miR-942 promotes cancer stem cell-like traits in esophageal squamous cell carcinoma through activation of Wnt/β-catenin signalling pathway

The Wnt/β-catenin signalling pathway is known to play a vital role in the maintenance of cancer stem cells (CSCs), which are reported to be the origine of malignant cancers, and result in poor prognosis of multiple kinds of cancer. Therefore, it is of great importance to illuminate the mechanism by which the Wnt/β-catenin pathway regulates the cancer stem cell-like traits in cancers. Here, we report that miR-942 is significantly upregulated in esophageal squamous cell carcinoma (ESCC), and miR-942 levels are associated with poor prognosis in ESCC patients. Overexpression of miR-942 promotes, whereas inhibition of miR-942 decreases, the tumor sphere formation, the CD90⁺ subpopulation cells and the expression of pluripotency associated markers. Moreover, in vivo assay shows that miR-942 overexpressing cells form larger tumors and display higher tumourigenesis. Furthermore, we demonstrate that miR-942 upregulates the Wnt/β-catenin signaling activity via directly targeting sFRP4, GSK3β and TLE1, which are multiple level negative regulators of the Wnt/β-catenin signaling cascade. In addition, our results indicate that c-myc directly binds to the miR-942 promoter and promotes its expression. Taken together, our findings establish an oncogenic role of miR-942 in ESCC and indicate that miR-942 might be an effective therapeutic target for ESCC.     

HIF2α is involved in the expansion of CXCR4-positive cancer stem-like cells in renal cell carcinoma

Background:

Hypoxia and the subsequent activation of hypoxia-inducible factor-2α (HIF2α) contribute to the progression of a variety of cancers. However, their role in the generation of renal cell carcinoma-derived stem cells has not been fully addressed.

Methods:

A sphere formation assay, cell proliferation, RT–PCR, western blot, FACS, immunohistochemistry and tumour xenograft were used to study the role of HIF2α.

Results:

Propagation of four renal cell carcinoma (RCC) cell lines (Caki-1, Caki-2, 786-O, 769-P) in anchorage-independent floating spheres led to the expansion of cells bearing the CXCR4 (CD184) surface marker. Inhibition of the CXCR4 pathway reduced sphere expansion. The enhanced self-renewal activity of the CXCR4-positive spheres was preceded by the upregulation of HIF2α. Knockdown of HIF2α abrogated CXCR4 expression and sphere formation. Finally, RCC-derived spheres showed an undifferentiated phenotype in vivo and formed subcutaneous tumours that highly expressed HIF2α and CXCR4. Inhibition of HIF2α abolished tumour growth in animal models.

Conclusions:

These results suggest that the generation of RCC-derived CSCs involves the activation of HIF2α and may provide a foundation for the development of new strategies to prevent the induction of CSCs in RCC.     

MiR-1207 overexpression promotes cancer stem cell–like traits in ovarian cancer by activating the Wnt/β-catenin signaling pathway

Wnt/β-catenin signaling pathway is strictly controlled by multiple negative regulators. However, how tumor cells override the negative regulatory effects to maintain constitutive activation of Wnt/β-catenin signaling, which is commonly observed in various cancers, remains puzzling. In current study, we reported that overexpression of miR-1207 in ovarian cancer activated Wnt/β-catenin signaling by directly targeting and suppressing secreted Frizzled-related protein 1 (SFRP1), AXIN2 and inhibitor of β-catenin and TCF-4 (ICAT), which are vital negative regulators of the Wnt/β-catenin pathway. We found that the expression of miR-1207 was ubiquitously upregulated in both ovarian cancer tissues and cells, which inversely correlated with patient overall survival. Furthermore, overexpression of miR-1207 enhanced, while silencing miR-1207 reduced, stem cell-like traits of ovarian cancer cells in vitro and in vivo, including tumor sphere formation capability and proportion of SP+ and CD133+ cells. Importantly, upregulating miR-1207 promoted, while silencing miR-1207 inhibited, the tumorigenicity of ovarian cancer cells. Hence, our results suggest that miR-1207 plays a vital role in promoting the cancer stem cell-like phenotype in ovarian cancer and might represent a potential target for anti-ovarian cancer therapy.     

