mitomycin, procarbazine, or dacacarbazine, slow the growth of the cancerous cell by forcing the replicating cell to repair the damaged DNA before the M-phase of the cell cycle, or may themselves cause sufficient damage to trigger apoptosis of the cancerous cell. Other events such as gene expression or transcription, protein translation, or methylation of the replicated DNA, for example, may also be interfered with by the varied arsenal of chemotherapeutic agents available to the clinician and help to trigger apoptotic processes within the cancerous cells. Alternately, a chemotherapeutic agent may enable the cancerous cell to be killed by aspects of the patient or test subject's humoral or acquired immune system, for example, the complement cascade or lymphocyte attack.

As used herein, a 'chemotherapeutic regimen' or 'chemotherapy' refers to the administration of at least one chemotherapy agent, to treat cancerous cells. Chemotherapy agents may be administered to a subject in a single bolus dose, or may be administered in smaller doses over time. A single chemotherapeutic agent may be used (single-agent therapy) or more than one agent may be used in combination (combination therapy). Chemotherapy may be used alone to treat some types of cancer. Alternatively, chemotherapy may be used in combination with other types of treatment, for example, radiotherapy or alternative therapies (for example immunotherapy) as described herein. Additionally, a chemosensitizer may be administered as a combination therapy with a chemotherapy agent, radiotherapy, or alternative therapies. As used herein, a 'chemotherapeutic agent' refers to a medicament that may be used to treat cancer, and generally has the ability to kill cancerous cells directly.

Examples of chemotherapeutic agents include alkylating agents, antimetabolites, natural products, hormones and antagonists, and miscellaneous agents. Examples of alternate names are indicated in brackets. Examples of alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine and thiotepa; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU) and streptozocin (streptozotocin); DNA synthesis antagonists such as estramustine phosphate; and triazines such as dacarbazine (DTIC, dimethyl- triazenoimidazolecarboxamide) and temozolomide. Examples of antimetabolites include folic acid analogs such as methotrexate (amethopterin); pyrimidine analogs such as fluorouracin (5- fluorouracil, 5-FU, 5FU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside) and gemcitabine; purine analogs such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG) and pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine and fludarabine; and topoisomerase inhibitors such as amsacrine. Examples of natural products include vinca alkaloids such as vinblastine (VLB) and vincristine; taxanes such as paclitaxel and docetaxel (Taxotere); epipodophyllotoxins such as etoposide and teniposide; camptothecins (CPT) such as topotecan and irinotecan; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin and tunicamycin (Tm); enzymes such as L- asparaginase; and biological response modifiers such as interferon alpha and interlelukin 2. Examples of hormones and antagonists include luteinising releasing hormone agonists such as buserelin; adrenocorticosteroids such as prednisone and related preparations; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol and related preparations; estrogen antagonists such as tamoxifen and anastrozole; androgens such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs such as leuprolide. Examples of miscellaneous agents include thalidomide; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin; anthracenediones such as mitoxantrone; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); adrenocortical suppressants such as mitotane (ο,ρ'-DDD) and aminoglutethimide; RXR agonists such as bexarotene; and tyrosine kinase inhibitors such as imatinib. Alternate names and trade-names of these and additional examples of chemotherapeutic agents, and their methods of use including dosing and administration regimens, will be known to a person versed in the art, and may be found in, for example "The Pharmacological basis of therapeutics", 10th edition. HARDMAN HG., LIMBIRD LE. editors. McGraw-Hill, New York, and in "Clinical Oncology", 3rd edition. Churchill Livingstone/ Elsevier Press, 2004. ABELOFF, MD. editor. In particular, suitable chemotherapeutic agents for use in accordance with the invention include, without limitation, nanoparticle albumin-bound paclitaxels.

As used herein, the term 'radiotherapeutic regimen' or 'radiotherapy' refers to the administration of radiation to kill cancerous cells. Radiation interacts with various molecules within the cell, but the primary target, which results in cell death is the deoxyribonucleic acid (DNA). However, radiotherapy often also results in damage to the cellular and nuclear membranes and other organelles. DNA damage usually involves single and double strand breaks in the sugar- phosphate backbone. Furthermore, there can be cross-linking of DNA and proteins, which can disrupt cell function. Depending on the radiation type, the mechanism of DNA damage may vary as does the relative biologic effectiveness. For example, heavy particles (i.e. protons, neutrons) damage DNA directly and have a greater relative biologic effectiveness. Electromagnetic radiation results in indirect ionization acting through short-lived, hydroxyl free radicals produced primarily by the ionization of cellular water. Clinical applications of radiation consist of external beam radiation (from an outside source) and brachytherapy (using a source of radiation implanted or inserted into the patient). External beam radiation consists of X-rays and/or gamma rays, while brachytherapy employs radioactive nuclei that decay and emit alpha particles, or beta particles along with a gamma ray.

Radiotherapy may further be used in combination chemotherapy, with the chemotherapeutic agent acting as a radiosensitizer. The choice of radiotherapy may be suited to an individual subject as determined by a skilled person at the point of care, taking into consideration the tissue and stage of the cancer. Examples of radiotherapy approaches to various cancers may be found in, for example "Clinical Oncology", 3rd edition. Churchill Livingstone/ Elsevier Press, 2004. ABELOFF, MD. (Editor).

