Metastatic colon cancer is a major cause of deaths among colorectal cancer (CRC) patients. Elevated expression of kallikrein 6 (KLK6), a member of a kallikrein subfamily of peptidase S1 family serine proteases, has been reported in CRC and is associated with low patient survival rates and poor disease prognosis. We knocked down KLK6 expression in HCT116 colon cancer cells to determine the significance of KLK6 expression for metastatic dissemination and to identify the KLK6-associated microRNAs (miRNAs) signaling networks in metastatic colon cancer. KLK6 suppression resulted in decreased cells invasion in vitro with a minimal effect on the cell growth and viability. In vivo, animals with orthotopic colon tumors deficient in KLK6 expression had the statistically significant increase in survival rates (P = .005) and decrease in incidence of distant metastases. We further performed the integrated miRNA and messenger RNA (mRNA) expression profiling to identify functional miRNA-mRNA interactions associated with KLK6-mediated invasiveness of colon cancer.Through bioinformatics analysis we identified and functionally validated the top two up-regulated miRNAs, miR-182 and miR-203, and one down-regulated miRNA, miRNA-181d, and their seven mRNA effectors. The established miRNA-mRNA interactions modulate cellular proliferation, differentiation and epithelial–mesenchymal transition (EMT) in KLK6-expressing colon cancer cells via the TGF-β signaling pathway and RAS-related GTP-binding proteins. We confirmed the potential tumor suppressive properties of miR-181d and miR-203 in KLK6-expressing HCT116 cells using Matrigel invasion assay. Our data provide experimental evidence that KLK6 controls metastasis formation in colon cancer via specific downstream network of miRNA-mRNA effectors.

The PGE2 pathway is important in inflammation-driven diseases and specific targeting of the inducible mPGES-1 is warranted due to the cardiovascular problems associated with the long-term use of COX-2 inhibitors. This review focuses on patents issued on methods of measuring mPGES-1 activity, on drugs targeting mPGES-1 and on other modulators of free extracellular PGE2 concentration. Perspectives and conclusions regarding the status of these drugs are also presented. Importantly, no selective inhibitors targeting mPGES-1 have been identified and, despite the high number of published patents, none of these drugs have yet made it to clinical trials.

Kallikrein-related peptidase 6 (KLK6) overexpression is commonly observed in primary tumors of colorectal cancer (CRC) patients and has been associated with tumor aggressiveness, metastasis, and poor prognosis. We previously established a unique contribution of KLK6 in colon cancer metastasis via a specific network of microRNAs and mRNAs. Here we evaluated the cellular functions of KLK6 protease in Caco-2 colon adenocarcinoma cell line after introduction of the enzymatically active or inactive form of the enzyme. We found that proteolytically active KLK6 increased Caco-2 cells invasiveness in vitro and decreased the animal survival in the orthotopic colon cancer model. The active KLK6 induced phosphorylation of SMAD 2/3 proteins leading to the altered expression of the epithelial-mesenchymal transition (EMT) markers. KLK6 overexpression also induced the RNA-binding protein LIN28B and high-mobility group AT-hook 2 (HMGA2) transcription factor, two essential regulators of cell invasion and metastasis. In the CRC patients, KLK6 protein levels were elevated in the non-cancerous distant and adjacent tissues, compared to their paired tumor tissues (p < 0.0001 and p = 0.0157, respectively). Patients with mutant K-RAS tumors had significantly higher level of KLK6 protein in the luminal surface of non-cancerous distant tissue, compared to the corresponding tissues of the patients with K-RAS wild type tumors (p ≤ 0.05). Furthermore, KLK6 and HMGA2 immunohistochemistry (IHC) scores in patients’ tumors and paired adjacent tissues positively correlated (Spearman correlation P < 0.01 and p = 0.03, respectively). These findings demonstrate the critical function of the KLK6 enzyme in colon cancer progression and its contribution to the signaling network in colon cancer.

This article reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel class of compounds capable of reducing production of PGE2 in HCA-7 human adenocarcinoma cells. A total of 28 analogs were synthesized, assayed for PGE2 reduction, and selected active compounds were evaluated for inhibitory activity against COX-2 in a cell free assay. Compound 2xxiv (R1 = H, R2 = p-CH3O) exhibited the most potent activity in cells (EC50 = 0.02 µM) and minimal inhibition of COX-2 activity (3% at 5 µM). Furthermore, the anti-tumor activity of analog 2vii was analyzed in xenograft mouse models exhibiting good anti-cancer activity.

Background: Kallikrein-related peptidase 6 (KLK6) belongs to the family of human tissue kallikrein genes, majority of which are shown to be differentially expressed in cancers. Clinical studies have demonstrated that upregulation of KLK6 in primary colorectal tumors and lymph nodes correlates with serosal invasion, liver metastasis and indicative of poor prognosis in patients. It has been reported that KLK6 protein is involved in regulation of the epithelial-mesenchymal transition (EMT) program in an organ-specific context. The aim in this study was to investigate contribution of KLK6 enzyme in the EMT during neoplastic transformation in the colon. Results: We expressed enzymatically active or inactive KLK6, using pcDNA3.1(+)preproKLK6 and pcDNA3.1(+)preproKLK6 Ser197Ala mutant plasmids, in Caco-2 colon cancer cell line, which has been characterized before as a very low KLK6 expresser with an undetectable secreted KLK6. Stable isogenic clones were selected and further evaluated for their ability to migrate and invade using in vitro assays and to metastasize in vivo using SCID orthotopic mouse model. We found no effect of KLK6 enzyme activity on migration of Caco-2 cells, expressing the empty vector (Caco-2 mock), and Caco-2 cells, expressing an enzymatically active KLK6 (Caco-2 KLK6 wt) or inactive KLK6 (Caco-2 KLK6 mut). But Caco-2 KLK6 wt cells demonstrated the invasive phenotype in Matrigel invasion assays (p&lt;0.001, compared to Caco-2 mock and Caco2 KLK6 mut cells). The Caco-2 mock and Caco-2 KLK6 mut cells, injected in SCID mice orthotopically, developed primary colon tumors but no metastatic lesions were identified. In contrast, Caco-2 KLK6 wt cells formed primary colon tumors and metastasized locally, although they failed to form the distant metastasis (lung and mesentery). Animals, growing the Caco-2 KLK6 wt tumors, displayed a significant decrease in their survival rates, compared to other groups (p=0.02). In Caco-2 KLK6 wt cells TGF-β protein expression and secretion was induced, which resulted in activation of TGF-β-SMAD2/3 signaling pathway. This phenotype was associated with the elevated expression of known regulator of the EMT, zinc-finger protein Snail. In addition, the expression of a high-mobility group AT-hook 2 (HMGA2) protein was induced in Caco-2 KLK6 wt cells. The HMGA2 expression is implicated in the EMT program, acting through the TGF-β signaling pathway and is associated with a poor survival in colorectal cancer. Conclusion. These findings demonstrate that KLK6 enzyme activity is required for colon cancer progression via induction of the EMT program. We identified the TGF-β- signaling pathway as a mechanism driving the EMT in colon cancer cells expressing KLK6 enzyme. Citation Format: Hwudaurw Chen, Earlphia Sells, Haiyan Cui, Ritu Pandey, George Pampalakis, Georgia Sotiropoulou, Thomas Doetschman, Natalia A. Ignatenko. Human tissue Kallikrein 6 enzyme activity regulates epithelial-mesenchymal transition in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 854. doi:10.1158/1538-7445.AM2017-854