We show here that inducible expression of Braf(V600E) off the endogenous Braf gene in mouse melanocytes stimulates skin hyperpigmentation and the appearance of nevi harboring senescent melanocytes. Additionally, approximately 70% of Braf(V600E) mice develop melanomas that reproduce many of the cardinal histological and molecular features of human melanoma and whose cells can colonize the lungs of nude mice. We show that the tumor suppressor p16(INK4a) is not required to induce melanocyte senescence and that its loss is not required for tumor progression, although it does regulate tumor penetrance and latency. Thus, we have developed a mouse model of melanoma driven by Braf(V600E) expressed at physiological levels that reflects the genetics and pathology of the human disease.

Basal-like breast cancers form a distinct subtype of breast cancer characterized by the expression of markers expressed in normal basal/myoepithelial cells. Breast cancers arising in carriers of germline BRCA1 mutations are predominately of basal-like type, suggesting that BRCA1 dysfunction may play a role in the pathogenesis of sporadic basal-like cancers. We analysed 37 sporadic breast cancers expressing the basal marker cytokeratin 5/6, and age-and grade-matched controls, for downregulation of BRCA1. Although BRCA1 promoter methylation was no more common in basal-like cancers (basal 14% vs controls 11%, P ¼ 0.72), BRCA1 messenger RNA expression was twofold lower in basal-like breast cancers compared to matched controls (P ¼ 0.008). ID4, a negative regulator of BRCA1, was expressed at 9.1-fold higher levels in basal-like breast cancer (Po0.0001), suggesting a potential mechanism of BRCA1 downregulation. BRCA1 downregulation correlated with the presence of multiple basal markers, revealing heterogeneity in the basal-like phenotype. Finally, we found that 63% of metaplastic breast cancers, a rare type of basal-like cancers, had BRCA1 methylation, in comparison to 12% of controls (Po0.0001). The high prevalence of BRCA1 dysfunction identified in this study could be exploited in the development of novel approaches to targeted treatment of basal-like breast cancer.

Aberrant b-catenin expression as determined by assessment of its subcellular localization constitutes a surrogate marker of Wnt signalling pathway activation and has been reported in a subset of breast cancers. The association of b-catenin/Wnt pathway activation with clinical outcome and the mechanisms leading to its activation in breast cancers still remain a matter of controversy. The aims of this study were to address the distribution of b-catenin expression in invasive breast cancers, the correlations between b-catenin expression and clinicopathological features and survival of breast cancer patients, and to determine whether aberrant b-catenin expression is driven by CTNNB1 (b-catenin encoding gene) activating mutations. Immunohistochemistry was performed on a tissue microarray containing 245 invasive breast carcinomas from uniformly treated patients, using two anti-b-catenin monoclonal antibodies. Selected samples were subjected to CTNNB1 exon 3 mutation analysis by direct gene sequencing. A good correlation between the two b-catenin antibodies was observed (Spearman's r 40.62, Po0.001). Respectively, 31 and 11% of the cases displayed lack/reduction of b-catenin membranous expression and nuclear accumulation. Complete lack of b-catenin expression was significantly associated with invasive lobular carcinoma histological type. Subgroup analysis of non-lobular cancers or non-lobular grade 3 carcinomas revealed that lack/reduction of b-catenin membranous expression and/or nuclear accumulation were significantly associated with oestrogen receptor negativity, absence of HER2 gene amplification and overexpression, lack/reduction of E-cadherin expression and tumours of triple-negative and basal-like phenotype. Univariate survival analysis revealed a significant association between b-catenin nuclear expression and shorter metastasis-free and overall survival in the whole cohort; however, b-catenin nuclear expression was not an independent predictor of outcome in multivariate analysis. No CTNNB1 mutations were identified in the 28 selected breast carcinomas analysed. In conclusion, b-catenin/Wnt pathway activation is preferentially found in triple-negative/basal-like breast carcinomas, is associated with poor clinical outcome and is unlikely to be driven by CTNNB1 mutations in breast cancer.

Purpose: Classic lobular carcinomas (CLC) account for 10% to 15% of all breast cancers. At the genetic level, CLCs show recurrent physical loss of chromosome16q coupled with the lack of E-cadherin (CDH1 gene) expression. However, little is known about the putative therapeutic targets for these tumors. The aim of this study was to characterize CLCs at the molecular genetic level and identify putative therapeutic targets. Experimental Design: We subjected 13 cases of CLC to a comprehensive molecular analysis including immunohistochemistry for E-cadherin, estrogen and progesterone receptors, HER2/ neu and p53; high-resolution comparative genomic hybridization (HR-CGH); microarray-based CGH (aCGH); and fluorescent and chromogenic in situ hybridization for CCND1 and FGFR1. Results: All cases lacked the expression of E-cadherin, p53, and HER2, and all but one case was positive for estrogen receptors. HR-CGH revealed recurrent gains on 1q and losses on 16q (both, 85%). aCGH showed a good agreement with but higher resolution and sensitivity than HR-CGH. Recurrent, high level gains at 11q13 (CCND1) and 8p12-p11.2 were identified in seven and six cases, respectively, and were validated with in situ hybridization. Examination of aCGH and the gene expression profile data of the cell lines, MDA-MB-134 and ZR-75-1, which harbor distinct gains of 8p12-p11.2, identified FGFR1 as a putative amplicon driver of 8p12-p11.2 amplification in MDA-MB-134. Inhibition of FGFR1 expression using small interfering RNA or a small-molecule chemical inhibitor showed that FGFR1 signaling contributes to the survival of MDA-MB-134 cells. Conclusions: Our findings suggest that receptor FGFR1 inhibitors may be useful as therapeutics in a subset of CLCs.