As used herein, the term 'alternative therapeutic regimen' or 'alternative therapy' may include for example, biologic response modifiers (including polypeptide-, carbohydrate-, and lipid- biologic response modifiers), toxins, lectins, antiangiogenic agents, receptor tyrosine kinase inhibitors (for example Iressa™ (gefitinib), Tarceva™ (erlotinib), Erbitux™ (cetuximab), imatinib mesilate (Gleevec™), proteosome inhibitors (for example bortezomib, Velcade™); VEGFR2 inhibitors such as PTK787 (ZK222584), aurora kinase inhibitors (for example ZM447439); mammalian target of rapamycin (mTOR) inhibitors, cyclooxygenase-2 (COX-2) inhibitors, rapamycin inhibitors (for example sirolimus, Rapamune™); farnesyltransferase inhibitors (for example tipifarnib, Zarnestra); matrix metalloproteinase inhibitors (for example BAY 12-9566; sulfated polysaccharide tecogalan); angiogenesis inhibitors (for example Avastin™ (bevacizumab); analogues of fumagillin such as TNP-4; carboxy amino triazole; BB-94 and BB-2516; thalidomide; interleukin-12; linomide; peptide fragments; and antibodies to vascular growth factors and vascular growth factor receptors); platelet derived growth factor receptor inhibitors, protein kinase C inhibitors, mitogen-activated kinase inhibitors, mitogen- activated protein kinase kinase inhibitors, Rous sarcoma virus transforming oncogene (SRC) inhibitors, histonedeacetylase inhibitors, small hypoxia- inducible factor inhibitors, hedgehog inhibitors, and TGF-β signalling inhibitors. Furthermore, an immunotherapeutic agent would also be considered an alternative therapeutic regimen. Examples include chemokines, chemotaxins, cytokines, interleukins, or tissue factor. Suitable immunotherapeutic agents also include serum or gamma globulin containing preformed antibodies; nonspecific immunostimulating adjuvants; active specific immunotherapy; and adoptive immunotherapy. In addition, alternative therapies may include other biological-based chemical entities such as polynucleotides, including antisense molecules, polypeptides, antibodies, gene therapy vectors and the like. Such alternative therapeutics may be administered alone or in combination, or in combination with other therapeutic regimens described herein. Alternate names and trade-names of these agents used in alternative therapeutic regimens and additional examples of agents used in alternative therapeutic regimens, and their methods of use including dosing and administration regimens, will be known to a physician versed in the art. Furthermore, methods of use of chemotherapeutic agents and other agents used in alternative therapeutic regimens in combination therapies, including dosing and administration regimens, will also be known to a person versed in the art.

In particular, suitable alternative therapeutic regimens include, without limitation, antibodies to molecules on the surface of cancer cells such as antibodies to Her2 (e.g., Trastuzumab), EGF or EGF Receptors, VEGF (e.g., Bevacizumab) or VEGF Receptors, CD20, and the like. The therapeutic agent may further comprise any antibody or antibody fragment which mediates one or more of complement activation, cell mediated cytotoxicity, inducing apoptosis, inducing cell death, and opsinization. For example, such an antibody fragment may be a complete or partial Fc domain.

As used herein, a 'chemosensitizer' or 'sensitizer' is a medicament that may enhance the therapeutic effect of a chemotherapeutic agent, radiotherapy treatment or alternative therapeutic regimen, and therefore improve efficacy of such treatment or agent. Chemosensitizers may be used to overcome a resistant phenotype or to allow for a lower dose of a chemotherapeutic agent, radiotherapy treatment or alternative therapeutic regimen (and with the lower dose, reduced side effects). The sensitivity or resistance of a tumor or cancerous cell to treatment may also be measured in an animal, such as a human or rodent, by, e.g., measuring the tumor size, tumor burden or incidence of metastases over a period of time. For example, about 2, about 3, about 4 or about 6 months for a human and about 2-4, about 3-5, or about 4-6 weeks for a mouse. A composition or a method of treatment may sensitize a tumor or cancerous cell's response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is about 10% or more, for example, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%), or more, to about 2- fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15- fold, about 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of a person versed in the art. The terms 'peptide,' 'polypeptide,' and 'protein' may be used interchangeably, and refer to a compound comprised of at least two amino acid residues covalently linked by peptide bonds or modified peptide bonds, for example peptide isosteres (modified peptide bonds) that may provide additional desired properties to the peptide, such as increased half-life. A peptide may comprise at least two amino acids. The amino acids comprising a peptide or protein described herein may also be modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It is understood that the same type of modification may be present in the same or varying degrees at several sites in a given peptide.

Examples of modifications to peptides may include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, Proteins- Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold F, Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in Posttranslational Covalent Modification of Proteins, B. C. Johnson, ed., Academic Press, New York, 1983; Seifter et al., Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. (1990) 182: 626-646 and Rattan et al. (1992), Protein Synthesis: Posttranslational Modifications and Aging, " Ann NY Acad Sci 663: 48-62.

As used herein, the term 'polynucleotide' includes RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non- natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), and modified linkages (e.g., alpha anomeric polynucleotides, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.

'Peptide nucleic acids' (PNA) as used herein refer to modified nucleic acids in which the sugar phosphate skeleton of a nucleic acid has been converted to an N-(2-aminoethyl)-glycine skeleton. Although the sugar-phosphate skeletons of DNA/RNA are subjected to a negative charge under neutral conditions resulting in electrostatic repulsion between complementary chains, the backbone structure of PNA does not inherently have a charge. Therefore, there is no electrostatic repulsion. Consequently, PNA has a higher ability to form double strands as compared with conventional nucleic acids, and has a high ability to recognize base sequences. Furthermore, PNAs are generally more robust than nucleic acids. PNAs may also be used in arrays and in other hybridization or other reactions as described above and herein for oligonucleotides.

As used herein, the term 'vector' refers to a polynucleotide compound used for introducing exogenous or endogenous polynucleotide into host cells. A vector comprises a nucleotide sequence, which may encode one or more polypeptide molecules. Plasmids, cosmids, viruses and bacteriophages, in a natural state or which have undergone recombinant engineering, are non-limiting examples of commonly used vectors to provide recombinant vectors comprising at least one desired isolated polynucleotide molecule.

