Veronica Castro

Cyfip1 is a putative invasion suppressor in epithelial cancers

Identification of bona fide tumor suppressors is often challenging because of the large number of genetic alterations present in most human cancers. To evaluate candidate genes present within chromosomal regions recurrently deleted in human cancers, we coupled high-resolution genomic analysis with a two-stage genetic study using RNA interference (RNAi). We found that Cyfip1, a subunit of the WAVE complex, which regulates cytoskeletal dynamics, is commonly deleted in human epithelial cancers. Reduced expression of CYFIP1 is commonly observed during invasion of epithelial tumors and is associated with poor prognosis in this setting. Silencing of Cyfip1 disturbed normal epithelial morphogenesis in vitro and cooperated with oncogenic Ras to produce invasive carcinomas in vivo. Mechanistically, we have linked alterations in WAVE-regulated actin dynamics with impaired cell-cell adhesion and cell-ECM interactions. Thus, we propose Cyfip1 as an invasion suppressor gene.

Integration of Genomic Data Enables Selective Discovery of Breast Cancer Drivers

Identifying driver genes in cancer remains a crucial bottleneck in therapeutic development and basic understanding of the disease. We developed Helios, an algorithm that integrates genomic data from primary tumors with data from functional RNAi screens to pinpoint driver genes within large recurrently amplified regions of DNA. Applying Helios to breast cancer data identified a set of candidate drivers highly enriched with known drivers (p < 10(-14)). Nine of ten top-scoring Helios genes are known drivers of breast cancer, and in vitro validation of 12 candidates predicted by Helios found ten conferred enhanced anchorage-independent growth, demonstrating Helioss exquisite sensitivity and specificity. We extensively characterized RSF-1, a driver identified by Helios whose amplification correlates with poor prognosis, and found increased tumorigenesis and metastasis in mouse models. We have demonstrated a powerful approach for identifying driver genes and how it can yield important insights into cancer.

The miR-424(322)/503 cluster orchestrates remodeling of the epithelium in the involuting mammary gland

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. Through the generation of a knockout 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 BCL-2 and IGF1R (insulin growth factor-1 receptor). Furthermore, we demonstrate that the expression of this microRNA cluster is regulated by TGF-β, a well-characterized regulator of mammary involution. Overall, our data suggest a model in which activation of the TGF-β pathway after weaning induces the transcription of miR-424(322)/503, which in turn down-regulates 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.

Inhibition of the autocrine IL-6–JAK2–STAT3–calprotectin axis as targeted therapy for HR−/HER2+ breast cancers

HER2-positive (HER2(+)) breast adenocarcinomas are a heterogeneous group in which hormone receptor (HR) status influences therapeutic decisions and patient outcome. By combining genome-wide RNAi screens with regulatory network analysis, we identified STAT3 as a critically activated master regulator of HR(-)/HER2(+) tumors, eliciting tumor dependency in these cells. Mechanistically, HR(-)/HER2(+) cells secrete high levels of the interleukin-6 (IL-6) cytokine, inducing the activation of STAT3, which in turn promotes a second autocrine stimulus to increase S100A8/9 complex (calprotectin) production and secretion. Increased calprotectin levels activate signaling pathways involved in proliferation and resistance. Importantly, we demonstrated that inhibition of the IL-6-Janus kinase 2 (JAK2)-STAT3-calprotectin axis with FDA-approved drugs, alone and in combination with HER2 inhibitors, reduced the tumorigenicity of HR(-)/HER2(+) breast cancers, opening novel targeted therapeutic opportunities.

The MicroRNA 424/503 Cluster Reduces CDC25A Expression during Cell Cycle Arrest Imposed by Transforming Growth Factor β in Mammary Epithelial Cells

ABSTRACT Recently, we demonstrated that the microRNA 424(322)/503 [miR-424(322)/503] cluster is transcriptionally controlled by transforming growth factor β (TGF-β) in the mammary epithelium. Induction of this microRNA cluster impacts mammary epithelium fate by regulating apoptosis and insulin-like growth factor 1 (IGF1) signaling. Here, we expanded our finding to demonstrate that miR-424(322)/503 is an integral component of the cell cycle arrest mediated by TGF-β. Mechanistically, we showed that after TGF-β exposure, increased levels of miR-424(322)/503 reduce the expression of the cell cycle regulator CDC25A. miR-424(322)/503-dependent posttranscriptional downregulation of CDC25A cooperates with previously described transcriptional repression of the CDC25A promoter and proteasome-mediated degradation to reduce the levels of CDC25A expression and to induce cell cycle arrest. We also provide evidence that the TGF-β/miR-424(322)/503 axis is part of the mechanism that regulates the proliferation of hormone receptor-positive (HR+) mammary epithelial cells in vivo.