TR4 nuclear receptor increases prostate cancer invasion via decreasing the miR-373-3p expression to alter TGFβR2/p-Smad3 signals

Testicular nuclear receptor 4 (TR4), a member of the nuclear receptor superfamily, may play important roles to modulate the metabolic diseases and prostate tumorigenesis. Here we found TR4 could increase prostate cancer (PCa) cell invasion. Mechanism dissection revealed that TR4 might increase PCa cell invasion via decreasing the miR-373-3p expression that resulted in the activation of the TGFβR2/p-Smad3 signals. The in vivo mouse model using orthotopically xenografted CWR22Rv1 cell line transfected with luciferase-reporter confirmed in vitro cell line studies showing TR4 increased PCa metastasis via decreasing the miR-373-3p expression. Together, these data suggest that TR4 may increase PCa metastasis via a newly identified signal and targeting these TR4/miR-473-3p/TGFβR2/p-Smad3 signals using TR4 antagonist or TR4-siRNA or miR-373-3p may allow us to develop a new potential therapeutic approach to better suppress PCa metastasis.     

Improving function of cytotoxic T‐lymphocytes by transforming growth factor‐β inhibitor in oral squamous cell carcinoma

Immunotherapy with immune‐checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor‐β (TGF‐β) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T‐cells (Tregs) and cancer‐associated fibroblasts and inhibiting the function of cytotoxic T‐lymphocytes (CTLs) and natural killer cells. TGF‐β may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF‐β on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T‐cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. Additionally, TGF‐β inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8⁺ T‐cell/Treg ratio and between TGFB1 mRNA expression and the Ki‐67 expression in CD8⁺ T‐cells, indicating that TGF‐β also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF‐β function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune‐checkpoint inhibitors and TGF‐β inhibitors, for OSCCs.     

Cyclin D1 cooperates with p21 to regulate TGFβ-mediated breast cancer cell migration and tumor local invasion

Introduction

Deregulation of the cell cycle machinery is often found in human cancers. Modulations in the cell cycle regulator function and expression result not only in proliferative advantages, but also lead to tumor progression and invasiveness of the cancer. In particular, cyclin D1 and p21 are often over-expressed in human cancers, correlating with high tumor grade, poor prognosis and increased metastasis. This prompted us to investigate the role of the cyclin D1/p21 signaling axis downstream of transforming growth factor beta (TGFβ) in breast cancer progression.

Methods

Cyclins mRNA and protein expressions were assessed by quantitative real-time PCR and Western blot in triple negative breast cancer cell lines. Co-localization and interaction between cyclin D1 and p21 were performed by immunocytochemistry and co-immunoprecipitation, respectively. Cell migration was assessed by wound healing and quantitative time-lapse imaging assays. In addition, the effects of cyclin D1 on cellular structure and actin organization were examined by staining with F-actin marker phalloidin and mesenchymal intermediate filament vimentin. Finally, a mammary fat pad xenograft mouse model was used to assess mammary tumor growth and local invasion.

Results

We found TGFβ to specifically up-regulate the expression of cyclin D1 in triple negative breast cancer cells. Induction of cyclin D1 is also required for TGFβ-mediated cell migration. Suppression of cyclin D1 expression not only resulted in a rounded and epithelial-like phenotype, but also prevented TGFβ-induced vimentin and F-actin co-localization at the cell edge as well as invadopodia formation. Furthermore, TGFβ promoted the nuclear co-localization and physical interaction between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins are required for the initial steps of tumor development, as double knockdown of these two molecules prevented primary tumor formation in a Xenograft mouse model. Moreover, the in vivo studies indicated that locally advanced features of the invasive tumors, including skeletal muscle, mammary fat pad and lymphovascular invasion, as well as ulcerated skin, were attenuated in the absence of cyclin D1 and p21.

