Coexpression of HOXA6 and PBX2 promotes metastasis in gastric cancer.

HOXA6 gene plays a role of the oncogene in various cancers. Nonetheless, its effect on gastric cancer (GC) occurrence and development is still unclear. We analysed whether HOXA6 interacts with the PBX2 protein using the STRING database. The molecular mechanism by which HOXA6 synergizes with PBX2 in GC metastasis is not fully understood. Here, we found that the expression of HOXA6 was increased in GC tissues and cell lines. The upregulation of HOXA6 was closely associated with differentiation, lymph node metastasis, AJCC stage, TNM stage, and poor survival outcome in GC patients based on tissue microarray (TMA) data. Moreover, the overexpression of HOXA6 promoted, whereas siRNA-mediated repression of HOXA6 inhibited, the cell proliferation, migration, and invasion of GC cells. Furthermore, HOXA6 could physically interact with and stabilize PBX2. In addition, HOXA6 and PBX2 expression was positively correlated in GC cells and tissue. HOXA6 and PBX2 suppression in GC cells also led to decreased migration and invasion potential in vitro. In vivo, HOXA6 was shown to cooperate with PBX2 to enhance cell metastasis via orthotopic implantation. These data indicate that HOXA6 promotes cell proliferation, migration, and invasion and that the HOXA6-PBX2 axis may be a useful biomarker for disease progression in GC.

The POU2F1/miR-4490/USP22 axis regulates cell proliferation and metastasis in gastric cancer.

PURPOSE: Growing evidence indicates that aberrant expression of microRNAs contributes to tumor development. However, the biological role of microRNA-4490 (miR-4490) in gastric cancer (GC) remains to be clarified. METHODS: To explore the function of miR-4490 in GC, we performed colony formation, EdU incorporation, qRT-PCR, Western blotting, in situ hybridization (ISH), immunohistochemistry (IHC), flow cytometry, ChIP and dual-luciferase reporter assays. In addition, the growth, migration and invasion capacities of GC cells were evaluated. RESULTS: We found that miR-4490 was significantly downregulated in primary GC samples and in GC-derived cell lines compared with normal controls, and that this expression level was negatively correlated with GC malignancy. Exogenous miR-4490 expression not only reduced cell cycle progression and proliferation, but also significantly inhibited GC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, we found that miR-4490 directly targets USP22, which mediates inhibition of GC cell proliferation and EMT-induced metastasis in vitro and in vivo. Moreover, we found through luciferase and ChIP assays that transcription factor POU2F1 can directly bind to POU2F1 binding sites within the miR-4490 and USP22 promoters and, by doing so, modulate their transcription. Spearman's correlation analysis revealed a positive correlation between USP22 and POU2F1 expression and negative correlations between miR-4490 and USP22 as well as miR-4490 and POU2F1 expression in primary GC tissues. CONCLUSION: Based on our results we conclude that miR-4490 acts as a tumor suppressor, and that the POU2F1/miR-4490/USP22 axis plays an important role in the regulation of growth, invasion and EMT of GC cells.

MicroRNA­500a­5p inhibits colorectal cancer cell invasion and epithelial­mesenchymal transition.

The development of malignant tumors is a series of complex processes, the majority of which have not been elucidated. The aim of the present study was to investigate the microRNAs (miRNAs/miR) that affect the migration and invasion abilities of CRC cells. Our previous reports have revealed that miR­500a­5p suppressed CRC cell growth and malignant transformation. The present study demonstrated that overexpression of miR­500a­5p reduced the expression of vimentin, while increasing the expression of E­cadherin. Inhibition of miR­500a­5p resulted in spindle­like morphological changes and reorganization of F­actin in CRC cells. Furthermore, miR­500a­5p attenuated the transforming growth factor­ß signaling pathway in EMT. Additionally, emodin inhibited the miR­500a­5p inhibitor and suppressed the EMT process. In animal models of metastasis using nude mice, EMT and LoVo cell metastasis was modulated by miR­500a­5p. Therefore, the findings of the present study demonstrated that miR­500a­5p is associated with a positive therapeutic outcome in terms of invasion/migration of CRC cells and mesenchymal­like cell changes.

HOXD9 promotes the growth, invasion and metastasis of gastric cancer cells by transcriptional activation of RUFY3.

BACKGROUND: The transcription factor HOXD9 is one of the members of the HOX family, which plays an important role in neoplastic processes. However, the role of HOXD9 in the growth and metastasis of gastric cancer (GC) remains to be elucidated. METHODS: In vitro functional role of HOXD9 and RURY3 in GC cells was determined using the TMA-based immunohistochemistry, western blot, EdU incorporation, gelatin zymography, luciferase, chromatin Immunoprecipitation (ChIP) and cell invasion assays. In vivo tumor growth and metastasis were conducted in nude mice. RESULTS: HOXD9 is overexpressed in GC cells and tissues. The high expression of HOXD9 was correlated with poor survival in GC patients. Functionally, HOXD9 expression significantly promoted the proliferation, invasion and migration of GC cells. Mechanically, HOXD9 directly associated with the RUFY3 promoter to increase the transcriptional activity of RUFY3. Inhibition of RUFY3 attenuated the proliferation, migration and invasiveness of HOXD9-overexpressing GC cells in vitro and in vivo. Moreover, both HOXD9 and RUFY3 were highly expressed in cancer cells but not in normal gastric tissues, with their expressions being positively correlated. CONCLUSIONS: The evidence presented here suggests that the HOXD9-RUFY3 axis promotes the development and progression of human GC.

Ubiquitin-specific protease 3 promotes cell migration and invasion by interacting with and deubiquitinating SUZ12 in gastric cancer.

