IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.

Breast cancers frequently progress or relapse during targeted therapy, but the molecular mechanisms that enable escape remain poorly understood. We elucidated genetic determinants underlying tumor escape in a transgenic mouse model of Wnt pathway-driven breast cancer, wherein targeted therapy is simulated by abrogating doxycycline-dependent Wnt1 transgene expression within established tumors. In mice with intact tumor suppressor pathways, tumors typically circumvented doxycycline withdrawal by reactivating Wnt signaling, either via aberrant (doxycycline-independent) Wnt1 transgene expression or via acquired somatic mutations in the gene encoding β-catenin. Germline introduction of mutant tumor suppressor alleles into the model altered the timing and mode of tumor escape. Relapses occurring in the context of null Ink4a/Arf alleles (disrupting both the p16 Ink4a and p19 Arf tumor suppressors) arose quickly and rarely reactivated the Wnt pathway. In addition, Ink4a/Arf-deficient relapses resembled p53-deficient relapses in that both displayed morphologic and molecular hallmarks of an epithelial-to-mesenchymal transition (EMT). Notably, Ink4a/Arf deficiency promoted relapse in the absence of gross genomic instability. Moreover, Ink4a/Arf-encoded proteins differed in their capacity to suppress oncogene independence. Isolated p19 Arf deficiency mirrored p53 deficiency in that both promoted rapid, EMT-associated mammary tumor escape, whereas isolated p16 Ink4a deficiency failed to accelerate relapse. Thus, p19 Arf /p53 pathway lesions may promote mammary cancer relapse even when inhibition of a targeted oncogenic signaling pathway remains in force. IntroductionBreast cancer research offers a clinically important venue for exploring resistance to targeted therapy. Antagonists of estrogen receptor-dependent (ER-dependent) and human epidermal growth factor receptor 2 (HER2-dependent) signaling are mainstays of modern breast cancer treatment that enhance cure rates when applied against early-stage disease and contribute to disease remissions when applied against late-stage disease (1, 2). Even so, potent targeted agents impose strong selective pressure that ultimately favors tumor escape, wherein treatment-resistant cancer cells survive and proliferate (3). Indeed, resistance to targeted agents, when not encountered de novo, routinely emerges during treatment (4, 5). As a result, targeted agents supplement traditional breast cancer treatment strategies but do not yet obviate the need for surgery, radiation, and cytotoxic chemotherapy. Moreover, incorporating targeted agents into routine clinical practice does not yet permit cure of advanced disease. Thus, tumor escape sets profound limits on the clinical usefulness of targeted therapy in breast cancer patients.In principle, tumors can escape growth constraints imposed by targeted therapy either by reactivating the targeted signaling pathway or by perturbing untargeted compensatory pathways. Both mechanisms appear capable of promoting tumor escape in breast cancer patients....

We have cloned a novel metastasis-suppressor gene (BRMS1) by differential display, comparing metastatic human breast carcinoma cell line MDA-MB-435 to its metastasissuppressed human chromosome 11 microcell hybrid. Screening of a murine cDNA library led to the identification of a 1.4 kb cDNA with a sequence revealing 85% homology to human BRMS1 within the open reading frame. The predicted protein sequence for the murine ortholog is 95% identical, suggesting that it is strongly conserved across these 2 species. The cloned cDNA was used to screen a murine strain SV129 BAC library to obtain brms1 genomic DNA. Three Metastasis-suppressor genes are a relatively newly described group of genes that, by definition, suppress the spread of a primary tumor to a discontinuous site without altering its growth. 1 There are currently 8 genes for which functional suppression of metastasis has been described: Nm23, KiSS1, Kai1, TIMPs, Maspin, MKK4 and BRMS1. BRMS1 was identified by us using differential display, comparing the metastatic human breast carcinoma cell line MDA-MB-435 and its metastasis-suppressed microcell hybrid bearing full-length human chromosome 11. 2 Transfection of full-length BRMS1 cDNA into highly metastatic breast-cancer cell lines MDA-MB-435 and MDA-MB-231 suppressed their metastatic ability without affecting tumorigenicity, meeting the functional definition of metastasis suppression. That the BRMS1 gene maps to 11q13.1-13.2, a region frequently altered in late-stage breast carcinoma, 3 provides further impetus to determine the mechanism by which this candidate metastasis suppressor functions. Despite being a predominantly nuclear protein, BRMS1 restores homotypic gap junctional intercellular communication in MDA-MB-435 and MDA-MB-231 cells. 4,5 However, its normal physiologic role is not known.Determination of the normal physiologic role of BRMS1 might be assisted by the availability of a murine ortholog. Therefore, in the studies presented here, we describe the isolation of brms1, the structural characterization of the brms1 genomic locus and the functionality of brms1 (i.e., ability to suppress metastasis in a series of mammary carcinoma cell lines). That the human and murine orthologs are biologically and molecularly similar provides justification for developing a brms1 knockout mouse and use of murine mammary tumor models for mechanistic studies in the future.