Title: Delivery of CD44 shRNA/Nanoparticles within Cancer Cells
Abstract: Our studies have shown that constitutive interactions between hyaluronan and CD44 on tumor cells induces various anti-apoptotic cell survival pathways through the formation of a multimeric signaling complex that contains activated receptor tyrosine kinases. Inhibition of the hyaluronan-CD44 interactions on tumor cells by hyaluronan-CD44 interaction antagonists suppresses these activities by disassembling the complex. Although the anti-tumor activity of hyaluronan-oligosaccharides, a hyaluronan-CD44 interaction antagonist, is effective in sensitizing tumor cells to chemotherapeutic agents and reducing tumor growth in xenografts, hyaluronan-oligosaccharide alone was not effective in reducing tumor progression in Apc Min/+ mice. We now show in vitro and in vivo that targeted inhibition of the expression of CD44v6 depletes the ability of the colon tumor cells to signal through hyaluronan-CD44v6 interactions. First, we cloned oligonucleotides coding CD44v6 shRNA into a conditionally silenced pSico vector. Second, using pSico-CD44v6 shRNA and a colon-specific Fabpl promoter-driven Cre recombinase expression vector packaged into transferrin-coated nanoparticles, we successfully delivered the CD44v6 shRNA within pre-neoplastic and neoplastic colon malignant cells. Third, using the Apc Min/+ mice model, we demonstrated that inhibition of the CD44v6 expression reduces the signaling through a hyaluronan/CD44v6-pErbB2-Cox-2 interaction pathway and reduced adenoma number and growth. Together, these data provide insight into the novel therapeutic strategies of short hairpin RNA/nanoparticle technology and its potential for silencing genes associated with colon tumor cells. Our studies have shown that constitutive interactions between hyaluronan and CD44 on tumor cells induces various anti-apoptotic cell survival pathways through the formation of a multimeric signaling complex that contains activated receptor tyrosine kinases. Inhibition of the hyaluronan-CD44 interactions on tumor cells by hyaluronan-CD44 interaction antagonists suppresses these activities by disassembling the complex. Although the anti-tumor activity of hyaluronan-oligosaccharides, a hyaluronan-CD44 interaction antagonist, is effective in sensitizing tumor cells to chemotherapeutic agents and reducing tumor growth in xenografts, hyaluronan-oligosaccharide alone was not effective in reducing tumor progression in Apc Min/+ mice. We now show in vitro and in vivo that targeted inhibition of the expression of CD44v6 depletes the ability of the colon tumor cells to signal through hyaluronan-CD44v6 interactions. First, we cloned oligonucleotides coding CD44v6 shRNA into a conditionally silenced pSico vector. Second, using pSico-CD44v6 shRNA and a colon-specific Fabpl promoter-driven Cre recombinase expression vector packaged into transferrin-coated nanoparticles, we successfully delivered the CD44v6 shRNA within pre-neoplastic and neoplastic colon malignant cells. Third, using the Apc Min/+ mice model, we demonstrated that inhibition of the CD44v6 expression reduces the signaling through a hyaluronan/CD44v6-pErbB2-Cox-2 interaction pathway and reduced adenoma number and growth. Together, these data provide insight into the novel therapeutic strategies of short hairpin RNA/nanoparticle technology and its potential for silencing genes associated with colon tumor cells. Extracellular matrix has a significant role in solid tumor growth (1Sprenger C.C. Plymate S.R. Reed M.J. Br. J. Cancer. 