There are also provided nucleic acid constructs comprising control elements and a nucleic acid molecule described herein operatively linked to the control elements (e.g., a suitable promoter) for expression of a polypeptide or a polypeptide herein described. Protein expression is dependent on the level of RNA transcription, which is in turn regulated by DNA signals. Similarly, translation of mRNA requires, at the very least, an AUG initiation codon, which is usually located within about 10 to about 100 nucleotides of the 5' end of the message. Sequences flanking the AUG initiator codon have been shown to influence its recognition by eukaryotic ribosomes, with conformity to a perfect Kozak consensus sequence resulting in optimal translation (see, e.g., Kozak, J. Molec. Biol. (1987) 196: 947-950). Also, successful expression of an exogenous nucleic acid in a cell can require post-translational modification of a resultant protein. Accordingly, the invention provides plasmids encoding polypeptides wherein the vector is, e.g., pCDNA3.1 or a derivative thereof.

The nucleic acid molecules described herein may comprise a coding region operatively linked to a suitable promoter, which promoter is preferably functional in eukaryotic cells. Viral promoters, such as, without limitation, the RSV promoter and the adenovirus major late promoter can be used in the invention. Suitable non-viral promoters include, but are not limited to, the phosphoglycerokinase (PGK) promoter and the elongation factor la promoter. Non-viral promoters are desirably human promoters. Additional suitable genetic elements, many of which are known in the art, also can be ligated to, attached to, or inserted into the inventive nucleic acid and constructs to provide additional functions, level of expression, or pattern of expression. The native promoters for expression of the SPARC family genes also can be used, in which event they are preferably not used in the chromosome naturally encoding them unless modified by a process that substantially changes that chromosome. Such substantially changed chromosomes can include chromosomes transfected and altered by a retroviral vector or similar process. Alternatively, such substantially changed chromosomes can comprise an artificial chromosome such as a HAC, YAC, or BAC.

In addition, the nucleic acid molecules described herein may be operatively linked to enhancers to facilitate transcription. Enhancers are cis-acting elements of DNA that stimulate the transcription of adjacent genes. Examples of enhancers, which confer a high level of transcription on linked genes in a number of different cell types from many species include, without limitation, the enhancers from SV40 and the RSV-LTR. Such enhancers can be combined with other enhancers, which have cell type-specific effects, or any enhancer may be used alone. To optimize protein production of the nucleic acid molecules described herein, the molecules may further comprise a polyadenylation site following the coding region of the nucleic acid molecule. Also, preferably all the proper transcription signals (and translation signals, where appropriate) will be correctly arranged such that the exogenous nucleic acid will be properly expressed in the cells into which it is introduced. If desired, the exogenous nucleic acid also can incorporate splice sites (i.e., splice acceptor and splice donor sites) to facilitate mRNA production while maintaining an in frame, full-length transcript. Moreover, the inventive nucleic acid molecules can further comprise the appropriate sequences for processing, secretion, intracellular localization, and the like.

The nucleic acid molecules can be inserted into any suitable vector. Suitable vectors include, without limitation, viral vectors. Suitable viral vectors include, without limitation, retroviral vectors, alpha viral, vaccinial, adenoviral, adeno-associated viral, herpes viral, and fowl pox viral vectors. The vectors preferably have a native or engineered capacity to transform eukaryotic cells, e.g., CHO-K1 cells. Additionally, the vectors useful in the context of the invention can be 'naked' nucleic acid vectors (i.e., vectors having little or no proteins, sugars, and/or lipids encapsulating them) such as plasmids or episomes, or the vectors can be complexed with other molecules. Other molecules that can be suitably combined with the inventive nucleic acids include without limitation viral coats, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties such as ligands, receptors, or antibodies that target cellular molecules.

Transfection vectors capable of expressing polynucleotides encoding fragments or the full length SPARC polypeptide sequence or fragments or variants thereof and/or the collagen-IV polypeptide sequence into cancer cells, may be used to transform cancer cells to sensitize them prior to or concomitantly with a chemotherapeutic agent.

Gene therapy is a medical intervention that involves modifying the genetic material of living cells to fight disease. Gene therapy is being studied in clinical trials (research studies with humans) for many different types of cancer and for other diseases. Accordingly, the invention further provides for an isolated nucleic acid molecule encoding a SPARC polypeptide suitable for use in 'gene therapy' (for example, Patil et al, AAPS J. 7(l):E61-77 (2005)). In general, a gene may be delivered to the cell using a 'vector' such as those disclosed herein. The most common types of vectors used in gene therapy are viruses. Viruses used as vectors in gene therapy are genetically disabled, whereby they are unable to reproduce themselves. Some gene therapy clinical trials rely on mouse retroviruses to deliver the desired gene. Other viruses used as vectors include adenoviruses, adeno-associated viruses, poxviruses, and the herpes virus. Suitable viral gene therapy vectors and modes of their administration in vivo and ex vivo are known in the art.

Gene therapy can be performed both ex vivo and in vivo. Typically, in ex vivo gene therapy clinical trials, cells from the subject's blood or bone marrow are removed and grown in the laboratory. The cells are exposed to the virus that is carrying the desired gene. The virus enters the cells, and the desired gene becomes part of the cells' DNA. The cells grow in the laboratory and are then returned to the patient by injection into a vein. Using in vivo gene therapy, vectors such as, e.g., viruses or liposomes may be used to deliver the desired gene to cells inside the patient's body. In the present application, the GRP78 inhibitor gene and SPARC gene may be delivered to a subject (in vivo) or to a subject's cells (ex vivo) either in the same vector or in separate vectors, such that both are produced by or near the cancer cells being targeted. Furthermore, the subject's blood could be treated with a chemo therapeutic agent either ex vivo or in vivo.

