Molecular Modulators of the Oncogenic Transcription Factor STAT3

Abstract

Since the neoplastic phenotype of a cell is largely driven by aberrant gene expression patterns, increasing attention has been focused on transcription factors that regulate critical mediators of tumorigenesis such as signal transducer and activator of transcription 3 (STAT3). Here we investigate how the inappropriate activation of STAT3 contributes to cancer pathogenesis and how it can be targeted therapeutically. As proteins that interact with STAT3 may be key in addressing these questions, we took three complementary approaches: a proteomics approach to identify novel STAT3-interacting proteins, a chemical biology approach to identify STAT3-interacting proteins critical for oncogenesis, and molecular analysis of a STAT3 structural domain known to mediate protein-protein interactions. First, we performed mass spectrometry on STAT3-containing complexes from breast cancer cells that have constitutively active STAT3 and are dependent on STAT3 function. We identified granulin as a novel STAT3-interacting protein that is critical to STAT3 transcriptional activity and STAT3-mediated tumorigenic phenotypes. Furthermore, granulin expression positively correlated with STAT3 gene expression signatures in breast cancer patients. We then applied this mass spectrometry approach to investigate the mechanism of two small molecules – ST3-01 and Pyrimethamine (Pyr) – identified by transcription-based reporter screens to inhibit STAT3 activity without altering its activation or nuclear localization. ST3-01 and Pyr reduced STAT3 interaction with the chromatin remodeler, BRG1, which we found to be necessary for STAT3 function. Next, we studied the role of the STAT3 N-terminal domain (NTD), which mediates interactions between two STAT3 dimers for cooperative DNA binding. We identified STAT3 target genes dependent on the NTD for transcriptional regulation. We then showed that NTD mutations which inhibit cooperative DNA binding reduced the induction of a subset of STAT3 target genes by decreasing STAT3 binding to their regulatory regions. These studies demonstrate that a proteomics approach can reveal critical modulators of transcription factor function. Moreover, our characterization of the impact of the STAT3 NTD on STAT3-dependent activity provides a deeper mechanistic understanding of STAT3 signaling as well as a structural template for drug design. Collectively, these insights into how STAT3 protein-protein interactions modulate its transcriptional function may guide future therapies that target this oncogenic signaling pathway.