Role of the E3 ubiquitin-ligase Hakai in intestinal inflammation and cancer bowel disease.

The E3 ubiquitin-ligases are important for cellular protein homeostasis and their deregulation is implicated in cancer. The E3 ubiquitin-ligase Hakai is involved in tumour progression and metastasis, through the regulation of the tumour suppressor E-cadherin. Hakai is overexpressed in colon cancer, however, the implication in colitis-associated cancer is unknown. Here, we investigated the potential role of Hakai in intestinal inflammation and cancer bowel disease. Several mouse models of colitis and associated cancer were used to analyse Hakai expression by immunohistochemistry. We also analysed Hakai expression in patients with inflamed colon biopsies from ulcerative colitis and Crohn's disease. By Hakai interactome analysis, it was identified Fatty Acid Synthase (FASN) as a novel Hakai-interacting protein. Moreover, we show that Hakai induces FASN ubiquitination and degradation via lysosome, thus regulating FASN-mediated lipid accumulation. An inverse expression of FASN and Hakai was detected in inflammatory AOM/DSS mouse model. In conclusion, Hakai regulates FASN ubiquitination and degradation, resulting in the regulation of FASN-mediated lipid accumulation, which is associated to the development of inflammatory bowel disease. The interaction between Hakai and FASN may be an important mechanism for the homeostasis of intestinal barrier function and in the pathogenesis of this disease.

Regulation of Epithelial-Mesenchymal Plasticity by the E3 Ubiquitin-Ligases in Cancer.

The epithelial-mesenchymal plasticity (EMP) is a process by which epithelial cells acquire the ability to dynamically switch between epithelial and mesenchymal phenotypic cellular states. Epithelial cell plasticity in the context of an epithelial-to-mesenchymal transition (EMT) confers increased cell motility, invasiveness and the ability to disseminate to distant sites and form metastasis. The modulation of molecularly defined targets involved in this process has become an attractive therapeutic strategy against cancer. Protein degradation carried out by ubiquitination has gained attention as it can selectively degrade proteins of interest. In the ubiquitination reaction, the E3 ubiquitin-ligases are responsible for the specific binding of ubiquitin to a small subset of target proteins, and are considered promising anticancer drug targets. In this review, we summarize the role of the E3 ubiquitin-ligases that control targeted protein degradation in cancer-EMT, and we highlight the potential use of the E3 ubiquitin-ligases as drug targets for the development of small-molecule drugs against cancer.

Hakin-1, a New Specific Small-Molecule Inhibitor for the E3 Ubiquitin-Ligase Hakai, Inhibits Carcinoma Growth and Progression.

The requirement of the E3 ubiquitin-ligase Hakai for the ubiquitination and subsequent degradation of E-cadherin has been associated with enhanced epithelial-to-mesenchymal transition (EMT), tumour progression and carcinoma metastasis. To date, most of the reported EMT-related inhibitors were not developed for anti-EMT purposes, but indirectly affect EMT. On the other hand, E3 ubiquitin-ligase enzymes have recently emerged as promising therapeutic targets, as their specific inhibition would prevent wider side effects. Given this background, a virtual screening was performed to identify novel specific inhibitors of Hakai, targeted against its phosphotyrosine-binding pocket, where phosphorylated-E-cadherin specifically binds. We selected a candidate inhibitor, Hakin-1, which showed an important effect on Hakai-induced ubiquitination. Hakin-1 also inhibited carcinoma growth and tumour progression both in vitro, in colorectal cancer cell lines, and in vivo, in a tumour xenograft mouse model, without apparent systemic toxicity in mice. Our results show for the first time that a small molecule putatively targeting the E3 ubiquitin-ligase Hakai inhibits Hakai-dependent ubiquitination of E-cadherin, having an impact on the EMT process. This represents an important step forward in a future development of an effective therapeutic drug to prevent or inhibit carcinoma tumour progression.

Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability.

The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90.

In Vitro Anti-proliferative and Anti-invasive Effect of Polysaccharide-rich Extracts from Trametes Versicolor and Grifola Frondosa in Colon Cancer Cells.

Colorectal cancer (CRC) is one of leading causes of mortality in western countries and novel treatment strategies are required. The medicinal application of mushrooms has been used in traditional medicine in many oriental countries. Polysaccharide-rich extracts obtained from certain medicinal mushroom species have shown antitumor effects in different experimental models. In the present study, we have developed polysaccharide-rich extracts from Trametes versicolor (TV) and Grifola frondosa (GF) fruit bodies. We aim to evaluate the anticancer effects of these polysaccharide-rich extracts in LoVo and HT-29 human colon cancer cells. The in vitro effects were determined by cytotoxicity assay, proliferation assay, wound healing assay and invasion assay. Moreover, the effect on anchorage independent-cell growth was also determined. Our results showed that TV and GF extracts did inhibit human colon cell proliferation and induce cytotoxicity. Furthermore, both fungal extracts significantly inhibited oncogenic potential, cell migration and invasion in colon cancer cells. In addition, extracts induce a more epithelial phenotype, observed by phase contrast images, together with an increase expression of the E-cadherin epithelial marker, detected by western-blotting analyses. Moreover, by using gelatin zymography assays, it was detected a decrease of MMP-2 enzyme activity, a crucial metalloproteinase important for the degradation of the extracellular matrix. Finally, the combination of the extracts with one the most clinical used agents for colorectal cancer, 5-fluorouracil, increases cell cytotoxicity. Taken together our results underscore a potential antitumor effect of polysaccharide-rich extracts obtained from TV and GF in human colon cancer cells lines. These finding may contribute to the reported health effects of fungal extracts.

Hakai overexpression effectively induces tumour progression and metastasis in vivo.

At early stages of carcinoma progression, epithelial cells undergo a program named epithelial-to-mesenchymal transition characterized by the loss of the major component of the adherens junctions, E-cadherin, which in consequence causes the disruption of cell-cell contacts. Hakai is an E3 ubiquitin-ligase that binds to E-cadherin in a phosphorylated-dependent manner and induces its degradation; thus modulating cell adhesions. Here, we show that Hakai expression is gradually increased in adenoma and in different TNM stages (I-IV) from colon adenocarcinomas compared to human colon healthy tissues. Moreover, we confirm that Hakai overexpression in epithelial cells drives transformation in cells, a mesenchymal and invasive phenotype, accompanied by the downregulation of E-cadherin and the upregulation of N-cadherin, and an increased proliferation and an oncogenic potential. More importantly, for the first time, we have studied the role of Hakai during cancer progression in vivo. We show that Hakai-transformed MDCK cells dramatically induce tumour growth and local invasion in nude mice and tumour cells exhibit a mesenchymal phenotype. Furthermore, we have detected the presence of micrometastasis in the lung mice, further confirming Hakai role during tumour metastasis in vivo. These results lead to the consideration of Hakai as a potential new therapeutic target to block tumour development and metastasis.

Proteomic Analysis of the E3 Ubiquitin-Ligase Hakai Highlights a Role in Plasticity of the Cytoskeleton Dynamics and in the Proteasome System.

Carcinoma, the most common type of cancer, arises from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell-cell contacts. E-cadherin is a tumor suppressor, and it is down-regulated during epithelial-to-mesenchymal transition (EMT); indeed, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis and finally degradation, leading the alterations of cell-cell contacts. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Moreover, a profound decreased expression in several proteasome subunits during Hakai-driven EMT was highlighted. Since proteasome inhibitors are becoming increasingly used in cancer treatment, our findings suggest that the E3 ubiquitin-ligase, such as Hakai, may be a better target than proteasome for using novel specific inhibitors in tumor subtypes that follow EMT.