Abstract PR11: Characterizing the mechanism and the clinical relevance of the synthetic lethal interaction between STAT3 inhibition and HER2 overexpression in breast cancers
Authors: Rodriguez-Barrueco Ruth , Yu Jiyang , Alvarez Mariano , Castro Veronica , Villagrasa Patricia , Marshall Netonia , Sun Ruoxi , Castro Laura , Califano Andrea 
Published: August 2014

Abstract
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.