BACKGROUND: The deubiquitinating enzyme ubiquitin-specific protease 3 (USP3) plays a crucial role in numerous biological processes. The aberrant expression of USP3 may have an important role in tumor development. However, the mechanism by which USP3 promotes gastric cancer (GC) metastasis remains largely unknown. METHODS: Effects of USP3 on the progression of GC in vivo and in vitro and the potential underlying mechanisms have been investigated utilizing proteomics, RT-PCR, western blotting, immunohistochemistry, immunofluorescence, cell invasion and migration assays and xenograft tumor models. RESULTS: USP3 expression was upregulated in GC compared with matched normal tissues and was predictive of poor survival. USP3 also promoted migration and epithelial-to-mesenchymal transition (EMT) in GC cells. Moreover, TGF-ß1 induced USP3 expression, and USP3 knockdown inhibited TGF-ß1-induced EMT. Furthermore, we utilized Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to identify differentially expressed proteins in USP3-overexpressing cells compared with control cells. Importantly, we found that SUZ12 is indispensable for USP3-mediated oncogenic activity in GC. We observed that USP3 interacted with and stabilized SUZ12 via deubiquitination. SUZ12 knockdown inhibited USP3-induced migration and invasion, as well as EMT in GC cells. Examination of clinical samples confirmed that USP3 expression was positively correlated with SUZ12 protein expression and that the levels of USP3 or SUZ12 protein were negatively correlated with the levels of E-cadherin protein. CONCLUSIONS: These findings identify USP3 as a critical regulator. The USP3-SUZ12 axis might promote tumor progression and could be a potential therapeutic candidate for human GC.

Coexpression of FOXK1 and vimentin promotes EMT, migration, and invasion in gastric cancer cells.

In human gastric cancer (GC), the upregulation of FOXK1 and vimentin is frequently observed in cancer cells and correlates with increased malignancy. We report that FOXK1 synergizes with vimentin to promote GC invasion and metastasis via the induction of epithelial-mesenchymal transition (EMT). We showed that higher expression levels of FOXK1 were significantly associated with GC development. FOXK1 can physically interact with and stabilize vimentin. Moreover, a positive correlation between the expression of FOXK1 and vimentin was found in GC cells. Higher expression levels of these two proteins were significantly associated with differentiation, lymph node metastasis, AJCC stage, and poorer prognosis. Furthermore, the coexpression of FOXK1 and vimentin enhances cell metastasis through the induction of EMT in GC cells. However, the siRNA-mediated repression of vimentin in FOXK1-overexpressing cells reversed the EMT-like phenotype and reduced GC cell migration and invasion in vitro and in vivo. Altogether, our findings suggest that the vimentin-FOXK1 axis provides new insights into the molecular mechanisms underlying EMT regulation during GC progression and metastasis.

The FOXK1-CCDC43 Axis Promotes the Invasion and Metastasis of Colorectal Cancer Cells.

BACKGROUND/AIMS: The CCDC43 gene is conserved in human, rhesus monkey, mouse and zebrafish. Bioinformatics studies have demonstrated the abnormal expression of CCDC43 gene in colorectal cancer (CRC). However, the role and molecular mechanism of CCDC43 in CRC remain unknown. METHODS: The functional role of CCDC43 and FOXK1 in epithelial-mesenchymal transition (EMT) was determined using immunohistochemistry, flow cytometry, western blot, EdU incorporation, luciferase, chromatin Immunoprecipitation (ChIP) and cell invasion assays. RESULTS: The CCDC43 gene was overexpressed in human CRC. High expression of CCDC43 protein was associated with tumor progression and poor prognosis in patients with CRC. Moreover, the induction of EMT by CCDC43 occurred through TGF-ß signaling. Furthermore, a positive correlation between the expression patterns of CCDC43 and FOXK1 was observed in CRC cells. Promoter assays demonstrated that FOXK1 directly bound and activated the human CCDC43 gene promoter. In addition, CCDC43 was necessary for FOXK1- mediated EMT and metastasis in vitro and vivo. Taken together, this work identified that CCDC43 promoted EMT and was a direct transcriptional target of FOXK1 in CRC cells. CONCLUSION: FOXK1-CCDC43 axis might be helpful to develop the drugs for the treatment of CRC.

Snail/FOXK1/Cyr61 Signaling Axis Regulates the Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

BACKGROUND/AIMS: Metastasis is the primary cause of colorectal cancer (CRC)-related death. However, the molecular mechanisms underlying metastasis in CRC remain unclear. METHODS: We evaluated mRNA and protein expression levels by quantitative real-time reverse transcription PCR, western blotting, immunofluorescence, tissue microarrays, and immunohistochemistry assays. We also assessed the migration and invasion abilities of CRC cells in vitro by wound healing assays, invasion and migration assays, western blot analysis, and immunofluorescence. Tumor metastasis was evaluated in nude mice in vivo. RESULTS: A positive correlation was observed between the expression patterns of Forkhead box k1 (FOXK1) and Snail in CRC. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that Snail directly bound to and activated the human FOXK1 gene promoter. Moreover, the Snail-FOXK1 axis promote epithelial mesenchymal transition (EMT)-mediated CRC cell invasion and metastasis. FOXK1 and Snail expression levels were correlated with tumor progression and served as significant predictors of overall survival in patients with CRC. Furthermore, overexpression of FOXK1 induced the EMT by upregulating the expression of cysteine-rich angiogenic inducer 61 (Cyr61). Luciferase assays showed that Cyr61 was a direct transcriptional target of FOXK1. Down regulation of Cyr61 decreased FOXK1-enhanced "CRC cell" migration, invasion, and metastasis. Additionally, FOXK1 expression was positively correlated with Cyr61 expression and was associated with poor prognosis. CONCLUSIONS: The Snail/FOXK1/Cyr61 signaling axis regulates the EMT and metastasis of CRC.