An important characteristic of multicellular organisms is the control that the tissue architecture exerts on the fate of individual cells. Epithelial cells sense their location through interactions with the extracellular matrix (ECM) and remove themselves by programmed cell death (anoikis) when those interactions are disturbed. Importantly, anoikis is a line of defense that has to be circumvented by cancerous epithelial cells to be able to leave their home environment and establish long distance metastases. Here, by combining a genome-wide RNAi screen and a novel algorithm to study copy number alterations (ISAR-DEL), we identify the Bridging Integrator-3 (BIN3) as a novel 8p21 tumor suppressor gene whose inactivation promotes escape from anoikis in epithelial cancers. Mechanistically, we link the tumor suppression function of BIN3 to its ability to relocate to the cell membrane after cell detachment and to induce a proapoptotic cascade. This death signaling is mediated by CDC42 activation of the P38-α stress pathway and the consequent accumulation of the apoptotic facilitator BimEL. Our results identify BIN3 as a novel epithelial tumor suppressor gene, provide novel insights on the mechanisms of attachment tumor suppressor checkpoint and highlight the importance of anoikis escape in driving cell transformation and metastasis in human cancer. Note: This abstract was not presented at the meeting. Citation Format: Netonia Marshall, Felix Sanchez, David Llobet, Ruth Rodriguez Barrueco, Veronica Castro, Dylan Kotlia, Maira Pires, Patricia Villagrasa, Preeti Putcha, Ramon Parsons, Dana Pe'er, Jose Silva. BIN3 is a novel 8p21 tumor suppressor gene that regulates the attachment checkpoint in epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 432. doi:10.1158/1538-7445.AM2014-432

IntroductionThe female mammary gland is a very dynamic organ that undergoes continuous tissue remodelling during adulthood. Although it is well established that the number of menstrual cycles and pregnancy increase the risk of breast cancer, the reasons are unclear. Clinical and experimental evidence indicates that improper involution plays a role in the development of this malignancy. Recently, we described the miR-424(322)/503 cluster as an important regulator of mammary epithelial involution after pregnancy and that miR-424(322)/503 is commonly lost in a subset of aggressive breast cancers. Through the use of a knockout mouse model, we demonstrated for the first time that loss of miR-424(322)/503 promotes breast tumorigenesis in vivo. Remarkably, we found that loss of miR-424(322)/503 promotes chemoresistance due to the up-regulation of two of its targets: BCL-2 and insulin-like growth factor-1 receptor (IGF1R). Importantly, targeted therapies blocking the aberrant activity of these targets restore sensitivity to chemotherapy.Material and methodsPatient data were assessed from METABRIC and TCGA corresponding to breast cancer samples with available whole-genome DNA CNAs, mRNA expression data, and clinicopathological data.Targets of the miR-424(322)/503 were validated by western blot, 3’UTR reporter assays and PAR-CLIP. Wnt localization and activity was assessed by cell fractionation and qRT-PCR.Results and discussionsTumours generated in the miR-424(322)/503 knock out mouse model present morphological and molecular characteristics of metaplasic squamous cell carcinoma (SSC) of the breast. These include the presence of a non-glandular component, spindle cells and expression of cytokeratin 6. Primary SSC of the breast is a very aggressive tumour with high metastatic activity and refractory to treatment. It has been reported that nearly all the primary metaplasic carcinomas present activation of Wnt pathway, and about 40% of them carry mutations in genes of the Wnt pathway. By analysing the expression data of over 3000 breast tumours, we have determined that the loss of mir-424(322)/503 contributes to the activation of Wnt/b-catenin pathway in SSC. We also show that the miR-424(322)/503 directly targets the Wnt receptor LRP-6, preventing the translocation of b-catenin to the nucleous and inhibiting the transcription of b-catenin-regulated genes.ConclusionMiR-424(322)/503 is a tumour suppressor in breast cancer and provide a link between mammary epithelial involution, tumorigenesis, and chemoresistance.

The mammary gland is a very dynamic organ that undergoes continuous remodeling. The critical regulators of this process are not fully understood. Here we identify the microRNA cluster miR-424(322)/503 as an important regulator of epithelial involution after pregnancy and tumorigenesis. Importantly, miR-424(322)/503 loci is deleted in 20% of basal and luminal B tumors. Through the generation of a knock-out mouse model, we found that regression of the secretory acini of the mammary gland was compromised in the absence of miR-424(322)/503. Mechanistically, we show that miR-424(322)/503 orchestrates cell life and death decisions by targeting CDC25A, BCL-2 and IGF1R. Furthermore, we demonstrate that the expression of this miR-cluster is regulated by TGF-β, a well characterized regulator of mammary involution. Overall, our data suggest a model where activation of the TGF-β pathway after weaning induces the transcription of the miR-424(322)/503 which in turn downregulates the expression of key genes. Here, we unveil a previously unknown, multilayered-regulation of epithelial tissue remodeling coordinated by the microRNA cluster miR-424(322)/503. Note: This abstract was not presented at the meeting. Citation Format: David Llobet Navas, Ruth Rodriguez Barrueco, Jose Silva. miR-424(322)/503 cluster orchestrates remodeling of the epithelium in the involuting mammary gland and plays a role in breast cancer development. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5214. doi:10.1158/1538-7445.AM2014-5214