2008; 98: 250-255Crossref PubMed Scopus (18) Google Scholar). Hyaluronan (HA) 3The abbreviations used are: HA, hyaluronan; Cox-2, cyclooxygenase-2; shRNA, short hairpin RNA; siRNA, silencing RNA; Tf, transferring; Tf-R, Tf receptor; PEG, polyethylene glycol; PEI, polyethyleneimine; Min, multiple intestinal neoplasia; CD44v, CD44 variant isoforms; EGFP, enhanced green fluorescent protein; RT, reverse transcription; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; FAP, familial adenomatous polyposis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Apc, adenomatous polyposis coli. is one of the constituents of extracellular matrix. HA is a high molecular weight glycosaminoglycan present in almost every tissue of vertebrates. It is concentrated in regions of high cell division and invasion. Like numerous extracellular matrix constituents, HA serves both structural and instructive roles in terms of cell signaling via HA receptors, mainly CD44, on the surface of most cells (2Markwald R.R. Fitzharris T.P. Bank H. Bernanke D.H. Dev. Biol. 1978; 62: 292-316Crossref PubMed Scopus (111) Google Scholar, 3Lee J.Y. Spicer A.P. Curr. Opin. Cell Biol. 2000; 12: 581-586Crossref PubMed Scopus (450) Google Scholar, 4Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1678) Google Scholar). However, when cells proliferate or migrate, e.g. in embryonic processes, tissue remodeling, inflammation, and diseases such as cancer and atherosclerosis, HA-induced signaling is activated (4Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1678) Google Scholar, 5Hascall V.C. Majors A.K. De La Motte C.A. Evanko S.P. Wang A. Drazba J.A. Strong S.A. Wight T.N. Biochim. Biophys. Acta. 2004; 1673: 3-12Crossref PubMed Scopus (228) Google Scholar, 6Adamia S. Maxwell C.A. 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The CD44 structure of normal cells is distinct from that of cancer cells, because under various physiological and pathological conditions, the local environmental pressure influences alternate splicing and post-translational modification to produce diversified CD44 molecules (8Naor D. Nedvetzki S. Golan I. Melnik L. Faitelson Y. Crit. Rev. Clin. Lab. Sci. 2002; 39: 527-579Crossref PubMed Scopus (456) Google Scholar, 12van Weering D.H. Baas P.D. Bos J.L. PCR Methods Appl. 1993; 3: 100-106Crossref PubMed Scopus (85) Google Scholar). This diversification allows the production of specific targeting agents that will be useful for both diagnosis and therapy. Overexpression of the variant high molecular weight isoforms CD44v4-v7 and CD44v6-v9 in human lymphomas, colorectal adenocarcinomas, endometrial cancer, papillary thyroid carcinoma, lung and breast cancer, and metastasizing rat adenocarcinomas (13Ponta H. Sherman L. Herrlich P.A. Nat. Rev. Mol. 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A. 1997; 94: 3336-3340Crossref PubMed Scopus (1328) Google Scholar) are overexpressed in carcinogen-induced tumors, and our recent study demonstrates that HA-CD44 interactions constitutively regulate COX-2-induced cell survival in normal epithelial cells and colon carcinoma cells (30Misra S. Hascall V.C. Berger F.G. Markwald R.R. Ghatak S. Connect. Tissue Res. 2008; 49: 219-224Crossref PubMed Scopus (56) Google Scholar, 31Misra S. Obeid L.M. Hannun Y.A. Minamisawa S. Berger F.G. Markwald R.R. Toole B.P. Ghatak S. J. Biol. Chem. 2008; 283: 14335-14344Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Our previous studies demonstrated that antagonists of HA-CD44 interactions, i.e. HA oligomers and overexpression of the ectodomain of CD44 (soluble CD44) (32Yu Q. Toole B.P. J. Biol. Chem. 1996; 271: 20603-20607Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) that acts as a competitive decoy by binding to endogenous HA, inhibit cell survival pathway activities, including activation of several receptor tyrosine kinases, namely ERBB2, EGFR, IGF1Rβ, c-MET, and PDGFRβ, in several types of malignant colon, breast, and prostate carcinoma cells (33Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 34Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In our recent study we demonstrated that elevated HA in normal intestinal epithelial cells (HIEC6-HAS2) regulates several properties required for the transformed phenotype. Increased HA in these cells regulates expression and enzymatic activity of COX-2, activation of ErbB2 