Neuropilin-1 Promotes Epithelial-to-Mesenchymal Transition by Stimulating Nuclear Factor-Kappa B and Is Associated with Poor Prognosis in Human Oral Squamous Cell Carcinoma

Background

The epithelial-to-mesenchymal transition (EMT) is a key process in carcinogenesis, invasion, and metastasis of oral squamous cell carcinoma (OSCC). In our previous studies, we found that neuropilin-1 (NRP1) is overexpressed in tongue squamous cell carcinoma and that this overexpression is associated with cell migration and invasion. Nuclear factor-kappa B (NF-κB) plays an essential role both in the induction and the maintenance of EMT and tumor metastasis. Therefore, we hypothesized that NRP1 induces EMT, and that NRP1-induced migration and invasion may be an important mechanism for promoting invasion and metastasis of OSCC through NF-κB activation.

Methods/Results

The variations in gene and protein expression and the changes in the biological behavior of OSCC cell lines transfected with a vector encoding NRP1, or the corresponding vector control, were evaluated. NRP1 overexpression promoted EMT and was associated with enhanced invasive and metastatic properties. Furthermore, the induction of EMT promoted the acquisition of some cancer stem cell (CSC)-like characteristics in OSCC cells. We addressed whether selective inhibition of NF-κB suppresses the NRP1-mediated EMT by treating cells with pyrrolidinedithiocarbamate ammonium (PDTC), an inhibitor of NF-κB. Immunohistochemical analysis of NRP1 in OSCC tissue samples further supported a key mediator role for NRP1 in tumor progression, lymph node metastasis, and indicated that NRP1 is a predictor for poor prognosis in OSCC patients.

Conclusion

Our results indicate that NRP1 may regulate the EMT process in OSCC cell lines through NF-κB activation, and that higher NRP1 expression levels are associated with lymph node metastasis and poor prognosis in OSCC patients. Further investigation of the role of NRP1 in tumorigenesis may help identify novel targets for the prevention and therapy of oral cancers.     

Naa10p and IKKα interaction regulates EMT in oral squamous cell carcinoma via TGF-β1/Smad pathway

Epithelial-mesenchymal transition (EMT) has been contributed to increase migration and invasion of cancer cells. However, the correlate of Naa10p and IKKα with EMT in oral squamous cell carcinoma (OSCC) is not yet fully understood. In our present study, we found N-α-acetyltransferase 10 protein (Naa10p) and IκB kinase α (IKKα) were abnormally abundant in oral squamous cell carcinoma (OSCC). Bioinformatic results indicate that the expression of Naa10p and IKKα is correlated with TGF-β1/Smad and EMT-related molecules. The Transwell migration, invasion, qRT-PCR and Western blot assay indicated that Naa10p repressed OSCC cell migration, invasion and EMT, whereas IKKα promoted TGF-β1–mediated OSCC cell migration, invasion and EMT. Mechanistically, Naa10p inhibited IKKα activation of Smad3 through the interaction with IKKα directly in OSCC cells after TGF-β1 stimulation. Notably, knockdown of Naa10p reversed the IKKα-induced change in the migration, invasion and EMT-related molecules in OSCC cells after TGF-β1 stimulation. These findings suggest that Naa10p interacted with IKKα mediates EMT in OSCC cells through TGF-β1/Smad, a novel pathway for preventing OSCC.     

Crosstalk between oral squamous cell carcinoma cells and cancer-associated fibroblasts via the TGF-β/SOX9 axis in cancer progression

Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial–mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-β1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-β1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-β/SOX9 axis.     

Crosstalk between oral squamous cell carcinoma cells and cancer-associated fibroblasts via the TGF-β/SOX9 axis in cancer progression

Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial–mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-β1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-β1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-β/SOX9 axis.     

Crosstalk between oral squamous cell carcinoma cells and cancer-associated fibroblasts via the TGF-β/SOX9 axis in cancer progression

Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial–mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-β1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-β1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-β/SOX9 axis.     

Naa10p and IKKα interaction regulates EMT in oral squamous cell carcinoma via TGF‐β1/Smad pathway

Epithelial‐mesenchymal transition (EMT) has been contributed to increase migration and invasion of cancer cells. However, the correlate of Naa10p and IKKα with EMT in oral squamous cell carcinoma (OSCC) is not yet fully understood. In our present study, we found N‐α‐acetyltransferase 10 protein (Naa10p) and IκB kinase α (IKKα) were abnormally abundant in oral squamous cell carcinoma (OSCC). Bioinformatic results indicate that the expression of Naa10p and IKKα is correlated with TGF‐β1/Smad and EMT‐related molecules. The Transwell migration, invasion, qRT‐PCR and Western blot assay indicated that Naa10p repressed OSCC cell migration, invasion and EMT, whereas IKKα promoted TGF‐β1–mediated OSCC cell migration, invasion and EMT. Mechanistically, Naa10p inhibited IKKα activation of Smad3 through the interaction with IKKα directly in OSCC cells after TGF‐β1 stimulation. Notably, knockdown of Naa10p reversed the IKKα‐induced change in the migration, invasion and EMT‐related molecules in OSCC cells after TGF‐β1 stimulation. These findings suggest that Naa10p interacted with IKKα mediates EMT in OSCC cells through TGF‐β1/Smad, a novel pathway for preventing OSCC.     

