Reticulocalbin3: A Ca2+ homeostasis regulator that promotes esophageal squamous cell carcinoma progression and cisplatin resistance.

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers worldwide. There is a critical need to identify new mechanisms that contribute to ESCC progression. Reticulocalbin3 (RCN3) is mainly located in the endoplasmic reticulum and Ca2+ -binding protein containing EF-hands. The function of RCN3 in tumor progression has not been clarified. We observed that the expression level of RCN3 was higher in ESCC tissues than in paired normal tissues. Overexpression of RCN3 was positively associated with tumor size, lymph node metastasis, TNM stage, lymphatic vessel infiltration, and poor outcome in patients with ESCC. Increased malignant phenotypes were observed in RCN3 overexpressing ESCC cells, whereas the opposite effects were achieved in RCN3-silenced cells. Reticulocalbin3 promoted the expression of MMP-2 and MMP-9 by regulating the inositol 1,4,5-trisphosphate receptor 1 (IP3R1)-Ca2+ -calcium/calmodulin-dependent protein kinase II-c-Jun signaling pathway. Reticulocalbin3 induced cisplatin resistance by regulating IP3R1/Ca2+ to maintain intracellular Ca2+ homeostasis and reduced reactive oxygen species in ESCC cells. Finally, the expression of RCN3 was regulated by hypoxia inducible factor-1α. Collectively, these data strongly support that RCN3 regulates Ca2+ homeostasis by targeting IP3R1 to promote the progression and platinum resistance of ESCC. Our studies suggest that RCN3 could serve as predictive factor of poor prognosis and potential therapeutic target for ESCC patients.

SRSF9 promotes colorectal cancer progression via stabilizing DSN1 mRNA in an m6A-related manner.

BACKGROUND: Serine/arginine-rich splicing factor 9 (SRSF9) is a classical RNA-binding protein that is essential for regulating gene expression programs through its interaction with target RNA. Whether SRSF9 plays an essential role in colorectal cancer (CRC) progression and can serve as a therapeutic target is largely unknown. Here, we highlight new findings on the role of SRSF9 in CRC progression and elucidate the underlying mechanism. METHODS: CRC cell lines and clinical tissue samples were used. qRT-PCR, Western blotting, immunohistochemistry (IHC), gain- and loss-of-function assays, animal xenograft model studies, bioinformatic analysis, methylated single-stranded RNA affinity assays, gene-specific m6A quantitative qRT-PCR, dual-luciferase reporter assays and RNA stability assays were performed in this study. RESULTS: The expression level of SRSF9 was higher in CRC cell lines than that in an immortal human intestinal epithelial cell line. Overexpression of SRSF9 was positively associated with lymph node metastasis and Dukes stage. Functionally, SRSF9 promoted cell proliferation, migration and invasion in vitro and xenograft growth. The results of bioinformatic analysis indicated that DSN1 was the downstream target of SRSF9. In CRC cells and clinical tissue samples, the expression of SRSF9 was positively associated with the expression of DSN1. Knockdown of DSN1 partially inhibited the SRSF9-induced phenotype in CRC cells. Mechanistically, we further found that SRSF9 is an m6A-binding protein and that m6A modifications were enriched in DSN1 mRNA in CRC cells. Two m6A modification sites (chr20:36773619-36773620 and chr20:36773645-chr20:36773646) in the SRSF9-binding region (chr20:36773597-36773736) of DSN1 mRNA were identified. SRSF9 binds to DSN1 in an m6A motif- and dose-dependent manner. SRSF9 modulates the expression of DSN1 in CRC cells. Such expression regulation was largely impaired upon methyltransferase METTL3 knockdown. Moreover, knockdown of SRSF9 accelerated DSN1 mRNA turnover, while overexpression of SRSF9 stabilized DSN1 mRNA in CRC cells. Such stabilizing was also weakened upon METTL3 knockdown. CONCLUSION: Overexpression of SRSF9 was associated with lymph node metastasis and Dukes stage in CRC. Knockdown of DSN1 eliminated the effects by SRSF9 overexpression in CRC. Our results indicated that SRSF9 functions as an m6A-binding protein (termed "reader") by enhancing the stability of DSN1 mRNA in m6A-related manner. Our study is the first to report that SRSF9-mediated m6A recognition has a crucial role in CRC progression, and highlights SRSF9 as a potential therapeutic target for CRC management.