and AKT, and translocates β-catenin to the nucleus (30Misra S. Hascall V.C. Berger F.G. Markwald R.R. Ghatak S. Connect. Tissue Res. 2008; 49: 219-224Crossref PubMed Scopus (56) Google Scholar, 31Misra S. Obeid L.M. Hannun Y.A. Minamisawa S. Berger F.G. Markwald R.R. Toole B.P. Ghatak S. J. Biol. Chem. 2008; 283: 14335-14344Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). To explore the mechanism of constitutive HA-CD44 interaction and the consequent outcomes in cancer cells, we have shown that all four types of reagents, namely HA-oligosaccharides, anti-CD44 antibody, soluble CD44, or CD44 siRNA, block signaling responses in a variety of tumor cell types and also block activation of receptor tyrosine kinases (30Misra S. Hascall V.C. Berger F.G. Markwald R.R. Ghatak S. Connect. Tissue Res. 2008; 49: 219-224Crossref PubMed Scopus (56) Google Scholar, 31Misra S. Obeid L.M. Hannun Y.A. Minamisawa S. Berger F.G. Markwald R.R. Toole B.P. Ghatak S. J. Biol. Chem. 2008; 283: 14335-14344Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 33Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 34Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Although the anti-tumor activity of HA-oligosaccharides is effective in sensitizing tumor cells to chemotherapeutic agents in cell culture models (34Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 35Misra S. Ghatak S. Toole B.P. J. Biol. Chem. 2005; 280: 20310-20315Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 36Misra S. Ghatak S. Zoltan-Jones A. Toole B.P. J. Biol. Chem. 2003; 278: 25285-25288Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and in reducing tumor growth in a subcutaneous in vivo xenograft model (37Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2002; 277: 38013-38020Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), HA-oligosaccharides alone are not effective in reducing tumor progression in an Apc Min/+ mice in vivo model. 4S. Misra, unpublished data. Thus, an alternative HA/CD44 antagonist is required to treat distant tumors. Because CD44 is present in the HA-induced signaling complex, we also used RNA interference to test the role of CD44 in these cancer cells (33Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 34Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). All cancer cell lines (HCT 116 and HCA7 colon cancer cells, C4-2 and LNCaP prostate cancer cells, and MCF7/Adr breast cancer cells and TA3St mouse mammary carcinoma cells) transfected with a 21-nucleotide small interfering RNA (siRNA) targeted to CD44 greatly decreased activation of cell survival proteins and multiple receptor tyrosine kinases as well as CD44 and COX-2 expression when compared with the above cells transfected with scrambled control siRNA (30Misra S. Hascall V.C. Berger F.G. Markwald R.R. Ghatak S. Connect. Tissue Res. 2008; 49: 219-224Crossref PubMed Scopus (56) Google Scholar, 31Misra S. Obeid L.M. Hannun Y.A. Minamisawa S. Berger F.G. Markwald R.R. Toole B.P. Ghatak S. J. Biol. Chem. 2008; 283: 14335-14344Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 34Misra S. Toole B.P. Ghatak S. J. Biol. Chem. 2006; 281: 34936-34941Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In mammalian cell culture, the transfection of 21-nucleotide double-stranded siRNA efficiently inhibits endogenous gene expression in a sequence-specific manner without induction of the interferon response (38Heidel J.D. Hu S. Liu X.F. Triche T.J. Davis M.E. Nat. Biotechnol. 2004; 22: 1579-1582Crossref PubMed Scopus (167) Google Scholar). However, the phenotypic changes induced by siRNAs only persist 1 week because of lack of transfer of siRNA or dilution of siRNA concentration after each cell division, which limits their utility for use in inhibiting tumor progression. The technique of using shRNA in an expression vector is an alternative strategy to stably suppress gene expression, and such constructs with well defined initiation and termination sites have been used to produce various small RNA species that inhibit the expression of genes with diverse functions in mammalian cell lines (39Paul C.P. Good P.D. Winer I. Engelke D.R. Nat. Biotechnol. 