Alternatively, a subject's cells (for example, blood or bone marrow cells) may be treated ex vivo to by exposing the cells to SPARC, a GRP78 inhibitor, and a chemotherapeutic agent to treat the cells prior to returning the cells to the subject. Alternatively, the subject's cells may be cultured with a GRP78 inhibitor and exposed to SPARC peptide/protein and exposed to a

chemotherapeutic agent.

Alternatively, the GRP78 inhibitor may be used in a pharmaceutical composition by combining the GRP78 inhibitor with SPARC protein and/or a chemotherapeutic agent. The GRP78 inhibitor may be combined with SPARC peptide/protein and administered to a tumor locally. The administration may be directly to a tumor or to a surgical site following tumor resection. The compositions described herein may also include one or more chemotherapeutic agent(s). The compositions described herein may also be viscous, in the form of a gel or paste. The compositions when administered may also have the added benefit that the SPARC/ GRP78 inhibitor and/or one or more chemotherapeutic agents may have a longer residence time at the desired site. Furthermore, such compositions may provide delayed release of the SPARC peptide/protein, the GRP78 inhibitor, and the one or more chemotherapeutic agents.

Accordingly, the effect of the composition may be extended beyond that of traditional systemic delivery and or topical administration with a less viscous composition.

Methods and Materials

Cells

MIP-101 cells are a colon carcinoma cell line, which are sensitive to chemotherapeutic treatments. SPARC over-expressing MIPlOl cells (MIP/SP), empty-vector control (MIP/ZEO), intrinsically high-SPARC-expressing cells (HCT1 16), low-SPARC-expressing cells (RKO), GRP78-expressing cells (MIP/78), and SPARC over-expressing/GRP78-expressing cells (MIP/SP/78) were also used. The cells were assessed for response to Tunicamycin (Tm); 5-FU; CPT; and Cetuximab treatments in terms of cell viability (i.e. MTT-assay) and induction of apoptosis (i.e. TUNEL-assay and caspase 3/7 activity).

Human colorectal cancer cells MIPlOl and RKO was maintained in DMEM medium

supplemented with 1% penicillin-streptomycin 1% kanamycin, 10% newborn calf serum, and 5% C0₂. MIP/Zeo and MIP/SP were stably transfected with empty vector or with SPARC and selected with Zeocin (Invitrogen™). For MIP/5FU and RKO/5FU, the media were

supplemented with 10 μΜ 5-FU. MIP/CPT and RKO/CPT, the media were supplemented with 50 μΜ CPT-11.

Cell viability (MTT assay)

Analysis of cell viability at 48 hours following treatment with 5FU and Tm was performed by MTT assay and showed no significant change in GRP78-overexpressing MIPlOl cells (MIP/78) compared to control (MIP or MIP/Zeo empty vector). However, overexpressing GRP78 in SPARC-overexpressing MIP/SP (MIP/SP/78) cells showed that the reduction in cell viability in Tm or 5FU-treated MIP/SP cells could be abolished in MIP/SP/78 cells (*P<0.05, **P<0.005). Cell viability was assessed by adding MTT reagent for 4 hour followed by addition of DMSO to develop the purple color formazan and measuring absorbance at 490 nM and 650 nM. The absorbance values at 690 nM were subtracted from the absorbance values 450 nM and percentage of viable cells were calculated based on the reading of treated cells /untreated cells (control) XI 00=% cell viability.

TUNEL-Assay

Detection of apoptosis by TUNEL assay in MIP, MIP/78, MIP/SP, MIP/SP/78 treated with 5FU (10 μΜ) and Tm (0.5 mg/ml) demonstrated a significant reduction in TUNEL-positive cells in GRP78- overexpressing cells (*P < 0.05, **P < 0.0057). The cells were seeded to achieve 60% confluence. After 24h, the cells were treated with 5FU (5 μΜ) and Tm (0.5 mg/ml) for 48 h. The suspension and attached cells were harvested, and fixed onto glass slides using a Shandon Cytospin™ at 2000 rpm for 10 min and stained as per the manufacturer's instructions

(Promega™). The number of TUNEL-positive cells was counted and averaged from four independent experiments were counted.

Caspase 3/7 Assay

Caspase 3/7 activity of MlP/Zeo, MIP/SP and HCT1 16 cells treated with Tm (0.5 g/ml) for 30 min. All three cell lines showed a significant increased in caspase 3/7 activity (*P < 0.05) indicating that the cells were undergoing apoptosis (FIGURE 3A).

Treatment with 5FU (5 μΜ) increased caspase 3/7 activity in MIP/SP compared to MlP/Zeo (*P value < 0.05). 5FU (5 μΜ) was added after 24 hours of cell seeding and cells were treated for 0, 1 and 3 hours. Cells were harvested at each time point (i.e. 0, 1, and 3 hours) (FIGURE 3C). Cell lysates were prepared using CHAPS lx and 20 μg of proteins were used in Caspase-Glo 3/7 Assay (Promega™), using 1 : 1 dilution of Caspase - Glo 3/7 Substrate. Relative luminescence units (RLU) were quantified using a VICTOR2 1420™ multi-label counter (PerkinElmer Life Sciences™). GRP78 siRNA Knockdown

Knockdown of GRP78 in MIP/SP cells reduced cell viability following treatment with 5FU (10 μΜ), CPT (10 μΜ), and Cetuximab (10 μg/ml) after 72 h (*P< 0.05) as compared to cells that were not treated with GRP78 siRNA. The siRNA transfection was performed using a pool of three GRP78 siRNAs (Thermo Scientific™).

