Yes-associated protein (YAP) is a well characterized transcriptional coactivator that interacts with various transcription factors and modulates their transcriptional activities. Phosphorylation of YAP by specific kinases regulates its cellular distribution and transcriptional activation functions. Sequestration of phosphorylated YAP in cytoplasm results in the reduction of transcription from its target genes. Since, YAP has been characterized as a coactivator of estrogen (ER) and progesterone (PR) receptors, we examined the immunohistochemical expression profile of YAP and correlation of YAP expression with that of ER and PR in normal (40 samples) and tumor breast (226 samples) from microarray tissue samples using immunohistochemistry. Here we show that YAP expression is significantly reduced in invasive carcinoma samples compared to normal breast tissues, which express high levels of YAP (YAP was positive for 45.1% of invasive carcinoma samples vs. 82.5% of normal samples p<.0001). Furthermore, our data shows that reduced expression of YAP in invasive carcinoma samples is significantly associated with ER negativity (YAP was negative for 59.9% in ER negative vs. 38.9% in ER positive invasive carcinoma samples, p=0.007) and PR negativity (YAP was negative for 60.1% in PR negative vs. 28.9% in PR positive, p=0.0004). Among invasive carcinoma samples, 42.9% were YAP, ER and PR negative, whereas only 7.5% were found to be YAP, ER and PR positive. On the contrary, 20 out of 23 (87%) normal breast tissues that were positive for ER and PR were also positive for YAP. These data suggest that YAP may act as a tumor suppressor in invasive breast carcinomas and it can also be used as a molecular marker for ER and PR negative breast tumors.

Breast cancer is the leading cause of cancer-related death in women worldwide. Expression of the WWOX tumor suppressor is absent or reduced in a large proportion of breast tumors suggesting that loss of WWOX may contribute to breast tumorigenesis. Wwox-deficient mice die by 3-4 weeks of age precluding adult tumor analysis. To evaluate the effect of WWOX-altered expression on mammary tumor formation, the Wwox-heterozygous allele was back crossed onto the C3H mammary tumor-susceptible genetic background (Wwox C3H þ /À) and incidence of mammary tumor formation was evaluated. Although 50% of the female Wwox C3H þ /À mice developed mammary carcinomas, only 7% of Wwox C3H þ / þ mice did. Intriguingly, mammary tumors in Wwox C3H þ /À mice frequently lost WWOX protein expression suggesting a genetic predisposition toward mammary tumorigenesis. Immunohistochemical staining of hormone receptors revealed loss of estrogen receptor-a (ER) and progesterone receptor in the majority of these tumors. In vitro, depletion of WWOX in MCF7 ER-positive cells led to reduced ER expression and reduced sensitivity to tamoxifen and estrogen treatment and was associated with enhanced survival and anchorageindependent growth. Finally, cDNA array analyses of murine normal mammary epithelial cells and mammary tumors identified 163 significantly downreguated and 129 upregulated genes in the tumors. The majority of differentially expressed genes were part of pathways involved in cellular movement, cell-to-cell signaling and interaction, cellular development, cellular growth and proliferation and cell death. These changes in gene expression of mouse mammary tumors in Wwox C3H þ /À mice resemble, at least in part, human breast cancer development. Our findings demonstrate the critical role that the WWOX tumor suppressor gene has in preventing tumorigenesis in breast cancer.

E6-associated protein (E6-AP) is a dual function protein. It acts as an E3 ubiquitin-protein ligase enzyme and coactivator of steroid hormone receptors such as estrogen (ERα) and progesterone (PR) receptors. It promotes the degradation of ERα and PR through the ubiquitin-proteasome pathway. Furthermore, it has been shown that the levels of E6-AP are inversely associated with that of ERα in human breast tumors. But the role of wild-type human E6-AP and its ubiquitin-protein ligase activity in mammary tumorigenesis is still unknown. To investigate this role, the authors utilized transgenic mice lines that specifically overexpress either the wild-type human E6-AP (E6-AP(WT)) or the ubiquitin-protein ligase defective E6-AP that contains C833S mutation (E6-AP(C833S)) in the mammary gland. To further substantiate the role of E6-AP in the development of breast tumorigenesis, it was also examined the expression of E6-AP in a large cohort of human breast cancer samples. The transgenic mice that overexpress wild-type E6-AP (E6-AP(WT)) fail to develop mammary tumors. Unlike the E6-AP(WT) mice, the E6-AP(C833S) mice that overexpress ubiquitin-protein ligase defective E6-AP protein develop mammary hyperplasia with a median latency of 18 months. These observations suggest that the inactivation of the ubiquitin-protein ligase function of E6-AP is sufficient to initiate the process of mammary tumor development. Furthermore, the data also suggests that E6-AP exerts its effects on target cells by modulating the protein levels and functions of ERα and PR. In addition, it was found in human breast cancer patients that the level of E6-AP is decreased in invasive breast tumors compared to normal breast tissue. Moreover, the authors also show that the survival patterns for E6-AP negative patients were worse compared to E6-AP positive patients. Taken together, these data suggests that E6-AP may act as a tumor suppressor in breast.