Twenty-four-well plates precoated with Development Factor Reduced Matrigel (BD Biosciences) were handled according to the manufacturers instructions

Twenty-four-well plates precoated with Development Factor Reduced Matrigel (BD Biosciences) were handled according to the manufacturers instructions. MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by B-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing B-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that B-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors. Introduction Breast cancer is the most frequently diagnosed cancer in women and is responsible for 411,000 deaths per year in women worldwide (1). Although clinical indices such as tumor size and grade and axillary lymph node metastases are useful prognostic factors in breast cancer, there is an urgent need to identify molecular characteristics of breast carcinomas that more accurately predict clinical outcome and guide specific therapies for individual patients (2). Recent gene expression profiling of human breast cancer CCT239065 has led to the identification of several subtypes of breast cancer with different clinical outcomes: 2 estrogen receptorCpositive (ER-positive) subtypes, a subtype with high expression CCT239065 CCT239065 of the erythroblastic leukemia viral oncogene homolog 2/HER-2 (ErbB2/HER-2) proto-oncogene, a normal breast-like subtype, and a basal-like subtype that expresses genes characteristic of basal epithelial cells ZPK and normal breast myoepithelial CCT239065 cells, such as cytokeratin 5 (CK5) and CK17, and does not express ER or ErbB2/HER-2 (3C6). Of these CCT239065 subtypes, the ErbB2/HER-2 and basal-like groups are associated with the shortest overall and relapse-free survival. Unlike the ErbB2/HER-2 subtype, the genes in the basal-like cluster responsible for these cells clinically aggressive behavior are unknown. Identification of these genes could lead to new targeted therapies for basal-like breast tumors, which account for 15C20% of breast cancer cases. By exploring existing breast cancer cDNA microarray data (3, 4), we observed that (was most consistently expressed in the basal-like tumors and the normal breast samples (Figure ?(Figure1A).1A). Consistent with these gene expression results, immunohistochemistry (IHC) revealed that B-crystallin protein was coexpressed with CK5/6 in 2 known basal-like breast tumors (Figure ?(Figure1,1, B and C) and was predominantly expressed in myoepithelial cells in normal breast tissue (Figure ?(Figure1D).1D). We next examined the expression of B-crystallin by IHC in a tissue microarray (TMA) of invasive breast carcinomas with linked clinical and pathological data (median follow-up, 10.8 yr; range, 0.3C20.0 yr) (17). Tumors were scored as strongly positive (Figure ?(Figure1E,1E, left), weakly positive (Figure ?(Figure1E,1E, middle) or negative (Figure ?(Figure1E,1E, right) for B-crystallin expression (IHC results for all tumors can be viewed at B-Crystallin was expressed in 39 of 361 (11%) breast carcinomas (25 tumors were weakly positive and 14 were strongly positive). Using an IHC surrogate to identify basal-like tumors (negative for ER and ErbB2/HER-2 and positive for EGFR/HER-1 and/or CK5/6) that was validated in an independent breast cancer series (18), we observed that B-crystallin was expressed in 18 of 40 basal-like breast carcinomas (45%) in the TMA, but only 17 of 288 (6%) nonbasal tumors expressed B-crystallin (= 8 10C10 by Fishers exact test). Kaplan-Meier analyses revealed that expression of B-crystallin in breast carcinomas was associated with shorter disease-specific survival (Figure ?(Figure1F,1F, left; 10-year disease-specific survival, 59% for B-crystallinCpositive tumors versus 74% for B-crystallinCnegative tumors; = 0.0054). In addition, the levels of expression of B-crystallin correlated inversely with disease-specific survival: strongly positive tumors were associated with shorter survival than were weakly positive tumors (Figure ?(Figure1F,1F, right; 10-year disease-specific survival, 46% for strongly positive tumors versus 66% for weakly positive tumors; = 0.0125). In contrast, expression of the related small heat shock protein Hsp27 was not associated with survival in this cohort (ref. 17 and data not shown), thereby underscoring the specificity of our observation. Multivariate Cox regression analysis revealed that B-crystallin expression predicted shorter survival (hazard ratio, 2.23; = 0.001) independent of tumor grade, lymph node status, and ER and ErbB2/HER-2 expression status (tumor size was not included because accurate measures were unavailable for many cases). These results indicate that B-crystallin is commonly expressed in basal-like breast carcinomas and is an independent predictor of poor clinical outcome. Open in a separate window Figure 1 B-Crystallin is expressed in the basal-like subtype of breast cancer and independently predicts shorter survival. (A) Expanded view of the basal epithelial gene expression cluster presented by Sorlie et al. (4) (red, above.