Uddalak Bharadwaj

Colorectal cancer (CRC) occurs with higher frequency in patients with inflammatory bowel disease (IBD) and has increased morbidity and mortality compared with CRC in patients in the general population. STAT3 has been implicated in CRC development, but strategies to target it have yet to be employed to prevent its occurrence in groups at high risk for CRC. Our group developed TTI-101, a small-molecule STAT3 inhibitor, which was effective in treating colitis in the dextran sodium sulfate (DSS) mouse model. In the current study, we examined the ability of TTI-101 to prevent CRC in the azoxymethane (AOM)-DSS mouse model of CRC. Mice received AOM followed by DSS and were treated with either TTI-101 or vehicle control for 10 weeks. While vehicle-treated AOM-DSS mice developed polyps and adenocarcinomas, TTI-101-treated AOM-DSS mice did not. Levels of activated STAT3 (pY-STAT3) were increased in both the epithelial and stromal compartments of adenocarcinomas vs. normal colon mucosa and correlated with adenocarcinoma burden. Pharmacologically relevant concentrations of TTI-101 were detected in plasma and colon; plasma levels correlated with colon levels and correlated inversely with adenocarcinoma number. Transcriptomic analyses revealed that TTI-101 normalized expression of AOM-DSS-induced CRC-associated genes in the colon, many of which are regulated by STAT3. The addition of DSS to AOM resulted in a distinct cecal microbiome diversity and composition that was muted by the addition of TTI-101. Thus, TTI-101 prevented colitis-associated CRC in the AOM-DSS model through targeting STAT3’s pro-oncogenic effects on the colon transcriptome and modulating colitis-associated dysbiosis; targeting STAT3 in patients with IBD merits consideration for CRC prevention, as well as IBD treatment.

Background and Aims: Inflammatory bowel disease (IBD) predisposes to colorectal cancer (CRC). In the current studies, we used the dextran sodium sulfate (DSS) murine model of colitis, which is widely used in preclinical studies, to determine the contribution of STAT3 to IBD. STAT3 has two isoforms: (STAT3 α; which has pro-inflammatory and anti-apoptotic functions, and STAT3β; which attenuates the effects of STAT3α). In the current study, we determined the contribution of STAT3 to IBD across all tissues by examining DSS-induced colitis in mice that express only STAT3α and in mice treated with TTI-101, a direct small-molecule inhibitor of both isoforms of STAT3. Methods: We examined mortality, weight loss, rectal bleeding, diarrhea, colon shortening, apoptosis of colonic CD4+ T-cells, and colon infiltration with IL-17-producing cells following 7-day administration of DSS (5%) to transgenic STAT3α knock-in (STAT3β-deficient; ΔβΔβ) mice and wild-type (WT) littermate cage control mice. We also examined the effect of TTI-101 on these endpoints in DSS-induced colitis in WT mice. Results: Each of the clinical manifestations of DSS-induced colitis examined was exacerbated in ΔβΔβ transgenic versus cage-control WT mice. Importantly, TTI-101 treatment of DSS-administered WT mice led to complete attenuation of each of the clinical manifestations and also led to increased apoptosis of colonic CD4+ T cells, reduced colon infiltration with IL-17-producing cells, and down-modulation of colon mRNA levels of STAT3-upregulated genes involved in inflammation, apoptosis resistance, and colorectal cancer metastases. Conclusions: Thus, small-molecule targeting of STAT3 may be of benefit in treating IBD and preventing IBD-associated colorectal cancer.

