Purpose: Intrahepatic cholangiocarcinoma is a fatal primary liver cancer resulting from diagnosis at an advanced stage. Understanding the mechanisms of drug resistance and metastasis of cholangiocarcinoma may improve the disease prognosis. Enhanced aldehyde dehydrogenase (ALDH) activity is suggested to be associated with increased drug resistance and the metastasis. This study aims to investigate the roles of the ALDH isoforms in cholangiocarcinoma. Experimental Design: Aldefluor assays, RT-PCR, and Western blot analysis were used to identify the major ALDH isoforms contributing to Aldefluor activity in human cholangiocarcinoma cell lines. We manipulated isoform expression in HuCCT1 cells to elucidate the role of ALDH1A3 in the malignant progression of these cells. Finally, we used immunohistochemical staining to evaluate the clinical significance of ALDH1A3 in 77 hepatectomized cholangiocarcinoma patients and an additional 31 patients with advanced cholangiocarcinoma who received gemcitabine-based therapy. Results: ALDHhigh cholangiocarcinoma cells not only migrated faster but were more resistant to gemcitabine. Among the 19 ALDH isoforms studied, ALDH1A3 was found to be the main contributor to Aldefluor activity. In addition, we also found that knockdown of ALDH1A3 expression in HuCCT1 cells markedly reduced not only their sensitivity to gemcitabine, which might be attributed to a decreased expression of ribonucleotide reductase M1, but also their migration. Most importantly, this enzyme was also identified as an independent poor prognostic factor for patients with intrahepatic cholangiocarcinoma, as well as a prognostic biomarker of gemcitabine-treated patients. Conclusions: ALDH1A3 plays an important role in enhancing malignant behavior of cholangiocarcinoma and serves as a new therapeutic target. Clin Cancer Res; 22(16); 4225–35. ©2016 AACR.

The identification of the molecular mechanisms controlling the degradation of regulatory proteins in mesenchymal stromal cells (MSC) may provide clues to promote MSC osteogenic differentiation and bone regeneration. Ubiquitin ligase-dependent degradation of proteins is an important process governing cell fate. In this study, we investigated the role of the E3 ubiquitin ligase c-Cbl in MSC osteoblast differentiation and identified the mechanisms involved in this effect. Using distinct shRNA targeting c-Cbl, we showed that c-Cbl silencing promotes osteoblast differentiation in murine and human MSC, as demonstrated by increased alkaline phosphatase activity, expression of phenotypic osteoblast marker genes (RUNX2, ALP, type 1 collagen), and matrix mineralization in vitro. Coimmunoprecipitation analyses showed that c-Cbl interacts with the transcription factor STAT5, and that STAT5 forms a complex with RUNX2, a master transcription factor controlling osteoblastogenesis. Silencing c-Cbl decreased c-Cblmediated STAT5 ubiquitination, increased STAT5 protein level and phosphorylation, and enhanced STAT5 and RUNX2 transcriptional activity. The expression of insulin like growth factor-1 (IGF-1), a target gene of STAT5, was increased by c-Cbl silencing in MSC and in bone marrow stromal cells isolated from c-Cbl deficient mice, suggesting that IGF-1 contributes to osteoblast differentiation induced by c-Cbl silencing in MSC. Consistent with these findings, pharmacological inhibition of STAT5 activity, or neutralization of IGF-1 activity, abrogated the positive effect of c-Cbl knockdown on MSC osteogenic differentiation. Taken together, the data provide a novel functional mechanism by which the ubiquitin ligase c-Cbl regulates the osteoblastic differentiation program in mesenchymal cells by controlling Cbl-mediated STAT5 degradation and activity.

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