CHOP siRNA Knockdown

Knockdown of CHOP by transient siRNA transfection followed by 5FU (6 h) treatment impaired activation of ER-stress proteins in MIP/SP cells compared to MIP/Zeo. CHOP shRNA was obtained from Applied Biological Materials Inc.™ (Cat#i065675c (CHOP siRNA target sequences X 4 - in iLenti™-GFP siRNA Expression Vector)). qRT-PCR

Various colorectal cancer cell lines (i.e. MIP, MIP/SP, MIP-5FU, MIP-CPT, RKO, RKO-5FU, RKO-CPT, and HCT1 16) were assessed for GRP78 mRNA levels by quantitative RT-PCR (qRT-PCR). RNA was isolated with TRIzol (Invitrogen™). Total RNA 1 μg was used to generate cDNA (Superscript III, Invitrogen™). PCR products were separated on a 1.5% agarose gel electrophoresis. ImageJ™ was used to quantify the relative expression levels of GRP78 to β- actin.

Immunobloting Analysis

Gene expression analysis of GRP78 expression was also carried out by immunoblot assay.

GRP78 protein levels were compared to beta-actin by immunoblot analysis in various cell types (i.e. MIP/ZEO, MIP/SP, HCT1 16, MIP101 , and RKO) and some cell types that are resistant to some chemotherapeutics (i.e. MIP-5FU, MIP-CPT, RKO-5FU, and RKO-CPT) (see FIGURE 1(B)).

Treatment with Tm (0.5 mg/ml) for 30 min, increased GRP78, IRE1 and SPARC expression in the membrane fraction, as compared to the cytoplasm and nucleus in MIP/SP cells (FIGURE 2B). These immunoblots used calnexin (a membrane bound protein) and actin as controls. Western blot analysis of proteins involved in ER stress signaling: MIP/SP (high SPARC expression) and HCT1 16 (moderate SPARC expression) showed early activation of GRP78; and PERK/p-PERK, respectively, following treatment with Tm, as compared to MIP/Zeo cells (low SPARC expression). Increasing CHOP expression was most pronounced in MIP/SP cells. Results demonstrate a greater induction of the ER- stress pathway in chemosensitive SPARC- overexpressing CRC cells (FIGURE 2C).

Similarly, FIGURES 3B and 3D, show Western blots of ER-stress induced proteins, GRP78, p- PERK, p-eIF2 in MIP/Zeo and MIP/SP after treatment with 5Fu (5 μΜ) at various time points and a knockdown of CHOP by transient siRNA transfection followed by 5FU (6 h) treatment impaired activation of ER-stress proteins in MIP/SP cells compared to MIP/Zeo.

Immunodetection was performed using antibodies against GRP78, Full PARP, Cleaved-PARP, and Caspase 8, and CHOP (1 :1000, Cell Signaling Technology™); p-PERK, PERK, p-eIF2, EIF2 (1 : 1000, Santa Cruz Biotechnology™); β-actin (1 : 1000, Abeam™) followed by incubation with the appropriate secondary antibody.

Co-Immuno-preciptation Analysis

Activation of signaling events in ER-stress pathway in SPARC- overexpressing CRC cells (MIP/SP) was investigated. Co-immuno-precipitation studies showed an interaction between GRP78 and SPARC in MIP/SP cells. For the immune-precipitation studies, antibodies against GRP78 (Cell Signaling Technology™) and anti-V5 antibody (to detect the SPARC-V5 fusion protein) were used.

Confocal Microscopy

Confocal images of SPARC-overexpressing MIP101 cells (MIP/SP) cells expressing GRP78 (red) and SPARC (green) following treatment with Tm (0.5 mg/ml) for 30 min (nuclear stain - DRAQ5 (blue). GRP78 and SPARC expression in normal human colon and colon cancer (x300). For confocal microscopy, cells were fixed with 2% paraformaldehyde for 20 min, rinsed with cold PBS, followed by 0.1% triton in PBS. The cells were blocked with 5% BSA for 30 min and incubated with anti-SPARC and anti-GRP78 (all at 1 :200) in 5% BSA for 1 h at room temperature. Cells were washed with PBS and incubated with Alexafluor™ goat anti-mouse and Alexafluor™ goat anti-rabbit (all at 1 :400) in 5% BSA for 1 h at room temperature. Draq5 (1 :5000, 5 min) was used for nuclear stain.

Tissue Microarray - GRP78 to SPARC Expression

Tumors from patients were obtained for tissue microarray construction. 4 μπι-thick sections were made from the TMA block and subsequently de-paraffinized in xylene and rehydrated. Sections were heated in citrate buffer for 15 minutes in a cooker for antigen retrieval.

Endogenous peroxidase activity was blocked using 0.3% H₂0₂ and washed with PBS for 10 minutes. Immuno-histochemical staining with all primary antibodies was carried out using Ultravision LP™ detection kit (Thermo Fisher Scientific™, Fremont, CA, USA). Sections were treated with Ultra V Block for 5 minutes to prevent nonspecific reaction with primary antibodies, then incubated at 4°C for 24 hours with primary antibodies, followed by incubation with a primary antibody enhancer for 10 minutes at room temperature. Subsequently sections were treated with HRP polymer for 15 minutes and the reaction product was developed using 3,3- diaminobenzidine tetrahydrochloride (Zymed™ - South San Francisco, CA, USA). The sections were counterstained with hematoxylin and mounted with Tissue-Tek Glas™ 6419 (Sakura Finetek™ - Torrance, CA, USA). Negative controls consisted of omission of the primary antibodies. Primary antibodies used in this study were directed against GRP78 (Cell Signalling), and SPARC (Hematologic Technologies Inc™). Staining expression scores were based on the number of tumor cells with positive staining and were scored by two independent pathologists who were blinded to clinic-pathological data. The two expression scores per sample were averaged, with the average representing the patient's final expression intensity. GRP78: SPARC expression was considered "low" if the ration was <1.5. GRP78:SPARC expression was considered "high" for ratios >1.5.