2002; 20: 505-508Crossref PubMed Scopus (753) Google Scholar). The conditional alteration of gene expression by the use of an shRNA expression vector holds potential promise for therapeutic approaches for silencing disease-causing genes provided that appropriate extracellular and intracellular nucleic acid delivery systems (vector systems) are available that offer efficient vehicles for stable complexation and protection of the nucleic acid. To reach the targeted tissue, vectors need to overcome a number of extracellular and intracellular barriers. Systemic targeting by viral vectors toward the desired tissue is difficult because the host immune responses activate viral clearance. Systemic administration of a large amount of adenovirus (e.g. into the liver) can be a serious health hazard that even caused the death of one patient (40Raper S.E. Chirmule N. Lee F.S. Wivel N.A. Bagg A. Gao G.P. Wilson J.M. Batshaw M.L. Mol. Genet. Metab. 2003; 80: 148-158Crossref PubMed Scopus (1179) Google Scholar). Nonviral vectors, such as positively charged PEI complexes, mediate unspecific interactions with non-target cells and blood components, which results in the rapid clearance from the circulation. These unfavorable effects can be minimized by “shielding” of the positive surface charge of the vectors with polyethylene glycol (PEG). PEGylation of PEI polyplexes can prevent the systemic degradation of the plasmid DNA and reduce the toxicity of polyplexes (41Kursa M. Walker G.F. Roessler V. Ogris M. Roedl W. Kircheis R. Wagner E. Bioconjugate Chem. 2003; 14: 222-231Crossref PubMed Scopus (295) Google Scholar). To increase the transfection efficiency of the shielded particles (plasmid DNA/PEG-PEI), different targeting ligands, such as peptide, growth factors and proteins, or antibodies, have been incorporated into the vectors (42Bellocq N.C. Pun S.H. Jensen G.S. Davis M.E. Bioconjugate Chem. 2003; 14: 1122-1132Crossref PubMed Scopus (327) Google Scholar). One such targeting ligands is transferrin (Tf), an iron-transporting protein that is recognized by Tf receptors (Tf-R) present at high levels in the tumor cells (42Bellocq N.C. Pun S.H. Jensen G.S. Davis M.E. Bioconjugate Chem. 2003; 14: 1122-1132Crossref PubMed Scopus (327) Google Scholar, 43Qian Z.M. Li H. Sun H. Ho K. Pharmacol. Rev. 2002; 54: 561-587Crossref PubMed Scopus (936) Google Scholar). In contrast, in nonproliferating cells, expression of Tf-R is low or undetectable. Association of Tf to polyplexes significantly enhances transfection efficiency by promoting the internalization of polyplexes (plasmid DNA/Tf-PEG-PEI (designated as nanoparticles throughout this study)) in dividing and nondividing cells (42Bellocq N.C. Pun S.H. Jensen G.S. Davis M.E. Bioconjugate Chem. 2003; 14: 1122-1132Crossref PubMed Scopus (327) Google Scholar). After cellular association of nanoparticles to the target cells, particles are internalized by receptor-mediated endocytosis (42Bellocq N.C. Pun S.H. Jensen G.S. Davis M.E. Bioconjugate Chem. 2003; 14: 1122-1132Crossref PubMed Scopus (327) Google Scholar). Fig. 1 illustrates that the uptake of nanoparticles carrying multiple functional domains (surface shielding particles Tf-PEG-PEI, tissue-specific promoter-driven Cre recombinase, and conditionally silenced plasmid) can overcome the intracellular barriers for successful delivery of the shRNA gene. We tested the effects of CD44v6 shRNA in vivo in a mouse model of human familial adenomatous polyposis (FAP) where adenomatous polyposis coli gene (Apc) is mutated. Most FAP patients carry truncation mutations in the N-terminal half (44Miyoshi Y. Ando H. Nagase H. Nishisho I. Horii A. Miki Y. Mori T. Utsunomiya J. Baba S. Petersen G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4452-4456Crossref