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Claudin-3 Gene Silencing with Sirna Suppresses Ovarian Tumor Growth and Metastasis

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Claudin-3 Gene Silencing with Sirna Suppresses Ovarian Tumor Growth and Metastasis Claudin-3 gene silencing with siRNA suppresses ovarian tumor growth and metastasis Yu-Hung Huanga, Yunhua Baoa, Weidan Penga, Michael Goldbergb, Kevin Loveb, David A. Bumcrotc, Geoffrey Colec, Robert Langerb,d,1, Daniel G. Andersonb,1, and Janet A. Sawickia,e,f,1 aLankenau Institute for Medical Research, 100 E Lancaster Avenue, Wynnewood, PA 19096-3450; bDavid H. Koch Institute for Integrative Cancer Research and dChemical Engineering Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Rm E25-342, Cambridge, MA 02139-4307; cAlnylam Pharmaceuticals, Inc., 300 Third Street, Cambridge, MA 02142; and eDepartment of Dermatology and Cutaneous Biology and fKimmel Cancer Center, Jefferson Medical School, Thomas Jefferson University, 233 S 10th Street, Philadelphia, PA 19107 Contributed by Robert Langer, December 30, 2008 (sent for review September 24, 2008) Claudin-3 (CLDN3) is a tight junction protein that is overexpressed and CLDN4 results in cell death, and significantly inhibits in 90% of ovarian tumors. Previous in vitro studies have indicated ovarian tumor growth in a mouse model (6). Given their high that CLDN3 overexpression promotes the migration, invasion, and expression in ovarian tumor cells and the possibility that their survival of ovarian cancer cells. Here, we investigated the efficacy overexpression may disrupt tight junction barrier function and of lipidoid-formulated CLDN3 siRNA in 3 different ovarian cancer contribute to tumorigenesis, targeting CLDN3 and CLDN4 models. Intratumoral injection of lipidoid/CLDN3 siRNA into using siRNA is an attractive option as a potential therapy for OVCAR-3 xenografts resulted in dramatic silencing of CLDN3, ovarian cancer. Indeed, in vitro siRNA inhibition of CLDN3 and significant reduction in cell proliferation, reduction in tumor CLDN4 expression reduced the invasive properties of ovarian growth, and a significant increase in the number of apoptotic cells. tumor cells (4). Intraperitoneal injection of lipidoid-formulated CLDN3 siRNA re- Here, we have developed a lipid-like delivery system for i.p. sulted in a substantial reduction in tumor burden in MISIIR/TAg delivery of siRNA to ovarian tumor tissue to test the therapeutic transgenic mice and mice bearing tumors derived from mouse efficacy of CLDN3 siRNA in mouse tumor models. We have ovarian surface epithelial cells. Ascites development was reduced tested this approach in 3 mouse models for ovarian cancer, in CLDN3 siRNA-treated mice, suggesting the treatment effectively ovarian xenografts in nude mice, MISIIR/TAg transgenic mice suppressed metastasis. Toxicity was not observed after multiple (7), and nude mice injected i.p. with mouse ovarian surface MEDICAL SCIENCES i.p. injections. Importantly, treatment of mice with nonimmunos- epithelial cells (MOSEC) that express firefly luciferase (ID8- timulatory 2؅-OMe modified CLDN3 siRNA was as effective in Fluc cells) (8). We have used a novel lipid-like molecule, 98N12-5 suppressing tumor growth as unmodifed siRNA. These results (1), to deliver the siRNA intratumorally and i.p. to mice. This suggest that lipidoid-formulated CLDN3 siRNA has potential as a molecule belongs to a new class of lipidoid molecules that has therapeutic for ovarian cancer. recently been shown to deliver siRNA safely and effectively to lung, liver, and peritoneal macrophages in 3 different species, lipidoid ͉ ovarian cancer ͉ cancer therapy including a nonhuman primate (9). In all 3 mouse models, tumor growth in CLDN3 siRNA- varian cancer has the highest mortality rate among gyne- treated mice was significantly reduced, compared with mice Ocologic malignancies, and ranks 4th as the most common treated with control siRNA. In some mice, tumors even re- cancer in women in the United States (1). Treatment of early gressed in size. Ascites development in the ID8-Fluc model was stage ovarian carcinoma improves the survival rate up to 90%. suppressed, suggesting that CLDN3 siRNA treatment was ef- fective at inhibiting metastasis. CLDN3 siRNA-treated mice However, most women have advanced stage metastatic cancer at displayed no obvious ill effects from the treatment. The fact that the time of diagnosis due to the asymptomatic nature of early nonimmunostimulatory modified CLDN3 siRNA suppresses stages of the disease and the lack of effective screening modal- tumor growth and stimulatory unmodified siRNA suggests that ities. The standard treatment for patients with advanced stage the therapeutic effects we observe are the result of suppression epithelial ovarian cancer is surgical debulking followed by che- of CLDN3 protein. These promising results suggest that lipidoid- motherapy with paclitaxel plus a platinum-based therapy (cis- delivery of CLDN3 siRNA warrants further development as a platin or carboplatin). Although Ϸ80% of patients receiving this new therapeutic option for ovarian cancer. therapeutic regimen have an initial favorable response, recurrent disease will occur in a majority of cases. New effective therapies Results are urgently needed for those patients with advanced-stage CLDN3 Expression in Human and Mouse Ovarian Tumor Cells. Western ovarian cancer who either do not respond to initial therapy or blot analysis of membrane proteins prepared from human develop recurrent disease. OVCAR-3 cells, human ovarian ascites cells, ovarian tumors Claudins are integral membrane proteins associated with tight from MISIIR/TAg transgenic mice, and ID8-Fluc cells identified junctions. Two members of the claudin protein family, claudin-3 (CLDN3) and CLDN4, are overexpressed in epithelial ovarian tumors relative to normal ovarian tissue (2–5). In fact, they are Author contributions: D.A.B., D.G.A., and J.A.S. designed research; Y.