ABSTRACT
BACKGROUND: Loss of the adenomatous polyposis coli (APC) tumor suppressor gene plays a significant role in colorectal carcinogenesis. One function of the APC gene product is to regulate beta-catenin, a protein that plays a role in cell adhesion and also regulates the activity of certain transcription factors. To more precisely delineate the role of beta-catenin signaling in colon cancer growth, we treated mice bearing APC-mutant SW480 colon cancer xenografts with antisense oligonucleotides (ODNs) directed against beta-catenin mRNA and examined effects on beta-catenin expression and tumor growth. METHODS: Balb/C nude mice underwent subcutaneous injection of 1 x 10(6) SW480 cells to establish tumor xenografts. In one experiment, tumors were allowed to grow for 7 days, after which time animals were randomized to undergo daily intraperitoneal injections of either antisense beta-catenin ODN at doses of 5, 10, or 20 mg/kg, scrambled sequence beta-catenin ODN control, or saline control for 7 days. Tumors were excised and homogenized, and tumor lysates subjected to gel electrophoresis and Western blotting for beta-catenin protein quantification. In a second experiment, tumor-bearing animals began receiving daily intraperitoneal injections of either antisense beta-catenin ODN at doses of 5, 10, or 20 mg/kg, scrambled sequence beta-catenin ODN control, or saline control. Tumor growth was quantitated by measuring tumor volumes twice weekly. A third experiment evaluated the antitumor effects of daily bolus dosing versus continuous infusion of beta-catenin antisense ODNs (20 mg/kg). RESULTS: Treatment of APC-mutant colorectal carcinoma xenografts with beta-catenin antisense resulted in a dose-dependent down-regulation in beta-catenin protein expression as shown by Western blotting. Treatment of tumor-bearing mice with antisense directed at beta-catenin also demonstrated a dose-dependent inhibition of tumor growth. There appears to be little difference in the antitumor effects of antisense ODNs administered by continuous infusion or bolus dosing schedules. CONCLUSIONS: beta-Catenin expression plays a critical role in the tumorigenic growth of APC-mutant colon cancer xenografts. Strategies targeting beta-catenin, including the use of antisense ODNs, may be of use in the treatment of human colon cancer.
Subject(s)
Carcinoma/metabolism , Carcinoma/pathology , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cytoskeletal Proteins/antagonists & inhibitors , Cytoskeletal Proteins/genetics , Oligonucleotides, Antisense/pharmacology , Trans-Activators , Animals , Carcinoma/genetics , Colonic Neoplasms/genetics , Dose-Response Relationship, Drug , Down-Regulation , Female , Genes, APC , Humans , Mice , Mice, Inbred BALB C , Mutation , Neoplasm Transplantation , Transplantation, Heterologous , Tumor Cells, Cultured/transplantation , beta CateninABSTRACT
Mutations involving the adenomatous polyposis coli (APC) tumor suppressorgene/beta-catenin signaling pathway have been identified in the majority of colon carcinomas. However, the role of aberrant beta-catenin signaling in the neoplastic growth of APC-mutant colon cancer cells has not been directly studied. To address this question, antisense oligonucleotides have been used to specifically down-regulate beta-catenin expression in APC-mutant human colon carcinoma cells. Antisense-mediated suppression of beta-catenin inhibits the in vitro proliferation, anchorage-independent growth, and cellular invasiveness of APC-mutant human colon carcinoma cells. The systemic administration of beta-catenin antisense oligonucleotides down-regulates beta-catenin expression in vivo in human colon cancer xenografts in nude mice. Such treatment inhibits the tumorigenic growth of colon cancer xenografts and can completely eradicate tumors in some treated animals. These studies formally demonstrate the critical role of beta-catenin signaling in the neoplastic growth of APC-mutant colon cancer cells and suggest that strategies targeting beta-catenin may be of use in the therapy of colon cancer.
Subject(s)
Colonic Neoplasms/pathology , Cytoskeletal Proteins/antagonists & inhibitors , Genes, APC/genetics , Trans-Activators , Animals , Cell Adhesion/physiology , Cell Division/physiology , Colonic Neoplasms/genetics , Cytoskeletal Proteins/biosynthesis , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/physiology , Down-Regulation/drug effects , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Mutation , Neoplasm Transplantation , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Signal Transduction/physiology , Thionucleotides/genetics , Thionucleotides/pharmacology , Transplantation, Heterologous , Tumor Cells, Cultured , beta CateninABSTRACT
Overexpression of the HER2/neu oncogene is observed in approximately 30% of human breast carcinoma specimens. HER2/neu overexpression is a negative prognostic factor in breast cancer patients. Cancer cells that overexpress HER2/neu may also be less sensitive to chemotherapy. In order to further define mechanisms by which HER2/neu overexpression drives neoplastic cell growth and chemoresistance, antisense oligonucleotides (ODNs) have been utilized to selectively down-regulate HER2/neu expression in human breast cancer cells. Such antisense ODNs suppress HER2/neu mRNA and protein levels in a dose-dependent, sequence-specific manner. Down-regulation of HER2/neu expression in HER2/neu overexpressing breast cancer cells inhibits cell cycle progression in G0/G1 and results in apoptotic cell death. In tissue culture studies, combined treatment of HER2/ neu overexpressing breast cancer cells with HER2/neu antisense ODNs and conventional chemotherapeutic agents results in synergistic inhibition of cancer cell growth and activation of apoptotic cell death mechanisms. These studies have been extended to demonstrate synergistic antitumor effects following systemic treatment with antisense ODNs plus doxorubicin in nude mice bearing human breast carcinoma xenografts. Collectively these findings demonstrate that HER2/neu overexpression stimulates anti-apoptotic cell survival mechanisms and suggest that HER2/neu antisense ODNs may be of use in cancer therapeutics.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/therapy , Gene Expression Regulation, Neoplastic/drug effects , Oligonucleotides, Antisense/pharmacology , Receptor, ErbB-2/genetics , Animals , Breast Neoplasms/genetics , Cell Division , Humans , Tumor Cells, CulturedABSTRACT
BACKGROUND: The HER2/neu oncogene is overexpressed in a substantial fraction of human tumors. HER2/neu overexpressing tumors may be intrinsically resistant to chemotherapy. The present study examined the ability of antisense-mediated downregulation of HER2/neu expression to enhance the antitumor effects of conventional chemotherapeutic agents against human tumor cells that overexpress HER2/neu. METHODS: The effects of HER2/neu antisense oligodeoxynucleotides (ODNs) on the growth inhibitory and proapoptotic activity of several distinct chemotherapeutic agents were examined in vitro. In vivo effects of HER2/neu antisense ODNs in combination with doxorubicin hydrochloride were assessed by examining the growth of human tumor xenografts implanted into nude mice. RESULTS: The proliferation of tumor cell lines that overexpress HER2/neu was inhibited by antisense ODNs in combination with conventional chemotherapeutic agents in an additive or synergistic fashion. Such combination therapy also demonstrated synergistic activation of apoptosis. HER2/neu antisense ODNs in combination with doxorubicin hydrochloride demonstrated synergistic antitumor effects in vivo as well. CONCLUSIONS: Downregulation of HER2/neu expression can enhance the sensitivity of human cancer cells, which overexpress HER2/neu to the cytotoxic effects of chemotherapy. Antisense ODNs targeting the HER2/neu gene may play a role in cancer therapy.