Downregulation of TUSC3 promotes EMT and hepatocellular carcinoma progression through LIPC/AKT axis.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and malignant tumors in the digestive tract. Tumor Suppressor Candidate 3 (TUSC3) is one subunit of the endoplasmic reticulum Oligosaccharyl transferase (OST) complex, which plays an important role in N-glycosylation during the protein folding process. However, the role of TUSC3 in the initiation and progression of HCC has not been mentioned yet. In the present study, we aim to investigate the effects of TUSC3 on the initiation and progression of HCC. METHODS: Immunohistochemical assay and qRT-PCR were used to detect the expression of TUSC3 and lipase C hepatic type (LIPC) in HCC tissue and cells. Loss-of-function and gain-of-function were applied to detect the function of TUSC3 and LIPC in vivo and in vitro. Immunofluorescence assay and co-immunoprecipitation were used to detect the relationship between TUSC3 and LPC. Western blot was applied to detect the expression of epithelial-mesenchymal transition (EMT) markers and the Akt signaling pathway. RESULTS: TUSC3 was aberrantly decreased in hepatocellular carcinoma tissues compared to the matched adjacent normal tissues, which resulted in bigger size of tumor (P = 0.001, Table 2), worse differentiation (P = 0.006, Table 2) and an advanced BCLC stage. Down-regulation of TUSC3 led to the enhanced proliferation and migration of hepatocellular carcinoma cells in vivo and vitro, whereas the opposite effect could be observed in the TUSC3-overexpression group. The analysis of TUSC3 microarray showed that LIPC, a glycoprotein primarily synthesized and secreted by hepatocytes, was a downstream target of TUSC3, and it negatively modulated the development of HCC. The morphological changes in HCC cells indicated that TUSC3 regulated the epithelial-mesenchymal transition (EMT). Mechanistically, TUSC3 inhibited EMT progression through the LIPC/AKT axis. CONCLUSION: Down-regulation of TUSC3 promotes EMT progression by activating AKT signaling via targeting LIPC in HCC, which is probably the possible mechanism driving TUSC3-deficient hepatocellular carcinoma cells toward a malignant phenotype.

TUSC3 induces drug resistance and cellular stemness via Hedgehog signaling pathway in colorectal cancer.

Tumor suppressor candidate 3 (TUSC3) is a coding gene responsible for N-glycosylation of many critical proteins. TUSC3 gene plays an oncogenic role in colorectal cancer (CRC), however, the role of TUSC3 in drug resistance of CRC is still unclear. The aim of this study is to investigate the biological function and molecular mechanism of TUSC3 in CRC drug resistance. The expression of TUSC3 in CRC is positively correlated to tumor stage in 90 paired clinical samples, and negatively associated with overall survival and disease-free survival of CRC patients. In vitro, TUSC3 promotes the formation of stemness and induces the drug resistance to 5-fluorouracil and cis-dichlorodiammineplatinum(II) in CRC cells. The tissue microarray assay and bioinformatic analysis indicate that TUSC3 may promote the expression of CD133 and ABCC1 via Hedgehog signaling pathway. Treatment of Hedgehog signaling pathway agonist or inhibitor in TUSC3-silenced or TUSC3-overexpressed cells reverse the effects of TUSC3 in cellular stemness phenotype and drug resistance. Meanwhile, coimmunoprecipitation and immunofluorescence assays indicate a tight relationship between TUSC3 and SMO protein. Our data suggest that TUSC3 promotes the formation of cellular stemness and induces drug resistance via Hedgehog signaling pathway in CRC.

LTBP1 promotes esophageal squamous cell carcinoma progression through epithelial-mesenchymal transition and cancer-associated fibroblasts transformation.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers worldwide. Due to its high morbidity and mortality rates, it is urgent to find a molecular target that contributes to esophageal carcinogenesis and progression. In this research, we aimed to investigate the functions of Latent transforming growth factor ß binding protein 1(LTBP1) in ESCC progression and elucidate the underlying mechanisms. METHODS: The tandem mass tag-based quantitative proteomic approach was applied to screen the differentially expressed proteins (DEPs) between 3 cases of ESCC tumor samples and paired normal tissues. Then the DEPs were validated in human ESCC tissues using western blot assays and GEPIA database respectively. The expression level of LTBP1 was detected in 152 cases of ESCC tissues and paired normal tissues. Loss-of-function assays were performed to detect the function of LTBP1 in vivo and in vitro. Immunofluorescence and Western blot assays were used to detect the expression of apoptosis, epithelial-mesenchymal transition (EMT) and cancer-associated fibroblasts (CAFs) markers. RESULTS: A total of 39 proteins were screened to be up-regulated (ratio > 2.0) in all three ESCC tissues. The results of immunohistochemistry assays indicated that the expression level of LTBP1 was higher in ESCC tissues than that in paired normal tissues (p < 0.001). Overexpression of LTBP1 was positively associated with lymphatic metastasis in ESCC (p = 0.002). Down-regulation of LTBP1 inhibited the invasion and migration as well as metastatic abilities in vitro and in vivo. It was also observed the down-regulation of LTBP1 not only decreased the mesenchymal phenotypes but also inhibited TGFß-induced EMT in ESCC cells. We further found that down-regulation of LTBP1 enhanced ESCC cells' sensitivity to 5-FU treatment. Inhibition of LTBP1 expression could also attenuate induction of CAFs transformation and restrain fibroblast express fibronectin (FN1) in ESCC cells. CONCLUSION: Overexpression of LTBP1 was associated with lymph node metastasis in ESCC. Our results indicated that LTBP1 not only increased the malignant behaviors of ESCC cells but also induced EMT and CAFs transformation. Our studies suggested an oncogenic role of LTBP1 in ESCC progression and it may serve as a potential therapeutic target for ESCC patients.

LIPG: an inflammation and cancer modulator.

Endothelial lipase (LIPG/EL) performs fundamental and vital roles in the human body, including cell composition, cytokine expression, and energy provision. Since LIPG predominantly functions as a phospholipase as well as presents low levels of triglyceride lipase activity, it plays an essential role in lipoprotein metabolism, and involves in the metabolic syndromes such as inflammatory response and atherosclerosis. Cytokines significantly affect LIPG expression in endothelial cells in many diseases. Recently, it is suggested that LIPG contributes to cancer initiation and progression, and LIPG attached increasing importance to its potential for future targeted therapy.