Conclusions

Thus, our findings highlight the cyclin D1/p21 signaling axis as a critical regulator of TGFβ-mediated tumor growth initiation and local tumor cell invasion, both in vitro and in vivo.     

miR-29b attenuates tumorigenicity and stemness maintenance in human glioblastoma multiforme by directly targeting BCL2L2

Glioblastoma multiforme (GBM) is the most common malignant brain tumor and exhibits aggressive and invasive behavior. We previously identified four miRNAs—miR-29b, 494, 193a-3p, and 30e—with enhanced expression in GBM following treatment of ionizing radiation by miRNA microarray analysis. In this study, we found that only miR-29b inhibited tumor cell migration and invasion by reducing MMP-2 activity via phospho-AKT/β-catenin signaling, and stimulated a more epithelial-like morphology. Moreover, miR-29b inhibits angiogenesis by attenuating tube formation and the expression of VEGF and Ang-2, and stemness maintenance in GBM cells, as demonstrated by decreasing neurosphere formation and cancer stem cell marker protein expression. These findings support the anti-tumor properties of miR-29b in human GBM cells. Furthermore, miR-29b expression was inversely proportional to that of BCL2L2 mRNA or protein in various cancer cell types. Interestingly, BCL2L2 mRNA is highly expressed in the mesenchymal type of GBM. To further elucidate the relationship between miR-29b and BCL2L2 in GBM, we performed co-transfection reporter assays and determined that miR-29b downregulates BCL2L2 expression by directly binding its 3′UTR. Finally, we confirmed that BCL2L2 repression is of central importance to miR-29b anti-tumor activity using functional assays to examine cell migration, invasion, angiogenesis, and stemness. From these data, we propose that miR-29b may be a useful therapeutic agent in GBM.     

Inhibition of colorectal cancer stem cell survival and invasive potential by hsa-miR-140-5p mediated suppression of Smad2 and autophagy

Colorectal cancer (CRC) is the third highest mortality cancer in the United States and frequently metastasizes to liver and lung. Smad2 is a key element downstream of the TGF-β signaling pathway to regulate cancer metastasis by promoting epithelial to mesenchymal transition and maintaining the cancer stem cell (CSC) phenotype. In this study, we show that hsa-miR-140-5p directly targets Smad2 and overexpression of hsa-miR-140-5p in CRC cell lines decreases Smad2 expression levels, leading decreased cell invasion and proliferation, and increasing cell cycle arrest. Ectopic expression of hsa-miR-140-5p in colorectal CSCs inhibited CSC growth and sphere formation in vitro by disrupting autophagy. We have systematically identified targets of hsa-miR-140-5p involved in autophagy. Furthermore, overexpression of hsa-miR-140-5p in CSCs abolished tumor formation and metastasis in vivo. In addition, there is a progressive loss of hsa-miR-140-5p expression from normal colorectal mucosa to primary tumor tissues, with further reduction in liver metastatic tissues. Higher hsa-miR-140 expression is significantly correlated with better survival in stage III and IV colorectal cancer patients. The functional and clinical significance of hsa-miR-140-5p suggests that it is a key regulator in CRC progression and metastasis, and may have potential as a novel therapeutic molecule to treat CRC.     

