Use of STAT6 Phosphorylation Inhibitor and Trimethylglycine as New Adjuvant Therapies for 5-Fluorouracil in Colitis-Associated Tumorigenesis.

Colorectal cancer (CRC) is one of the most widespread and deadly types of neoplasia around the world, where the inflammatory microenvironment has critical importance in the process of tumor growth, metastasis, and drug resistance. Despite its limited effectiveness, 5-fluorouracil (5-FU) is the main drug utilized for CRC treatment. The combination of 5-FU with other agents modestly increases its effectiveness in patients. Here, we evaluated the anti-inflammatory Trimethylglycine and the Signal transducer and activator of transcription (STAT6) inhibitor AS1517499, as possible adjuvants to 5-FU in already established cancers, using a model of colitis-associated colon cancer (CAC). We found that these adjuvant therapies induced a remarkable reduction of tumor growth when administrated together with 5-FU, correlating with a reduction in STAT6-phosphorylation. This reduction upgraded the effect of 5-FU by increasing both levels of apoptosis and markers of cell adhesion such as E-cadherin, whereas decreased epithelial-mesenchymal transition markers were associated with aggressive phenotypes and drug resistance, such as ß-catenin nuclear translocation and Zinc finger protein SNAI1 (SNAI1). Additionally, Il-10, Tgf-ß, and Il-17a, critical pro-tumorigenic cytokines, were downmodulated in the colon by these adjuvant therapies. In vitro assays on human colon cancer cells showed that Trimethylglycine also reduced STAT6-phosphorylation. Our study is relatively unique in focusing on the effects of the combined administration of AS1517499 and Trimethylglycine together with 5-FU on already established CAC which synergizes to markedly reduce the colon tumor load. Together, these data point to STAT6 as a valuable target for adjuvant therapy in colon cancer.

Helminth-derived molecules inhibit colitis-associated colon cancer development through NF-κB and STAT3 regulation.

Inflammation is currently considered a hallmark of cancer and plays a decisive role in different stages of tumorigenesis, including initiation, promotion, progression, metastasis and resistance to antitumor therapies. Colorectal cancer is a disease widely associated with local chronic inflammation. Additionally, extrinsic factors such as infection may beneficially or detrimentally alter cancer progression. Several reports have noted the ability of various parasitic infections to modulate cancer development, favoring tumor progression in many cases and inhibiting tumorigenesis in others. The aim of our study was to determine the effects of excreted/secreted products of the helminth Taenia crassiceps (TcES) as a treatment in a murine model of colitis-associated colon cancer (CAC). Here, we found that after inducing CAC, treatment with TcES was able to reduce inflammatory cytokines such as IL-1ß, TNF-α, IL-33 and IL-17 and significantly attenuate colon tumorigenesis. This effect was associated with the inhibition of signal transducer and activator of transcription 3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation. Furthermore, we determined that TcES interfered with LPS-induced NF-κB p65 activation in human colonic epithelial cell lines in a Raf-1 proto-oncogene-dependent manner. Moreover, in three-dimensional cultures, TcES promoted reorganization of the actin cytoskeleton, altering cell morphology and forming colonospheres, features associated with a low grade of aggressiveness. Our study demonstrates a remarkable effect of helminth-derived molecules on suppressing ongoing colorectal cancer by downregulating proinflammatory and protumorigenic signaling pathways.

Early and Partial Reduction in CD4+Foxp3+ Regulatory T Cells during Colitis-Associated Colon Cancer Induces CD4+ and CD8+ T Cell Activation Inhibiting Tumorigenesis.

Colorectal cancer (CRC) is the second most commonly diagnosed cancer in women and the third in men in North America and Europe. CRC is associated with inflammatory responses in which intestinal pathology is caused by different cell populations including a T cell dysregulation that concludes in an imbalance between activated T (Tact) and regulatory T (Treg) cells. Treg cells are CD4+Foxp3+ cells that actively suppress pathological and physiological immune responses, contributing to the maintenance of immune homeostasis. A tumor-promoting function for Treg cells has been suggested in CRC, but the kinetics of Treg cells during CRC development are poorly known. Therefore, using a mouse model of colitis-associated colon cancer (CAC) induced by azoxymethane and dextran sodium sulfate, we observed the dynamic and differential kinetics of Treg cells in blood, spleen and mesenteric lymph nodes (MLNs) as CAC progresses, highlighting a significant reduction in Treg cells in blood and spleen during early CAC development, whereas increasing percentages of Treg cells were detected in late stages in MLNs. Interestingly, when Treg cells were decreased, Tact cells were increased and vice versa. Treg cells from late stages of CAC displayed an activated phenotype by expressing PD1, CD127 and Tim-3, suggesting an increased suppressive capacity. Suppression assays showed that T-CD4+ and T-CD8+ cells were suppressed more efficiently by MLN Treg cells from CAC animals. Finally, an antibody-mediated reduction in Treg cells during early CAC development resulted in a better prognostic value, because animals showed a reduction in tumor progression associated with an increased percentage of activated CD4+CD25+Foxp3- and CD8+CD25+ T cells in MLNs, suggesting that Treg cells suppress T cell activation at early steps during CAC development.

Lack of STAT6 Attenuates Inflammation and Drives Protection against Early Steps of Colitis-Associated Colon Cancer.

Colitis-associated colon cancer (CAC) is one of the most common malignant neoplasms and a leading cause of death. The immunologic factors associated with CAC development are not completely understood. Signal transducer and activator of transcription 6 (STAT6) is part of an important signaling pathway for modulating intestinal immune function and homeostasis. However, the role of STAT6 in colon cancer progression is unclear. Following CAC induction in wild-type (WT) and STAT6-deficient mice (STAT6-/-), we found that 70% of STAT6-/- mice were tumor-free after 8 weeks, whereas 100% of WT mice developed tumors. STAT6-/- mice displayed fewer and smaller colorectal tumors than WT mice; this reduced tumorigenicity was associated with decreased proliferation and increased apoptosis in the colonic mucosa in the early steps of tumor progression. STAT6-/- mice also exhibited reduced inflammation, diminished concentrations COX2 and nuclear ß-catenin protein in the colon, and decreased mRNA expression of IL17A and TNFα, but increased IL10 expression when compared with WT mice. Impaired mucosal expression of CCL9, CCL25, and CXCR2 was also observed. In addition, the number of circulating CD11b+Ly6ChiCCR2+ monocytes and CD11b+Ly6ClowLy6G+ granulocytes was both decreased in a STAT6-dependent manner. Finally, WT mice receiving a STAT6 inhibitor in vivo confirmed a significant reduction in tumor load as well as less intense signs of CAC. Our results demonstrate that STAT6 is critical in the early steps of CAC development for modulating inflammatory responses and controlling cell recruitment and proliferation. Thus, STAT6 may represent a promising target for CAC treatment. Cancer Immunol Res; 5(5); 385-96. ©2017 AACR.