HER2 is a receptor tyrosine kinase found overexpressed in 20-40% of breast tumors correlating with a malignant phenotype and worse prognosis. Different targeted therapies have been developed to specifically inhibit its activity such the monoclonal antibodies Trastuzumab and Pertuzumab or the small tyrosine kinase inhibitor Lapatinib. Unfortunately, a large number of these patient tumors eventually progress, acquiring resistance.. Our main goal is to identify which pathways have become essential for HER2 overexpressing cells but are not needed for normal cells to survive. To determine this, we performed a pooled genome-wide shRNA screen that provides us with a list of HER2 synthetic lethal genes. By combining the analysis from RNAi loss-of-function screens with system biology interactome models, we have recently found that the activation of the JAK/STAT pathway is essential for HER2 mediated transformation, as we demostrate by knocking-down STAT3 expression either in vitro or in orthotopic mouse models. To investigate the mechanism mediating STAT3 activation, we compared the expression profile of parental and HER2 activated cells and found a strong upregulation of IL6 and its canonical receptor that was associated with an increase of IL6 in the media. Overall, our data shows that overexpression of HER2 in breast cancer cell lines leads to an increase in the expression and secretion of IL6, generating an autocrine loop that keeps JAK/STAT3 pathway constitutively active. To further study the transcriptional program regulated by STAT3 in HER2+ breast cancer cell lines, we analyzed the RNA profile in different conditions by using expression microarrays. Interestingly, our results included STAT3 as well as SOCS3, previously described targets of STAT3, but also two S100 family members: S100A8 and S100A9. Our data shows that the overexpression of these two genes is also essential for the transformation of HER2+ cells in vitro and in orthotopic mouse models. In order to translate our results to the clinical setting, we searched for compounds that could inhibit STAT3 activity in vivo. Ruxolitinib is a small-molecule inhibitor of JAK1 and JAK2 approved by the FDA for the treatment of patients with myelofibrosis and other myeloproliferative disorders, so we decieded to assess if Ruxolitnib was effective at inhibiting STAT3 signaling in our model. In fact, treatment with Ruxolitinib impairs in vitro transformation of cells that overexpress HER2 as shown by soft agar assays and 3D cultures in matrigel. More interestingly, oral administration of Ruxolitinib to mice significantly reduces the growth of the tumors implanted in the mammary fat pad. In summary, JAK/STAT pathway activation is essential for HER2-associated transformation suggesting this pathway inhibition as a promising therapeutic approach in HER2 positive tumors that do not respond to the conventional treatment. Note: This abstract was not presented at the meeting. Citation Format: Ruth Rodriguez Barrueco, Jiyang Yu, Mariano Alvarez, Veronica Castro, Netonia Marshall, Ruoxi Su, Laura Castro, Matthew Maurer, Andrea Califano, Jose M Silva. Characterizing the mechanism and the clinical relevance of the synthetic lethal interaction between STAT3 inhibition and HER2 overexpression in breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2761. doi:10.1158/1538-7445.AM2014-2761

The generation of multidimensional datasets characterizing genetic, epigenetic, and functional properties of large normal and tumor-related samples is creating unique opportunities for the systems-level dissection of mechanisms associated with malignant phenotypes. Integrated data sets may lead to more efficient and specific approaches for the elucidation of therapeutic targets and biomarkers and for the identification of specific small molecule inhibitors. However, the power of these computational methods to study human cancer from an unbiased global perspective is severely compromised by the low through-put approaches necessary to functionally validate the in silico predictions. During the last years, RNA interference (RNAi) emerged as a very powerful genetic strategy to functionally interrogate the entire genome by loss-of-function studies. This cellular process can be experimentally controlled to knock down the expression of any specific gene. Our group has pioneered the development of RNAi-based genetic tools to translate RNAi studies to mammalian systems generating a shRNA-mir pooled lentiviral library that allows performing functional studies at genome-wide level. ErbB2 is a receptor tyrosine kinase found overexpressed in 20-40% of breast tumors correlating with a malignant phenotype and worse prognosis. Different targeted therapies have been developed to specifically inhibit its activity such the monoclonal antibody Herceptin, or the small tyrosine kinase inhibitor Lapatinib. Despite of the success of these inhibitors in the treatment of tumors that overexpress ErbB2, about 30% of them become resistant making necessary to look for alternative targets in these cases. The combination of the state-of-the-art computational analysis and RNAi-based technology toward system biology studies must represent a powerful tool to dissect the regulatory networks that govern the homeostasis of breast tumors. Applying this combined approach to an overexpressing ErbB2 breast cancer model we have identified the genes that are critical in those cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5137. doi:1538-7445.AM2012-5137