STAT3 mutations, predominantly in the DNA-binding domain (DBD) and Src-homology 2 domain (SH2D), cause rare cases of immunodeficiency, malignancy, and autoimmunity. The exact mechanisms by which these mutations abrogate or enhance STAT3 function are not completely understood. Here, we examined how loss-of-function (LOF) and gain-of-function (GOF) STAT3 mutations within the DBD and SH2D affect monomer and homodimer protein stability as well as their effect on key STAT3 activation events, including recruitment to phosphotyrosine (pY) sites within peptide hormone receptors, tyrosine phosphorylation at Y705, dimerization, nuclear translocation, and DNA binding. The DBD LOF mutants showed reduced DNA binding when homodimerized, whereas the DBD GOF mutants showed increased DNA binding. DBD LOF and GOF mutants showed minimal changes in other STAT3 functions or in monomer or homodimer protein stability. However, SH2D LOF mutants demonstrated reduced conformational stability as either monomers or homodimers, leading to decreased pY-peptide recruitment, tyrosine phosphorylation, dimerization, nuclear localization, and DNA binding. In contrast, cancer-causing SH2D GOF mutants showed increased STAT3 homodimer stability, which increased their DNA binding. Of note, a small-molecule inhibitor of STAT3 that targets the tyrosine phosphopeptide–binding pocket within the STAT3 SH2D potently inhibited cell proliferation driven by STAT3 SH2D GOF mutants. These findings indicate that the stability of STAT3 protein monomer and homodimer is critical for the pathogenesis of diseases caused by SH2D LOF and GOF mutations and suggest that agents that modulate STAT3 monomer and/or homodimer protein stability may have therapeutic value in diseases caused by these mutations.

Signal transducer and activator of transcription (STAT) 3 plays a central role in the host response to injury. It is activated rapidly within cells by many cytokines, most notably those in the IL-6 family, leading to pro-proliferative and pro-survival programs that assist the host in regaining homeostasis. With persistent activation, however, chronic inflammation and fibrosis ensue, leading to a number of debilitating diseases. This review summarizes advances in our understanding of the role of STAT3 and its targeting in diseases marked by chronic inflammation and/or fibrosis with a focus on those with the largest unmet medical need.

Pancreatic cancer is the fourth leading cause of cancer death with a 5-year survival less than 5 percent despite rigorous interventions. This largely is due to its late presentation, aggressive metastasis, and a lack of effective adjuvant therapies. Cytokines have been studied in many tumor types, where they have been shown to be an important influence in cancer cell behavior and to have potential as tumor markers, therapeutic targets, or as treatments themselves. Recently, the roles cytokines play in pancreatic cancer have become the subject of intense investigation. However, the story is complicated, largely because of the pleiotropic and overlapping nature of cytokine functions. This article attempts to provide a focused review of recent discoveries in this area by organizing the material along the pathophysiologic tasks a cancer cell must accomplish to achieve malignancy. We examined how cytokines act to create a microenvironment conducive to tumor cell survival and growth, discussed how cytokines affect proliferation, invasion, metastasis, and apoptosis in pancreatic cancer cells, and we summarized how this knowledge has been applied either to target cytokines or use them therapeutically.

We have studied variation at 24 microsatellite markers among 50 individuals from each of three endogamous groups, Bhargavas, Chaturvedis, and non-Bhargava, non-Chaturvedi Brahmins of Uttar Pradesh, India. The number of alleles at the loci tested varied from 4 to 11, with an average of 6 at each locus. Heterozygosity was found to be quite high at all loci in the three subpopulations. It varied between 0.44 to 0.84 among Bhargavas (average 0.6510), 0.44 to 0.80 among Chaturvedis(average 0.6633±), and 0.42 to 0.85 among Brahmins (average 6.694±). Hardy-Weinberg equilibrium analysis revealed that these populations are under genetic equilibrium at almost all the loci tested. Comparisons of allele frequency between Bhargavasand Chaturvedis showed that they differed significantly at 14 short tandem repeat (STR) markers (p < 0.001), while Chaturvedis and Brahmins differed at 6 (p < 0.05) and Brahmins and Bhargavas at 8 (p < 0.05). Average FIS and FST for the 24 STR markers was -0.02 and 0.013, respectively. We used both unweighted pair group with arithmetic mean and principal components analysis to evaluate genetic distances among the three groups. Our results revealed that although there were differences at particular allele frequencies between Bhargavas vs. Brahmins, Bhargavas vs. Chaturvedis, and Brahmins vs. Chaturvedis, these differences were not statistically significant when combined over all 24 STR markers between Chaturvedis vs. Brahmins and Bhargavas vs. Brahmins. The genetic distance analysis revealed that Bhargavas are slightly apart from the other two populations.