EXAMPLES

EXAMPLE 1: SPARC and GRP78 expression in colorectal cancer cells

FIGURE 1(A) shows GRP78 mRNA and protein expression in MIP, MIP/SP, MIP-5FU, MIP- CPT, RKO, RKO-5FU, RKO-CPT, and HCT1 16 (for mRNA) and FIGURE 1(B) MIP/ZEO, MIP/SP, HCT1 16, MIP101, MIP-5FU, MIP-CPT, RKO, RKO-5FU, and RKO-CPT (for protein). The protein expression was compared to beta-actin as a control. These results show an increase in GRP78 mRNA in both the non-resistant high-SPARC-expressing cells (i.e. MIP-SP) and all the low-SPARC-expressing cells (i.e. RKO, RKO-5FU, and RKO-CPT). Similarly, all the low-SPARC-expressing cells (i.e. RKO, RKO-5FU, and RKO-CPT) showed more intense staining of GRP78 than the cells expressing more SPARC, with the exception of the MIP-ZEO cells (empty vector control).

Confocal analysis of MIP/SP/78 cells were examined before and following Tm (0.5 mg/ml) treatment for 30 min and showed a marked decrease in SPARC expression in the Tm treated cells as compared to untreated cells (images not shown). Similarly, GRP78 and SPARC expression were examined by confocal analysis in normal human colon and colon cancer cells, wherein the tumor cells showed an increase in SPARC expression as compared to normal noncancerous colon cells (images not shown). The interaction between SPARC and GRP78, confirmed by co-IP studies (below), co-localization of SPARC and GRP78 was observed following Tm treatment, and appeared to be localized to the ER in CRC MIP/SP cells following treatment with tunicamycin.

EXAMPLE 2: Activation of signaling events in ER-stress pathway in SPARC- overexpressing CRC cells

The results in FIGURE 2 demonstrate a greater induction of the ER-stress pathway in

chemosensitive SPARC-overexpressing CRC cells (i.e. MIP/SP; and HTCl 16) than in MIP-ZEO cells. In FIGURE 2A, the co-immunoprecipitation studies show an interaction between GRP78 and SPARC in MIP/SP cells. In FIGURE 2B, treatment with Tm (0.5 mg/ml) for 30 min, increased GRP78, IREl and SPARC expression in the membrane fraction, as compared to the cytoplasm and nucleus of the CRC cells as compared to the controls, calnexin (a membrane bound protein) and actin. In FIGURE 2C, shows a Western blot analysis of proteins involved in ER stress signalling. MIP/SP (high SPARC expression) and HCT1 16 (moderate SPARC expression) showed early activation of GRP78; and PERK p-PERK, respectively, following treatment with Tm, as compared to MIP/Zeo cells (low SPARC expression). Increasing CHOP expression was most pronounced in MIP/SP cells. These results demonstrate a greater induction of the ER- stress pathway in chemosensitive SPARC-overexpressing CRC cells

Furthermore, in FIGURE 3 A, MIP/ZEO, MIP/SP and HCT1 16 were treated with Tm (0.5 g/ml) for 30 min or control DMSO prior to a caspase 3/7 assay. All three cell lines showed a significant increase in caspase 3/7 activity (*P <0.05) as compared to the DMSO control. In FIGURE 3B, shows a Western blot of ER-stress induced proteins, GRP78, p-PERK, p-eIF2 in MIP/ZEO and MIP/SP after treatment with 5Fu at various time points (0, 1 , 3, and 6 hours post treatment). FIGURE 3C, shows that treatment with 5FU (5 μΜ) increased caspase 3/7 activity in MIP/SP compared to MIP/ZEO (*P value < 0.05) at 0, 1, and 3 hours post treatment. In

FIGURE 3D, a knockdown of CHOP by transient siRNA transfection followed by 5FU treatment showed impaired activation of ER-stress proteins in MIP/SP cells as compared to MIP/ZEO cells.

EXAMPLE 3: SPARC inhibits GRP78-mediated cell survival in CRC cells

In order to understand the interaction between SPARC and GRP78, with regards to CRC cell survival, cell survival assays and apoptosis assays were carried out on various cell lines (MIP, MIP-ZEO, MIP-78, MIP-SP, and MIP-SP-78), that variably produce SPARC and GRP78, as shown in FIGURE 4.

As shown in FIGURE 4A, cell viability at 48 hours following treatment with 5FU and Tm by MTT assay showed no significant change in GRP78-overexpressing MIP101 cells (MIP/78) compared to control (MIP or MIP/ZEO empty vector). However, overexpressing GRP78 in SPARC-overexpressing MIP/SP (MIP/SP/78) cells showed that the reduction in cell viability in Tm or 5FU-treated MIP/SP cells could be abolished in MIP/SP/78 cells (*P<0.05, **P<0.005). Furthermore, FIGURE 4B shows that a knockdown of GRP78 in MIP/SP cells reduced cell viability following treatment with 5FU (10 μΜ), CPT (10 μΜ), and Cetuximab (10 μg/ml) after 72 h (*P< 0.05). FIGURE 4C, shows that apoptosis detected by TUNEL assay in MIP, MIP/78, MIP/SP, MIP/SP/78 treated with 5FU (10 μΜ) and Tm (0.5 mg/ml) showed a significant reduction in TUNEL-positive cells in GRP78-overexpressing cells (*P < 0.05, **P < 0.0057). The results shown in FIGURE 4 suggest that the interaction of SPARC and a GRP78 inhibitor may have a synergistic effect, since the benefit from the combination of SPARC with a GRP78 inhibitor is greater than the added individual benefits derived from SPARC alone and a GRP78 inhibitor alone.

EXAMPLE 4: Relative gene expression levels of GRP78 to SPARC in various MIP colorectal cancer cell lines

FIGURE 5 shows the relative gene expression level of GRP78 to SPARC for MIP-101 , MIP- ZEO, MIP-SP, and MIP-5FU cells. The ration of GRP78 to SPARC was highest in the 5FU chemoresistant CRC cell line (MIP-5FU) and lowest in the chemosensitive SPARC- overexpressing cell line (MIP-SP).