PubMed Scopus (518) Google Scholar). Several mouse models of FAP were constructed either by chemical mutagenesis of the Apc gene at codon 850 (Apc Min/+ mouse) or by homologous recombination in embryonic stem cells (knock-out strains) such as Apc 1638N, Apc 1638T, Apc 1309, Apc Δ716, and Apc Δ474 (45Fodde R. Smits R. Trends Mol. Med. 2001; 7: 369-373Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Germ line mutagenesis of C57BL/6J (B6) males was done by N-ethyl N-nitrosourea, and the offspring of mutated B6 × AKR/J (AKR) females yielded mice that carry 100% of an autosomal dominant mutation at codon 850 in the Apc gene. These five strains have mutated Apc alleles that encode truncated Apc proteins of 850, 1638, 1309, 716, and 474 amino acids, respectively. These variable truncated products form dimers with wild-type Apc protein and show variable dominant negative activities. As a result, the number of polyps varies in different strains. For example, the number of polyps at 16 weeks of age are ∼100 in Apc Min/+, Apc Δ474, and Apc 1309 mice. A much greater number of polyps (300-400) are found in Apc Δ716 mice, whereas much fewer polyps (∼10) are found in Apc 638N mice (46Sasai H. Masaki M. Wakitani K. Carcinogenesis. 2000; 21: 953-958Crossref PubMed Google Scholar). These mutations in the Apc allele and the phenotype variability observed in FAP patients allowed the establishment of genotype-phenotype correlations at the Apc locus resulting in multiple intestinal adenomas throughout the length of the small and large intestine (47Moser A.R. Pitot H.C. Dove W.F. Science. 1990; 247: 322-324Crossref PubMed Scopus (1312) Google Scholar). Interestingly, most polyps are found in the small intestine, although a small but significant number of polyps develop in the colon, a phenotype different from human FAP. Despite this caveat, the Apc Min/+ mouse offers the prospect to study intestinal tumors that are of the same genetic background as the host. Strong up-regulations of CD44, including both CD44s and CD44v6 encoded epitopes, and of Cox-2 were observed in aberrant crypt foci in Apc Min/+ mice (48Wielenga V.J. van der Neut R. Offerhaus G.J. Pals S.T. Adv. Cancer Res. 2000; 77: 169-187Crossref PubMed Google Scholar, 49Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2284) Google Scholar). Furthermore, CD44v proteins can form multimeric complexes in the plasma membrane, which dramatically enhances their HA binding capacity (11Sleeman J. Rudy W. Hofmann M. Moll J. Herrlich P. Ponta H. J. Cell Biol. 1996; 135: 1139-1150Crossref PubMed Scopus (121) Google Scholar). Moreover, our results with Apc Min/+ mice confirm earlier findings that Tf-R is present at high levels in the tumor cells (42Bellocq N.C. Pun S.H. Jensen G.S. Davis M.E. Bioconjugate Chem. 2003; 14: 1122-1132Crossref PubMed Scopus (327) Google Scholar), which is crucial for active targeting of Tf-R by Tf-mediated CD44v6 shRNA delivery in tumor cells. For these reasons, the Apc Min/+ mouse model can be used for modulating adenoma growth by using HA-CD44v6 interaction antagonists as therapeutic agents in vivo. In this study we tested whether HA regulates Cox-2 via its effects on a CD44v6 → ErbB2 → Cox-2 axis in colon cancer cells. More importantly we provide evidence that systemic application of (pSico-CD44v6 shRNA plus pFabpl-Cre)/nanoparticles in the Apc Min/+ mouse model reduces intestinal tumor growth by perturbing CD44v6 containing isoform expression. Apc 10.1, an intestinal cell line, was derived from Apc Min/+ mice and retains the host heterozygous Apc genotype (50De Giovanni C. Landuzzi L. Nicoletti G. Astolfi A. Croci S. Micaroni M. Nanni P. Lollini P.L. Int. J. Cancer. 2004; 109: 200-206Crossref PubMed Scopus (17) Google Scholar). HCA7 clone 29 colon carcinoma cells were obtained from ECCA (United Kingdom). HT-29 cells were obtained from ATCC (Manassas, VA). Apc Min/+ mice (47Moser A.R. Pitot H.C. Dove W.F. Science. 