Tumor escape in a Wnt1-dependent mouse breast cancer model is enabled by p19Arf/p53 pathway lesions but not p16 Ink4a loss.

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 beta-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.

Dormant Wnt-initiated mammary cancer can participate in reconstituting functional mammary glands.

The minimal residual disease foci that beget breast cancer relapse after a period of disease dormancy remain uncharacterized despite their enormous clinical importance. To model dormant breast cancer in vivo, we employed a transgenic mouse model in which Wnt1-initiated mammary cancer is doxycycline dependent. After regression of Wnt-dependent cancers, subclinical disease lesions were propagated in vivo using classical tissue recombination techniques. Surprisingly, outgrowths derived from dormant malignant tissue reconstituted morphologically normal ductal trees in wild-type mammary fat pads. Whereas hyperplasia-derived outgrowths remained benign, outgrowths derived from dormant malignancy underwent a morphological transition suggesting single-step transformation following reactivation of Wnt signaling and rapidly yielded invasive mammary tumors. Remarkably, outgrowths derived from dormant malignancy could be serially propagated in vivo and retained the potential to undergo lobuloalveolar differentiation in response to hormones of pregnancy. Matching somatic H-Ras mutations shared by antecedent tumors and descendant mammary ductal outgrowths confirmed their clonal relatedness. Thus, propagation of epithelium that possesses a latent malignant growth program reveals impressive regenerative and developmental potential, supporting the notion that dormant mammary cancers harbor transformed mammary progenitor cells. Our results define an experimental paradigm for elucidating biological properties of dormant malignancy.

Suppression of murine mammary carcinoma metastasis by the murine ortholog of breast cancer metastasis suppressor 1 (Brms1).

The murine ortholog (Brms1) of human breast cancer metastasis suppressor 1 shares 95% identity to the human metastasis suppressor, BRMS1, in amino acid structure. We tested Brms1 for suppression of metastasis of mouse mammary carcinoma cell line 4T1 in syngenic BALB/c mice, using orthotopic (mammary fat pad) injection as well as intravenous injection. As observed for BRMS1, transfection with Brms1 did not inhibit 4T1 primary tumor formation, but significantly suppressed lung colonization. We also show that Brms1 protein interacts with histone deacetylases, indicating involvement of Brms1 in murine Sin3-HDAC complex, like its human counterpart. Thus, because of similarities with its human ortholog, the results suggest that Brms1 will be useful as a model for studying mechanism of action of BRMS1.

A small molecule antagonist of the alpha(v)beta3 integrin suppresses MDA-MB-435 skeletal metastasis.

INTRODUCTION: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases. MATERIALS AND METHODS: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded. RESULTS AND CONCLUSIONS: 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.

Ubiquitous Brms1 expression is critical for mammary carcinoma metastasis suppression via promotion of apoptosis.

Morbidity and mortality of breast cancer patients are drastically increased when primary tumor cells are able to spread to distant sites and proliferate to become secondary lesions. Effective treatment of metastatic disease has been limited; therefore, an increased molecular understanding to identify biomarkers and therapeutic targets is needed. Breast cancer metastasis suppressor 1 (BRMS1) suppresses development of pulmonary metastases when expressed in a variety of cancer types, including metastatic mammary carcinoma. Little is known of Brms1 function throughout the initiation and progression of mammary carcinoma. The goal of this study was to investigate mechanisms of Brms1-mediated metastasis suppression in transgenic mice that express Brms1 using polyoma middle T oncogene-induced models. Brms1 expression did not significantly alter growth of the primary tumors. When expressed ubiquitously using a ß-actin promoter, Brms1 suppressed pulmonary metastasis and promoted apoptosis of tumor cells located in the lungs but not in the mammary glands. Surprisingly, selective expression of Brms1 in the mammary gland using the MMTV promoter did not significantly block metastasis nor did it promote apoptosis in the mammary glands or lung, despite MMTV-induced expression within the lungs. These results strongly suggest that cell type-specific over-expression of Brms1 is important for Brms1-mediated metastasis suppression.

Identification and characterization of the murine ortholog (brms1) of breast-cancer metastasis suppressor 1 (BRMS1).

We have cloned a novel metastasis-suppressor gene (BRMS1) by differential display, comparing metastatic human breast carcinoma cell line MDA-MB-435 to its metastasis-suppressed 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 BAC clones [226(I4), 226(H4) and 239(N7)] were confirmed to encode the entire brms1 gene. Detailed analysis of BAC clone 226(I4) shows that the gene spans 8.5 kb and, like the human gene, is organized into 10 exons and 9 introns. While the exons share a high degree of homology, there are greater differences when comparing intron structures between the human and murine genes. The 5' upstream region shares about 64% homology with its human counterpart, retaining several of the many putative regulatory elements. Like the human genomic BRMS1, the murine ortholog of the iGnT gene is found upstream of brms1 and the murine ortholog of the RIN1 gene is found downstream of brms1. brms1 was then tested for suppression of metastasis of mouse mammary carcinoma cell line 66cl4 in syngeneic BALB/c mice. Transfection with brms1 did not inhibit 66cl4 primary tumor formation but significantly suppressed its metastatic capability. This suggests that the murine ortholog functions similarly to BRMS1.