Our results demonstrate that MBCs show a basal-like phenotype, regardless of the type of metaplastic elements. Moreover, as these neoplasms frequently overexpress EGFR (57%), patients with MBC may benefit from treatment with anti-EGFR drugs.

Despite treatment with standard dose anthracycline-based chemotherapy, the clinical outcome of TN and BL cancers remains poor. Alternative chemotherapeutic regimens and/or novel therapeutic approaches are warranted. Although a significant phenotypic overlap exists between TN and basal-like tumours, the TN phenotype is not an ideal surrogate marker for basal-like breast cancers.

Purpose: To assess the prevalence of defective homologous recombination (HR)-based DNA repair in sporadic primary breast cancers, examine the clincopathologic features that correlate with defective HR and the relationship with neoadjuvant chemotherapy response.Experimental Design: We examined a cohort of 68 patients with sporadic primary breast cancer who received neoadjuvant anthracylcine-based chemotherapy, with core biopsies taken 24 hours after the first cycle of chemotherapy. We assessed RAD51 focus formation, a marker of HR competence, by immunofluorescence in postchemotherapy biopsies along with geminin as a marker of proliferative cells. We assessed the RAD51 score as the proportion of proliferative cells with RAD51 foci.Results: A low RAD51 score was present in 26% of cases (15/57, 95% CI: 15%-40%). Low RAD51 score correlated with high histologic grade (P ¼ 0.031) and high baseline Ki67 (P ¼ 0.005). Low RAD51 score was more frequent in triple-negative breast cancers than in ER-and/or HER2-positive breast cancer (67% vs. 19% respectively; P ¼ 0.0036). Low RAD51 score was strongly predictive of pathologic complete response (pathCR) to chemotherapy, with 33% low RAD51 score cancers achieving pathCR compared with 3% of other cancers (P ¼ 0.011).Conclusions: Our results suggest that defective HR, as indicated by low RAD51 score, may be one of the factors that underlie sensitivity to anthracycline-based chemotherapy. Defective HR is frequent in triple-negative breast cancer, but it is also present in a subset of other subtypes, identifying breast cancers that may benefit from therapies that target defective HR such as PARP inhibitors. Clin Cancer Res; 16(24); 6159-68. Ó2010 AACR.DNA double-strand break repair by homologous recombination (HR) is required for the repair of DNA damage arising from many currently used DNA-damaging chemotherapy agents (1), as well as DNA single-strand breaks that are generated by inhibitors of poly(ADP ribose) polymerase 1 (PARP; ref. 2). The tumor suppressor genes BRCA1 and BRCA2 encode proteins that are required for HR (3), and tumors arising in carriers of germline mutations in BRCA1/BRCA2 are defective in HR and, consequently, are highly sensitive to DNA-damaging chemotherapy (4, 5) and PARP inhibitors (6). Increasing evidence suggests that a proportion of sporadic breast cancers may also have defects in HR (1). Sporadic basallike breast cancers have low BRCA1 expression (7-9) and BRCA1 promoter methylation is found in approximately 10% of sporadic breast cancers including nonbasal tumors (7). Recently, it has also been proposed that loss of PTEN expression may also lead to a defect in HR (3,10,11).In this study, we set out to develop a functional assay for HR with the purpose of assessing the prevalence of a functional defect in HR in sporadic breast cancer and examine whether defective HR correlated with clinicopathologic features and sensitivity to chemotherapy. The protein RAD51 is a DNA recombinase and key component of HR (12). In response to DNA double-strand bre...

Purpose:The distribution and significance of caveolin1 (CAV1) expression in different breast cell types and role in breast carcinogenesis remain poorly understood. Both tumor-suppressive and oncogenic roles have been proposed for this protein. The aims of this study were to characterize the distribution of CAV1 in normal breast, benign breast lesions, breast cancer precursors, and metaplastic breast carcinomas; to assess the prognostic significance of CAV1expression in invasive breast carcinomas; and to define whether CAV1 gene amplification is the underlying genetic mechanism driving CAV1overexpression in breast carcinomas. Experimental Design: CAV1 distribution in frozen and paraffin-embedded whole tissue sections of normal breast was evaluated using immunohistochemistry, immunofluorescence, and immunoelectron microscopy. CAV1 expression was immunohistochemically analyzed in benign lesions, breast cancer precursors, and metaplastic breast carcinomas and in a cohort of 245 invasive breast carcinomas from patients treated with surgery followed by anthracyclinebased chemotherapy. In 25 cases, CAV1 gene amplification was assessed by chromogenic in situ hybridization. Results: In normal breast, CAV1 was expressed in myoepithelial cells, endothelial cells, and a subset of fibroblasts. Luminal epithelial cells showed negligible staining. CAV1was expressed in 90% of 39 metaplastic breast carcinomas and in 9.4% of 245 invasive breast cancers. In the later cohort, CAV1 expression was significantly associated with 'basal-like' immunophenotype and with shorter disease-free and overall survival on univariate analysis. CAV1 gene amplification was found in 13% of cases with strong CAV1expression. Conclusions:The concurrent CAV1amplification and overexpression call into question its tumorsuppressive effects in basal-like breast carcinomas.