ILK overexpression in human CRC associates with EMT and CSC traits, contributing to tumor progression and chemoresistance.

Background/Aims: Nesfatin-1, processed from nucleobindin-2 (NUCB2), is a potent anorexigenic peptide being expressed in rodent hypothalamic nuclei and involved in the regulation of feeding behavior and body weight in animals. The present study aimed to investigate NUCB2/nesfatin-1 protein expression in the human hypothalamus as well as its correlation with body weight. Methods: Sections of hypothalamus and adjacent cholinergic basal forebrain nuclei, including the nucleus basalis of Meynert (NBM) and the diagonal band of Broca (DBB), from 25 autopsy cases (17 males, 8 females; 8 lean, 9 overweight, 8 obese) were examined using immunohistochemistry and double immunofluorescence labeling. Results: Prominent NUCB2/nesfatin-1 immunoexpression was detected in supraoptic, paraventricular, and infundibular nuclei, lateral hypothalamic area (LHA)/perifornical region, and NBM/DBB. NUCB2/nesfatin-1 was found to extensively colocalize with (a) oxytocin and vasopressin in paraventricular and supraoptic nuclei, (b) melanin-concentrating hormone in the LHA, and (c) cocaine- and amphetamine-regulated transcript in infundibular and paraventricular nuclei and LHA. Interestingly, in the LHA, NUCB2/nesfatin-1 protein expression was significantly decreased in obese, compared with lean (p < 0.01) and overweight (p < 0.05) subjects. Conclusions: The findings of the present study are suggestive of a potential role for NUCB2/nesfatin-1 as an integral regulator of food intake and energy homeostasis in the human hypothalamus. In the LHA, an appetite- and reward-related brain area, reduced NUCB2/nesfatin-1 immunoexpression may contribute to dysregulation of homeostatic and/or hedonic feeding behavior and obesity. NUCB2/nesfatin-1 localization in NBM/DBB might imply its participation in the neuronal circuitry controlling cognitive influences on food intake and give impetus towards unraveling additional biological actions of NUCB2/nesfatin-1 in human neuronal networks.

Cofilin phospho-regulation is important for actin filament turnover and is implicated in cancer. Phosphorylation of cofilin is mediated by LIM kinases (LIMKs) and dephosphorylation by Slingshot phosphatases (SSH). LIMKs and SSH promote cancer cell invasion and metastasis and represent novel anti-cancer targets. However, little is known regarding LIMK/cofilin and SSH in human colorectal cancer (CRC). In this study, we aimed to address their expression and significance in human CRC. We evaluated expression of non-phosphorylated (active) and phosphorylated cofilin, LIMK1, LIMK2, and SSH1 by immunohistochemistry in 143 human CRC samples in relation to clinicopathologic parameters, response of metastatic disease to chemotherapy, and epithelial-mesenchymal transition (EMT) markers β-catenin, E-cadherin, and ZEB. We show that active cofilin, LIMK1, LIMK2, and SSH1 are overexpressed in human CRC and are associated with tumor progression parameters. SSH1 is an independent predictor of lymph node metastasis by multivariate analysis. LIMK1 and SSH1 expression is also higher in non-responders to chemotherapy, and SSH1 is shown by multivariate analysis to independently predict response of metastatic disease to chemotherapy. Active cofilin, LIMK1, LIMK2, and SSH1 also correlated with the EMT markers examined. In addition, immunofluorescence analysis showed increased expression of active cofilin, LIMK1, LIMK2, and SSH1 in HT29 colon cancer cells resistant to 5-fluorouracil compared to parental HT29 cells. Our results suggest that F-actin regulators LIMK/cofilin pathway and SSH1 are associated with CRC progression and chemoresistance representing promising tumor biomarkers and therapeutic targets in CRC.

This study aims to address the role of focal adhesion proteins α- and β-parvin in human colorectal carcinoma (CRC). Expression of α- and β-parvin was examined by immunohistochemistry and real-time RT-PCR in a series of human CRC. Parvins were overexpressed in CRC and their expression correlated significantly with tumor invasion, lymph node metastasis, and disease stage. A significant positive correlation of parvins protein expression with overexpression of integrin-linked kinase, p-AKT, and nuclear β-catenin, as well as with downregulation of E-cadherin was also observed. In conclusion, overexpression of α- and β-parvin seems to be implicated in human colorectal cancer progression.

Geminin, a DNA replication licensing inhibitor, ensures faithful DNA replication in vertebrates. Several studies have shown that geminin depletion in vitro results in rereplication and DNA damage, whereas increased expression of geminin has been observed in human cancers. However, conditional inactivation of geminin during embryogenesis has not revealed any detectable DNA replication defects. In order to examine its role in vivo, we conditionally inactivated geminin in the murine colon and lung, and assessed chemically induced carcinogenesis. We show here that mice lacking geminin develop a significantly higher number of tumors and bear a larger tumor burden than sham-treated controls in urethane-induced lung and azoxymethane/dextran sodium sulfate-induced colon carcinogenesis. Survival is also significantly reduced in mice lacking geminin during lung carcinogenesis. A significant increase in the total number and grade of lesions (hyperplasias, adenomas, and carcinomas) was also confirmed by hematoxylin and eosin staining. Moreover, increased genomic aberrations, identified by increased ATR and γH2AX expression, was detected with immunohistochemistry analysis. In addition, we analyzed geminin expression in human colon cancer, and found increased expression, as well as a positive correlation with ATM/ATR levels and a non-monotonic association with γH2AX. Taken together, our data demonstrate that geminin acts as a tumor suppressor by safeguarding genome stability, whereas its overexpression is also associated with genomic instability. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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Chromatin licensing and DNA replication factor 1 (CDT1), a protein of the pre-replicative complex, is essential for loading the minichromosome maintenance complex (MCM) helicases onto the origins of DNA replication. While several studies have shown that dysregulation of CDT1 expression causes re-replication and DNA damage in cell lines, and CDT1 is highly expressed in several human cancers, whether CDT1 deregulation is sufficient to enhance tumorigenesis in vivo is currently unclear. To delineate its role in vivo, we overexpressed Cdt1 in the mouse colon and induced carcinogenesis using azoxymethane/dextran sodium sulfate (AOM/DSS). Here, we show that mice overexpressing Cdt1 develop a significantly higher number of tumors with increased tumor size, and more severe dysplastic changes (high-grade dysplasia), compared with control mice under the same treatment. These tumors exhibited an increased growth rate, while cells overexpressing Cdt1 loaded greater amounts of Mcm2 onto chromatin, demonstrating origin overlicensing. Adenomas overexpressing Cdt1 showed activation of the DNA damage response (DDR), apoptosis, formation of micronuclei, and chromosome segregation errors, indicating that aberrant expression of Cdt1 results in increased genomic and chromosomal instability in vivo, favoring cancer development. In line with these results, high-level expression of CDT1 in human colorectal cancer tissue specimens and colorectal cancer cell lines correlated significantly with increased origin licensing, activation of the DDR, and microsatellite instability (MSI).