Espinoza, Ignacio

Abstract MN Background: Breast cancer is the second leading cause of cancer death of women worldwide. One of the major challenges for a better prognosis of breast cancer patients is the ineffectiveness of chemotherapy and drug resistance. To investigate the mechanisms of drug resistance, resistant cancer cell lines have been established with various anticancer agents. We found that FASN, an important metabolic enzyme overexpressed in aggressive carcinomas, is t responsible in part for the drug resistance phenotype of breast cancer cells. Our findings show that blockage of FASN augments the cytotoxicity of the anti-mitotic drug Taxol in breast cancer cells. Moreover, C75 and Taxol have a synergistic effect both drugs are combined. The in vivo effect of C75 and Taxol and the mechanism by which blockage of FASN sensitizes the cells to chemotherapy are unknown. Material and Methods: In this study first we show the effect of C75 plus Taxol in breast tumor growth. BT474 cells were injected into the mammary fat pad of nude mice to generate the tumors. When the tumors were measurable we treated the mice with C75, Taxol or with a combination of both drugs. Second we studied the lipid rafts disruption after treatments by sucrose gradient and immunofluorescence. Results: We found that C75 plus Taxol extensively inhibited the tumor growth 9 fold and reduced the tumor size in 4 fold comparedn to the control mice (no treatement). In comparison C75 alone reduced the tumor growth in 1.5 fold only; and Taxol 2.5 fold compared to the control mice. These results show for first time that blockage of FASN (with C75) sensitizes breast cancer tumors to Taxol treatment in vivo. In addition, we studied the possible mechanism involved in the sensitization of breast cancer cells to Taxol by FASN blockage. Previous studies reported that blockage of FASN or Taxol treatment decrease the sphingolipids synthesis. Sphingolipids and cholesterol are the most abundant fatty acid in the lipid rafts in the plasma membrane. Disruption of lipid rafts has been shown to induce apoptosis in different carcinoma cell lines. Based in these findings, we studied lipid rafts in BT474 cells treated with C75, Taxol or combination of both with sucrose gradient and immunofluorescence microscopy. Discussion: Our results show that C75 and Taxol disrupt the lipid rafts. Thesefindings show that FASN is a new mechanism of drug resistance and may be an ideal target for chemosensitization in breast cancer chemotherapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-03-01.

Abstract Estrogen receptors (ERs) play key roles in breast cancer development and influence treatment outcome in breast cancer patients. More than 60% of human breast cancer tumors express ER and many of those are Estrogen (E2) dependent. ER positive tumors are considered to have a good prognosis; however some tumors escape to E2 requirements. Thus, the identification of molecules that regulate ER function may facilitate development of breast cancer treatment strategies. The pro-angiogenic factor CYR61 induces breast cancer cells to bypass the normal estrogen requirements for growth and acquire an estrogen-independent and antiestrogen resistant phenotype. However, the mechanism by which CYR61 regulates the hormonal response in breast cancer is not well understood. We over-expressed CYR61 in the ER positive cells MCF-7 (MCF-7/CYR61). Our results demonstrated that CYR61 induces constitutive transcriptional activity of the ER, which is independent of E2. Moreover, we demonstrated that CYR61 co-localizes with the ER in the nucleus and casually CYR61 transcriptional activity is regulated, at least in part by FOXO3a binding site. In search of the mechanism by which CYR61 that modulates hormonal response we discovered that CYR61 and FOXO3a interact in the presence of E2. This functional interplay among CYR61/ER/FOXO3a most likely plays a critical role in blocking hormone-response of breast cancer growth in vitro and in vivo. Furthermore, these studies demonstrate that CYR61 and FOXO3a are novel therapeutic targets to halt hormone-independent breast carcinomas. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5145.

Abstract The growth factor Heregulin (HRG) is expressed in about 30% of breast cancer tumors, and induces tumorigenicity and metastasis of breast cancer cells. We have demonstrated previously that HRG overexpression renders breast cancer cells resistance to the microtubule-interfering agent Taxol, a drug of choice for the treatment of metastatic breast cancer. The mechanism by which HRG induces Taxol resistance is largely unknown. It is also known that triple negative breast cancer tumors do express high levels of HRG and unfortunately they do not respond to HRG. Our studies were aimed at targeting HRG with the goal of achieving a therapeutic target as well as restoring the response to Taxol, while preventing the formation of metastasis. Thus, we knocked-down HRG expression in three different breast cancer cell lines: MDA-MB-23, HS578T and BT549. Our data demonstrates that HRG expression is an absolute requirement for the anchorage-independent growth for triple negative breast cancer cells, since none of the breast cancer cells MDA-MB-231, HS578T and BT549 stable expressing the silencing (shRNA) for HRG, were capable of forming colony in soft agar. Furthermore, these cells, not only no longer were not anchorage-independent were no longer resistant to Taxol, to the contrary the shRNA/HRG cells were exquisitely sensitive to Taxol, to induce growth inhibition and apoptosis. More importantly, we observed that the disorganized structured observed in 3D matrigel culture observed for triple negative cells, was completely abolished once HRG was knockdown and a very organized structure. These characteristics resembled an EMT (epithelial-mesenchymal epithelial transition (MET). This should be deemed a potential target in developing therapies for triple negative breast carcinomas. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1142.

Abstract CYR61 render human breast cancer cells highly resistance to the microtubule-interfering agent Taxol, a drug of choice for the treatment of metastatic breast cancer. The angiogenic factor CYR61 and its receptor avβ3 are markedly up-regulated in very aggressive breast carcinomas. Integrin signaling has been shown to modulate cancer cell responses to chemotherapy agents. Thus, the signal transduction pathways initiated by integrin may be a potential target for inhibiting cell survival during cytotoxic drug exposure. However, the molecular mechanism for CYR61-induces cytoprotection still unknown. Our data demonstrates that functional blockade of avβ3 with a synthetic chemical peptidomimetic based upon the αvβ3 the RGD (Arg-Gly-Asp) motif, is specifically cytotoxic towards CYR61-overexpressing breast cancer cells and that pharmacological interference of the CYR61/αvβ3 interaction restores Taxol efficacy. This data suggests that a previously unrecognized CYR61/αvβ3-driven cellular signaling actively modulates cell growth, apoptosis and chemosensitivity. To better understand the mechanism by which CYR61 induces Taxol resistance, we studied a series of molecular events and reveal that CYR61 prevents Taxol induction of p53 accumulation, blocks p21WAF1/CIP1 overexpression, and furthermore we demonstrated that CYR61 induces JNK activation. These observations suggest that increased expression of CYR61 could promote a chemoresistance by suppressing a specific pro-apoptotic signaling while still given chemotherapeutic treatments to induce cell damage. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1139.