EXAMPLE 5: A comparison of CRC patients having either high and low expression levels of GRP78 to SPARC as a function of survival over time

In FIGURE 5, a plot shows a comparison of subjects having a high GRP78 to SPARC expression level to subjects having a low GRP78 to SPARC expression level, with regards to their survival probability over time. Subjects having a low GRP78 to SPARC expression level had a significantly longer overall survival than subjects with a high GRP78 to SPARC expression level. Accordingly, suggesting that the interplay between GRP78 and SPARC levels in significant in determining patient survival and that treating patients to produce a low GRP78 to SPARC ratio may be beneficial to the patient/subject.

Overexpression of GRP78 in chemosensitive MIP/SP cells resulted in an attenuation of drug sensitivity by increasing cell viability by 50%, while decreasing apoptosis by 50% in response to 5-FU in comparison to empty vector control transfected MIP/SP cells. Not surprisingly, the reverse was observed following GRP78 knock-down with siRNA, with a reduction in cell viability of 50%. Overexpression of GRP78 protects cells from ER-stress by association with ER sensors and re-establishment of homeostasis for normal ER function. Interestingly, exposure of MIP/SP cells to tm promoted ER-stress mediated apoptosis by upregulating pPERK, p-eIF2a and CHOP, while the opposite was observed in cells that under-express SPARC. This study reveals a novel interaction between SPARC and GRP78, and demonstrates that SPARC'S ability to reverse chemotherapy resistance in vitro and in vivo to also involve an attenuation of GRP78's ability to induce drug resistance by promoting ER-stress mediated apoptosis.

The results described herein, show a clear interaction between GRP78 and SPARC during endoplasmic recticulum (ER) stress. The present application shows that activation of the UPR pathway (following exposure to tunicamycin and 5FU) occurs more rapidly in CRC cells overexpressing SPARC, resulting in a greater induction of apoptosis. Furthermore, the reduction of GRP78 levels sensitizes CRC cells to chemothrapy, which augments apoptosis in chemosensitive SPARC-overexpressing cells.

In summary, the novel interaction between SPARC and GRP78 and our findings demonstrating SPARC' S ability to attenuate the cytoprotective function of GRP78 may offer new treatment options for CRC by combining SPARC therapy with GRP78 inhibitors to enhance tumor regression.

Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to an embodiment of the present invention. Any priority document(s) and all publications, including but not limited to patents and patent applications, cited in this specification are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings.

Claims

CLAIMS What is claimed is:

1. A method of sensitizing a cancer cell, the method comprising: contacting the cancer cell with a SPARC polypeptide and a GRP78 inhibitor.

2. The method of claim 1, wherein the contacting of the cancer cell is ex vivo.

3. The method of claim 1 , wherein the contacting of the cancer cell is in vivo.

4. The method of claim 1, 2, or 3, wherein the method, further comprises contacting the cancer cell with a chemotherapeutic agent.

5. The method of any one of claims 1-4, wherein the SPARC polypeptide is provided via gene therapy.

6. The method of any one of claims 1-5, wherein the GRP78 inhibitor is provided via gene therapy.

7. The method of any one of claims 1-4, wherein the SPARC polypeptide is provided in a pharmaceutical composition.

8. The method of any one of claims 1-4, wherein the GRP78 inhibitor is provided in a

pharmaceutical composition.

9. The method of any one of claims 1-8, wherein the SPARC polypeptide comprises the full length protein.

10. The method of any one of claims 1-8, wherein the SPARC polypeptide comprises a

sensitizing fragment or variant.

1 1. The method of any one of claims 1- 4, and 7-10, wherein the SPARC polypeptide and the GRP78 inhibitor form a pharmaceutical composition.

12. The method of any one of claims 1- 4, and 7-1 1, wherein the SPARC polypeptide, the GRP78 inhibitor, and the chemotherapeutic agent form a pharmaceutical composition.

13. The method of any one of claims 1-12, wherein the GRP78 inhibitor is selected from one or more of the following:

(a) a GRP78 inhibitory nucleic acid molecule;

(b) an antibody or antibody fragment thereof that specifically binds to a GRP78 polypeptide;

(c) an immunogenic composition or vaccine which comprises a GRP78 polypeptide;

(d) a vector encoding an inhibitory polypeptide or an inhibitory nucleic acid molecule; (e) a polypeptide; and

(f) a small molecule.

14. The method of claim 13, wherein the GRP78 inhibitory nucleic acid molecule is selected from an antisense oligonucleotide, a ribozyme, and an RNA interference (RNAi) molecule.

15. The method of claim 14, wherein the RNAi is selected from one or more of microRNA (miRNA); small interfering (siRNA); short-hairpin RNA (shRNA); primary-microRNA (pri-miRNA); asymmetric interfering RNA (aiRNA); small internally segmented RNS (sisiRNA); RNA-DNA chimeric duplex; trans-kingdom RNA (tkRNA); tRNA-shRNA; tandem siRNA (tsiRNA); tandem hairpin RNA (thRNA); pri-miRNA mimic cluster; and transcriptional gene silencing (TGS).

16. The method of claim 13, wherein the small molecule is selected from versipelostatin

(VST), a glycosylated derivative of versipelostatin, a versipelostatin analogue,

geldanamycin, and epigallocatechin Gallate (EGCG).

17. The method of any one of claims 1-16, wherein the cancer cell is a colorectal cancer cell.

18. The method of claim 17, wherein the colorectal cancer cell is chemotherapy resistant.

19. A composition comprising a SPARC polypeptide and a GRP78 inhibitor, wherein the

composition sensitizes a cancer cell to a chemotherapeutic agent.