The SOX17/miR-371-5p/SOX2 axis inhibits EMT,stem cell properties and metastasis in colorectal cancer

Cancer stem cells (CSCs) and EMT-type cells, which share molecular characteristics with CSCs, have been believed to play critical roles in tumor metastasis. Although much progress has been garnered in elucidating the molecular pathways that trigger EMT, stemness and metastasis, a number of key mechanistic gaps remain elusive. In the study, miR-371-5p was obviously down-regulated in primary CRC tissues compared with matched adjacent normal mucosa and correlated significantly with differentiation, tumor size, lymphatic and liver metastases. MiR-371-5p could attenuate proliferation, invasion in vitro and metastasis in vivo in CRC cells. It also suppressed EMT by regulating Wnt/β-catenin signaling and strongly decreased the CRC stemness phenotypes. Moreover, demethylation of SOX17 induced miR-371-5p expression and consequently suppressed its direct target SOX2 in CRC cells. MiR-371-5p was necessary for SOX17 mediated cancer-related traits and SOX2 was a functional target of miR-371-5p. A positive relationship between SOX17 and miR-371-5p expression and a negative one between miR-371-5p and SOX2 expression were observed in CRC cell lines and tissues. In conclusion, we identified miR-371-5p as an important “oncosuppressor” in CRC progression and elucidated a novel mechanism of the SOX17/miR-371-5p/SOX2 axis in the regulation of EMT, stemness and metastasis, which may be a potential therapeutic target.     

MicroRNA-9 promotes tumor metastasis via repressing E-cadherin in esophageal squamous cell carcinoma

MicroRNAs (miRNAs) play a critical role in development and progression of cancers. Deregulation of MicroRNA-9 (miR-9) has been documented in many types of cancers but their role in the development of esophageal squamous cell carcinoma (ESCC) has not been studied. This study aimed to investigate the effect of miR-9 in esophageal cancer metastasis. The up-regulation of miR-9 was frequently detected in primary ESCC tumor tissue, which was significantly associated with clinical progression (P = 0.022), lymph node metastasis (P = 0.007) and poor overall survival (P < 0.001). Functional study demonstrated that miR-9 promoted cell migration and tumor metastasis, which were effectively inhibited when expression of miR-9 was silenced. Moreover, we demonstrated that miR-9 interacted with the 3′-untranslated region of E-cadherin and down-regulated its expression, which induced β-catenin nuclear translocation and subsequently up-regulated c-myc and CD44 expression. In addition, miR-9 induced epithelial-mesenchymal transition (EMT) in ESCC, a key event in tumor metastasis. Taken together, our study demonstrates that miR-9 plays an important role in ESCC metastasis by activating β-catenin pathway and inducing EMT via targeting E-cadherin. Our study also suggests miR-9 can be served as a new independent prognostic marker and/or as a novel potential therapeutic target for ESCC.     

MIIP inhibits clear cell renal cell carcinoma proliferation and angiogenesis via negative modulation of the HIF-2α-CYR61 axis

Objective: In various cancers, migration and invasion inhibitory protein(MIIP) is expressed at low level and is involved in cancer pathogenesis. Herein, we sought to explore the function of MIIP in clear cell renal cell carcinoma(cc RCC).Methods: CCK-8, colony formation, cell cycle, and endothelial cell tube formation assays were performed to evaluate the roles of MIIP in cc RCC proliferation and angiogenesis. To explore the underlying mechanism, we conducted RNA-sequencing, GSEA, q RTPCR, Weste...     

A switch from CD44+ cell to EMT cell drives the metastasis of prostate cancer

Epithelial–mesenchymal transition (EMT) has been linked to cancer stem-like (CD44+) cell in the prostate cancer (PCa) metastasis. However, the molecular mechanism remains elusive. Here, we found EMT contributed to metastasis in PCa patients failed in androgen deprivation therapy (ADT). Castration TRAMP model also proved PCa treated with ADT promoted EMT with increased CD44+ stem-like cells. Switched CD44+ cell to EMT cell is a key step for luminal PCa cell metastasis. Our results also suggested ADT might go through promoting TGFβ1-CD44 signaling to enhance swift to EMT. Targeting CD44 with salinomycin and siRNA could inhibit cell transition and decrease PCa invasion. Together, cancer stem-like (CD44+) cells could be the initiator cells of EMT modulated by TGFβ1-CD44 signaling. Combined therapy of ADT with anti-CD44 may become a new potential therapeutic approach to battle later stage PCa.