The protein STAT3 (Signal Transducer and Activator of Transcription 3) plays a central role in a range of physiologic process, and when subverted in cancer, can be a central mediator of malignant cellular function. This protein resides at the critical junction between intracellular signaling events and the regulation of genes involved in apoptosis, differentiation, and cell proliferation. The present research has intended to investigate the role of STAT3 in the nuclear compartment 1) as transcription factor in the regulation of a new promoter sequence; 2) as protein hub in modulation of enhanceosomes STAT3-specific in response to different pathways; 3) as protein involved in onset and progression of human prostate cancer.
STAT3 acts as transcription factor in the regulatory region of TPX2 gene coding for a protein involved in the complex process of mitosis, and was identified as one of the microtubule-associated proteins. An inspection of the 5'-flanking region of the human BCL2L1 gene, coding for Bcl-xL, has many potentially binding sites for STAT proteins, but none of these correspond closely to the consensus sequence of Stat3. We identified a high-affinity binding sites for Stat3 located at -4305/-4297 base pairs from the transcription start of TPX2 and had a TTCCCGGAA sequence, which is identical to the sequence bound by activated Stat3 in the promoter of gene CDKN1A, coding for the protein p21WAF1/CIP1. By reporter gene assay, conducted in M14 cells treated with specific phosphopeptidic inhibitor of STAT3, we showed that this protein is recruited on the TPX2 gene promoter and regulated in vivo TPX2 expression.
Whereas STAT3 is tyrosine phosphorylated by three types of kinases, it uses a precise sequence of functional actions by multiple coactivator complexes and post-translational modifications (PTMs) for mediating gene activation. Considering that we found a different set of STAT3-associated proteins in tumoral cell lines in which the activation of STAT3 can be mediated by different pathways, we could speculate not only a tumor-specific, but also a signal-dependent composition of enhanceosome STAT3-specific. Following CoIP, ChIP and RT2-PCR assays, we hypotized a functional interplay between PARP-1 STAT3-associated proteins when the transcription factor was phosphorylated by Src-kinases or after EGF stimulation, instead the association with CBP/P300 is IL-6 inducted by JAK kinases.
In the study of STAT3 partners that affect its function, we demonstrated the importance of PTMs of STAT3 in prostate cancer. In this work we evaluated in parallel, by immunoblotting analysis, the variation of phosphorylation, acetylation and gluthationylation of STAT3 in cell lines and in human prostate tumor (FFPE). To investigate how differences in PTMs of STAT3 can influence gene expression and interactions with coactivators, immunoblottig analysis, Co-IP and ChIP experiments have been conducted in LNCaP and PC3 cells treated with IL-6 and H2O2, to simulate a physiological response to citokines or to oxidative stress. It has been shown that S-gluthationylation of STAT3 and the recruitment of coactivators such ERp57 and Ref-1, two protein involved in redox modification, is a response to oxidative stress, associated with a more advanced state of disease