20. The composition of claim 19, wherein the composition further comprises a

chemotherapeutic agent.

21. The composition of claim 19 or 20, wherein the SPARC polypeptide comprises the full length protein.

22. The composition of claim 19 or 20, wherein the SPARC polypeptide comprises a

sensitizing fragment or variant thereof.

23. The composition of any one of claims 19-22, wherein the composition is formulated as a gel or paste.

24. A pharmaceutical composition for treating colorectal cancer, comprising a SPARC

polypeptide and a GRP78 inhibitor.

25. The pharmaceutical composition of claim 24, wherein the composition is formulated as a gel or paste.

26. Use of a composition comprising a SPARC polypeptide and a GRP78 inhibitor for

sensitizing a cancer cell.

27. Use of a composition comprising a SPARC polypeptide and a GRP78 inhibitor and a pharmaceutically acceptable carrier for sensitizing a cancer cell.

28. Use of a SPARC polypeptide and a GRP78 inhibitor for sensitizing a cancer cell.

29. Use of a SPARC polypeptide and a GRP78 inhibitor in the manufacture of a medicament for sensitizing a cancer cell.

30. The use of any one of claims 26-29, wherein the cancer cell is a colorectal cancer cell.

31. The use of claim 30, wherein the colorectal cancer cell is chemotherapy resistant.

32. A commercial package comprising (a) a SPARC polypeptide and a GRP78 inhibitor; and (b) instructions for the use thereof for sensitizing a cancer cell.

33. A commercial package comprising (a) a pharmaceutical composition comprising a SPARC polypeptide, a GRP78 inhibitor and a pharmaceutically acceptable carrier; and (b) instructions for the use thereof for sensitizing a cancer cell.

34. A vector, the vector comprising a SPARC polypeptide and a GRP78 inhibitor encoding polynucleotides.

35. A method of sensitizing a CRC cell comprising contacting the cancer cell with a SPARC polypeptide and an agent that inhibits GRP78 in said cancer cells.

36. A method of sensitizing a tumor in an animal, the method comprising: (a) measuring the level of SPARC polypeptide and GRP78 polypeptide in said tumor or the level of SPARC mRNA and GRP78 mRNA in said tumor, to determine the ratio of GRP78 to SPARC polypeptide or mRNA in said tumor; and (b) administering either a SPARC polypeptide; or a GRP78 inhibitor; or both a SPARC polypeptide and a GRP78 inhibitor, to establish a low GRP78 to SPARC ratio in the animal.

37. The method of claim 36, wherein steps (a) and (b) are repeated until a low GRP78 to

SPARC ratio is established.

38. The method of claim 36 and 37, wherein the low GRP78 to SPARC ratio is <1.5.

39. The method of claim 36, 37, or 38, wherein the SPARC polypeptide is measured by

immunohistochemistry, western-blot or mass spectroscopy.

40. The method of any one of claims 36-39, wherein the contacting the cancer cell is ex vivo.

41. The method of any one of claims 36-39, wherein the contacting the cancer cell is in vivo.

42. The method of any one of claims 36-41 , wherein the method, further comprises contacting the cancer cell with a chemotherapeutic agent.

43. The method of any one of claims 36-42, wherein the SPARC polypeptide is provided via gene therapy.

44. The method of any one of claims 36-43, wherein the GRP78 inhibitor is provided via gene therapy.

45. The method of any one of claims 36-42, wherein the SPARC polypeptide is provided in a pharmaceutical composition.

46. The method of any one of claims 36-42, wherein the GRP78 inhibitor is provided in a pharmaceutical composition.

47. The method of any one of claims 36-46, wherein the SPARC polypeptide comprises the full length protein.

48. The method of any one of claims 36-46, wherein the SPARC polypeptide comprises a sensitizing fragment or variant.

49. The method of any one of claims 36-42, and 45-48, wherein the SPARC polypeptide and the GRP78 inhibitor form a pharmaceutical composition.

50. The method of any one of claims 36-42, and 45-49, wherein the SPARC polypeptide, the GRP78 inhibitor, and the chemotherapeutic agent form a pharmaceutical composition.

51. The method of any one of claims 36-50, wherein the GRP78 inhibitor is selected from one or more of the following:

(a) a GRP78 inhibitory nucleic acid molecule;

(b) an antibody or antibody fragment thereof that specifically binds to a GRP78 polypeptide;

(c) an immunogenic composition or vaccine which comprises a GRP78 polypeptide;

(d) a vector encoding an inhibitory polypeptide or an inhibitory nucleic acid molecule;

(e) a polypeptide; and

(f) a small molecule.

52. The method of claims 51, wherein the GRP78 inhibitory nucleic acid molecule is selected from an antisense oligonucleotide, a ribozymes, and an RNA interference (RNAi) molecule.

53. The method of claim 52, wherein the RNAi is selected from one or more of microRNA (miRNA); small interfering (siRNA); short-hairpin RNA (shRNA); primary-microRNA (pri-miRNA); asymmetric interfering RNA (aiRNA); small internally segmented RNS (sisiRNA); RNA-DNA chimeric duplex; trans-kingdom RNA (tkRNA); tRNA-shRNA; tandem siRNA (tsiRNA); tandem hairpin RNA (thRNA); pri-miRNA mimic cluster; and transcriptional gene silencing (TGS).

54. The method of claim 52, wherein the small molecule is selected from versipelostatin (VST), a glycosylated derivative of versipelostatin, a versipelostatin analogue,

geldanamycin, and epigallocatechin Gallate (EGCG).

55. The method of any one of claims 36-54, wherein the cancer cell is a colorectal cancer cell.

56. The method of claim 55, wherein the colorectal cancer cell is chemotherapy resistant.

57. The method of any one of claims 36-56, wherein the animal is a human.