1990; 247: 322-324Crossref PubMed Scopus (1312) Google Scholar) were obtained from the Colon Cancer Center, COBRE, at the University of South Carolina, Columbia, and The Jackson Laboratories (Bar Harbor, ME). The gene-switch reporter plasmid pSV(EGFP)/β-galactosidase (51Kaczmarczyk S.J. Green J.E. Nucleic Acids Res. 2001; 29: E56Crossref PubMed Scopus (52) Google Scholar) was obtained from Addgene Inc. (Cambridge, MA). The promoter of the liver form of the fatty acid-binding protein (Fabpl)-Cre plasmid (52Saam J.R. Gordon J.I. J. Biol. Chem. 1999; 274: 38071-38082Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) was a gift from Dr. J. I. Gordon (Washington University School of Medicine, St. Louis, MO). The COX-2-luc construct was a gift from Dr. R. DuBois (Vanderbilt-Ingram Cancer Center, Nashville, TN). The conditional silencing vector pSico and the conditional inactivating vector pSicoR (53Ventura A. Meissner A. Dillon C.P. McManus M. Sharp P.A. Van Parijs L. Jaenisch R. Jacks T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10380-10385Crossref PubMed Scopus (513) Google Scholar) were gifts from Dr. T. Jacks (Massachusetts Institute of Technology, Cambridge, MA). X-Gal was purchased from Invitrogen. The pSV-β-galactosidase plasmid (designated as pSV-β-gal throughout the paper) was purchased from Promega. Anti-COX-2 antibody, anti-human Tf-R antibody (cross-reacts with mouse Tf-R), and anti-human CD44 antibody were purchased from Santa Cruz Biotechnology; anti-phospho-ErbB2/HER2 (Y1248) antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY); and monoclonal anti-β-actin clone AC-15 antibody was purchased from Sigma. The restriction enzymes HpaI, XhoI, SacII, NotI, XhoI, and XbaI were purchased from New England Biolabs (Beverly, MA). Chemically competent DH5α cells were purchased from Invitrogen. Sephacryl S200 was purchased from Amersham Biosciences. ECL reagents were from Santa Cruz Biotechnology. The mouse HAS2 cDNA construct pCI-neo-HAS2 was obtained from Dr. A. Spicer (University of California, Davis, CA). pCI-neo and pSV-β-galactosidase plasmids were from Promega. Transferrin (molecular mass of 77 kDa) was purchased from Sigma; branched chain PEI (average molecular mass of 25 kDa) was from Sigma; and N-hydroxysuccinimide/PEG/maleimide (molecular mass of 3.40 kDa) was from Pierce. All other reagents were of analytical reagent grade. The siRNA transfections were done at 100 pmol using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Cells were transfected with the CD44v6 siRNA (scrambled (scr) siRNA as control) or plasmids (corresponding vector plasmid as control) in 6-well plates with cells at 70-90% confluence. The cells were then incubated at 37 °C in 5% CO2 for 24 h, replated in 150-mm dishes, and allowed to grow for 48 h in complete medium. We prepared a double-stranded cassette for CD44v6 shRNA following instructions from the Dr. Tyler Jacks laboratory (Massachusetts Institute of Technology). Using the CD44v6 siRNA sequence as described (54Cheng C. Yaffe M.B. Sharp P.A. Genes Dev. 2006; 20: 1715-1720Crossref PubMed Scopus (116) Google Scholar), sense and antisense oligonucleotides for the double-stranded cassette were designed. These oligonucleotides were synthesized and purified by Integrated DNA Technologies (Coralville, IA). The pSico and pSicoR vectors (Fig. 2) were linearized by digesting with HpaI and XhoI restriction enzymes, and the purified linear vectors gave no colonies when transfected with competent DH5α. These linearized vectors were ligated to the double-stranded oligonucleotide cassette. Transformation of DH5α was done with the products of ligation following the manufacturer's instructions. The plasmids were prepared from cultures grown from ampicillin-resistant colonies using the Qiagen kit. The purified plasmids were checked for the insert by digesting with SacII-NotI (for pSico-CD44v6 shRNA) and with XhoI-XbaI (for pSicoR-CD44v6 shRNA). Parallel control digestions were