-H.H., Y.B., W.P., G.C., among the most highly expressed proteins in ovarian tumors. and J.A.S. performed research; M.G., K.L., D.A.B., R.L., and D.G.A. contributed new re- High amounts of CLDN3 and CLDN4 are associated with agents/analytic tools; Y.-H.H., G.C., R.L., D.G.A., and J.A.S. analyzed data; and D.G.A., G.C., increased cellular motility and survival of ovarian tumor cells, and J.A.S. wrote the paper. and an increase in matrix metalloproteinase type 2 (MMP-2) (4). Conflict of interest statement: Janet Sawicki and Robert Langer have sponsored research grants from Alnylam. Robert Langer is also a consultant to Alnylam. David Bumcrot and These observations implicate a role for CLDN3 and CLDN4 in Geoffrey Cole are employed by Alnylam. ovarian tumorigenesis and metastasis, and suggest their impor- 1To whom correspondence may be addressed. E-mail: [email protected], tance as target proteins for development of new diagnostic and [email protected], or [email protected]. therapeutic reagents. CLDN3 and CLDN4 have also been This article contains supporting information online at www.pnas.org/cgi/content/full/ identified as receptors for cytotoxic Clostridium perfringens 0813348106/DCSupplemental. enterotoxin (CPE). Binding of CPE to cells that express CLDN3 © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813348106 PNAS Early Edition ͉ 1of5 Downloaded by guest on September 30, 2021 A B C D 1 2 1 2 1 2 3 4 1 2 3 4 E 1 2 3 4 5 CLDN3 β-actin Fig. 1. Western blot analysis of CLDN3 expression in human and mouse tumor cells and in CLDN3 siRNA-treated human OVCAR-3 cells. (A) Membrane proteins prepared from LLC PK1 cells (lane1, positive control) and from OVCAR-3 cells (lane 2). (A–C) CLDN3 fluoresces green and ␤-actin, the loading control, fluoresces red. (B) Membrane proteins prepared from LLC PK1 cells (lane 1, positive control) and from human ovarian ascites cells (lane 2). (C) LLC PK1 cells (lane 1, positive control); membrane proteins from 3 different ovarian tumors from MISIIR/TAg mice (lanes 2–4). (D) Membrane proteins from OVCAR-3 cells (lane 1, positive control) and ES2 cells (lane 2, negative contol). Cytoplasmic proteins (lane 3) and membrane proteins (lane 4) from ID8-Fluc cells. (E) Small interfering RNA analysis; OVCAR-3 membrane proteins (lane 1, positive control); ES2 membrane proteins (lane 2, negative control); mock transfected OVCAR-3 cells (lane 3); OVCAR-3 cells, control siRNA treated (lane 4); OVCAR-3 cells, CLDN3 siRNA treated (lane 5). CLDN3 (Upper); ␤-actin loading control (Lower). a 22-kDa protein corresponding to CLDN3 (Fig. 1 A–D). Membrane proteins prepared from LLC PK1, a renal epithelial cell line known to express CLDN3, served as a positive control (10). Carryover of ␤-actin in membrane protein preparations allowed for its convenient use as a control for protein loading on gels. CLDN3 expression was not detected in ES2 cells, another human ovarian cancer cell line (Fig. 1D, lane 2). The lack of expression in ES2 cells is consistent with previous reports that showed similar results in HOSE-B and A2780) (3). All of the Fig. 2. CLDN3 siRNA treatment of OVCAR-3 xenografts. (A) Fold increase human and mouse tumor cells that we tested expressed CLDN3 (mean Ϯ SD) in tumor volume at various times after intratumoral injection of in membrane protein preparations. We did not observe CLDN3 hCLDN3 siRNA, Fluc siRNA, or PBS (measured against baseline tumor volume expression in cytoplasmic protein preparations of ID8-Fluc cells at time 0). Orange triangles indicate times of injections. (B) Western blot (Fig. 1C, lanes 3 and 4). Based on these results, we chose to use analysis of CLDN3 protein in OVCAR-3 xenografts injected with hCLDN3 siRNA, Fluc siRNA, or PBS. Lanes 1 and 2, tumors before any treatment; lanes 2–7, OVCAR-3 cells to generate xenografts for evaluating therapeu- individual tumors after 4 intratumoral injections of siRNA or PBS; lane 8, tic efficacy of siRNA knockdown of CLDN3. membrane proteins from ES2 cells (negative control); lane 9, membrane We confirmed by Western blot analysis of membrane proteins proteins from OVCAR-3 cells (positive control). (C) Sections of xenografts prepared from OVCAR-3 cells grown in vitro that the human harvested on day 13 and immunostained for CLDN3 and PCNA, or TUNEL (h)CLDN3 siRNA sequence we selected as a potential thera- stained (with same-section DAPI nuclear stain on the right).
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