Abstract 432: BIN3 is a novel 8p21 tumor suppressor gene that regulates the attachment checkpoint in epithelial cells

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 Pe9er, 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

Abstract 2761: Characterizing the mechanism and the clinical relevance of the synthetic lethal interaction between STAT3 inhibition and HER2 overexpression in breast cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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

Abstract PR11: Characterizing the mechanism and the clinical relevance of the synthetic lethal interaction between STAT3 inhibition and HER2 overexpression in breast cancers

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. Thus, there is the need to find alternative tumor targets to develop specific and more efficient treatments for these patients. In a tumor cell, multiple regulatory networks have been rearranged in order to adapt to specific genomic abnormalities. Due to this divergence, transformed cells may acquire vulnerabilities that create opportunities for therapeutic intervention. 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. Signal transducers and activators of transcription (STATs) are latent transcription factors that reside in the cytoplasm until they are phosphorylated and activated by Janus kinases (JAKs). STATs then dimerize and enter the nucleus, where they can activate or repress transcription of their target genes. 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. This abstract is also presented as Poster B35. Citation Format: Ruth Rodriguez-Barrueco, Jiyang Yu, Mariano Alvarez, Veronica Castro, Patricia Villagrasa, Netonia Marshall, Ruoxi Sun, Laura Castro, Andrea Califano. 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 AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR11.

Abstract 3168: Helios identifies novel oncogenes in breast cancer by integrating genomic characterization of primary tumors and functional shRNA-screens.

Genomic profiling of tumors has uncovered a staggering diversity of recurrent aberrations. However, inferring functionally important driver genes from this data remains difficult_particularly in the case of copy-number aberrations (CNAs) that often span many genes. Genome-wide functional shRNA screens have been a useful orthogonal approach for discovering drivers. The integration of observational data from primary tumors with functional data on cell lines provides an unprecedented opportunity for the identification of driver genes. Unfortunately, most current analysis is limited to naive intersection of top scoring candidates and thus more powerful computational methods are needed. We have developed Helios, a novel Bayesian algorithm that integrates genomic data from primary tumors with functional shRNA screens in cell lines, gaining unprecedented sensitivity and specificity in identifying drivers. Applying Helios to TCGA breast cancer data led to the recapitulation of many known oncogenes as well as to the identification and validation of two novel oncogenes involved in chromatin regulation. Importantly, many of the drivers pinpointed by Helios were not identified on the basis of any one data type alone. Helios uses shRNA data in a novel fashion by employing a new score measuring oncogene addiction, a phenotype associated with many key cancer drivers. It integrates this with CNA, sequence mutation, and RNA expression data into a single probabilistic score for each gene which is then used to assess the most-likely driver gene in a region of recurrent CNA. We applied Helios to TCGA breast cancer data paired with two independent genome-wide shRNA screens on breast cancer cell lines. This identified many previously known oncogenes including FOXA1, ERBB2, PIK3CA, CCND1, IGF1R, BCL2, CDK4, ESR1, MYC, EGFR, GAB1, CCNE1, FGFR2, FGFR3, MYC as the top-scoring candidates in their respective amplified regions. We validated a number of novel predictions in vitro and selected two candidate oncogenes involved in chromatin remodeling for in depth follow up. The contribution of both genes to cancer was confirmed in vitro and in vivo, enhancing colony formation in agar and increasing tumor size in mouse models. One novel oncogene showed evidence of association with invasion and metastasis in a lung-cancer model. Another novel oncogene resides in a frequently amplified region in several epithelial cancers such as lung, bladder, stomach and ovarian carcinomas. Taken together, we have demonstrated that Helios is a powerful “in-silico” screen that can accelerate discovery of driver mutations in cancer. Citation Format: Felix Sanchez Garcia, Patricia Villagrasa, Junji Matsui, Bo-Juen Chen, Dylan Kotliar, Veronica Castro, Jose M. Silva, Dana Pe9er. Helios identifies novel oncogenes in breast cancer by integrating genomic characterization of primary tumors and functional shRNA-screens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3168. doi:10.1158/1538-7445.AM2013-3168