Up-regulated miR155 Reverses the Epithelial-mesenchymal Transition Induced by EGF and Increases Chemo-sensitivity to Cisplatin in Human Caski Cervical Cancer Cells

The epithelial-mesenchymal transition (EMT) induced by EGF promotes cervical cancer progression; however, the mechanisms underlying the EGF-induced EMT remain unclear. In this study, we reported that miR155 overexpression suppressed EGF-induced EMT, decreased migration/invasion capacities, inhibited cell proliferation and increased the chemo-sensitivity to DDP in human Caski cervical cancer cells. Further, the overexpression of miR155 increased TP53 expression but reduced SMAD2, and CCND1 expression levels. These data suggest that miR155 negatively regulates EGF-induced EMT. We conclude that miR155 does not act as an oncogene but as a tumour suppressor in Caski cells.     

HDAC2 promotes the EMT of colorectal cancer cells and via the modular scaffold function of ENSG00000274093.1

Histone deacetylase 2 (HDAC2), a member of the Histone deacetylase family, plays a vital role in various carcinomas. In this study, we identified that HDAC2 expression levels are associated with liver metastasis, higher T stages and poor prognosis in colorectal cancer. HDAC2 down-regulation via lentivirus-mediated expression of HDAC2-targeting shRNA reduced the in vitro migration and invasion ability of HCT116 cell as well as their liver metastasis in nude mouse xenografts. Mechanistically, HDAC2 promotes epithelial-mesenchymal transition (EMT) in colorectal cancer cells by combining HDAC1 with EZH2 (a key histone methyltransferase), possibly through the modular scaffold function of a new lncRNA, ENSG00000274093.1. HDAC2 thus appears to promote CRC cell migration and invasion through binding HDAC1 and EZH2 via ENSG00000274093.1.     

PKM2-regulated STAT3 promotes esophageal squamous cell carcinoma progression via TGF-β1-induced EMT

Recent studies have demonstrated pleiotropic roles of pyruvate kinase isoenzyme type M2 (PKM2) in tumor progression. However, the precise mechanisms underlying the effects of PKM2 on esophageal squamous cell carcinoma (ESCC) metastasis and transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) remain to be established. In this study, we observed upregulation of PKM2 in ESCC tissues that was markedly associated with lymph node metastasis and poor prognosis. High PKM2 expression in tumor tissues frequently coincided with the high pSTAT3Tyr705 expression and low E-cadherin expression. Furthermore, altered PKM2 expression was significantly associated with proliferation, migration, and invasion of ESCC cells, in addition to expression patterns of EMT markers (Snail, E-cadherin, and vimentin) and pSTAT3^Tyr705/STAT3 ratio. Overexpression of STAT3 significantly attenuated the effects of PKM2 knockdown on cell proliferation and motility as well as expression of pSTAT3 ^Tyr705 and EMT markers. Consistently, stable short hairpin RNA (shRNA)-mediated silencing of PKM2 reversed the effects of TGF-β1 treatment, specifically, upregulation of PKM2, phosphorylation of STAT3 at Tyr705, and increased EMT, migration, and invasion. We propose that PKM2 regulates cell proliferation, migration, and invasion via phosphorylation of STAT3 through TGF-β1-induced EMT. Our findings collectively provide mechanistic insights into the tumor-promoting role of PKM2, supporting its prognostic value and the therapeutic utility of PKM2 inhibitors as potential antitumor agents in ESCC.     

Tumor‐suppressive roles of ΔNp63β‐miR‐205 axis in epithelial–mesenchymal transition of oral squamous cell carcinoma via targeting ZEB1 and ZEB2

We previously revealed that epithelial‐to‐mesenchymal transition (EMT) was mediated by ΔNp63β, a splicing variant of ΔNp63, in oral squamous cell carcinoma (OSCC). Recent studies have highlighted the involvement of microRNA (miRNA) in EMT of cancer cells, though the mechanism remains unclear. To identify miRNAs responsible for ΔNp63β‐mediated EMT, miRNA microarray analyses were performed by ΔNp63β‐overexpression in OSCC cells; SQUU‐B, which lacks ΔNp63 expression and displays EMT phenotypes. miRNAs microarray analyses revealed miR‐205 was the most up‐regulated following ΔNp63β‐overexpression. In OSCC cells, miR‐205 expression was positively associated with ΔNp63 and negatively with zinc‐finger E‐box binding homeobox (ZEB) 1 and ZEB2, potential targets of miR‐205. miR‐205 overexpression by miR‐205 mimic transfection into SQUU‐B cells led to decreasing ZEB1, ZEB2, and mesenchymal markers, increasing epithelial markers, and reducing cell motilities, suggesting inhibition of EMT phenotype. Interestingly, the results opposite to this phenomenon were obtained by transfection of miR‐205 inhibitor into OSCC cells, which express ΔNp63 and miR‐205. Furthermore, target protector analyses revealed direct regulation by miR‐205 of ZEB1 and ZEB2 expression. These results showed tumor‐suppressive roles of ΔNp63β and miR‐205 by inhibiting EMT thorough modulating ZEB1 and ZEB2 expression in OSCC.     

Long non‐coding RNA LOC554202 modulates chordoma cell proliferation and invasion by recruiting EZH2 and regulating miR‐31 expression

Objectives

Chordoma is a rare malignant bone tumour arising from notochordal remnants. Long non‐coding RNA LOC554202, as the host gene of miR‐31, contributes to various cancer developments. However, little is known about the biological function of LOC554202 in chordoma. Here, the relationship between LncRNA LOC554202, miR‐31 and EZH2 was elucidated in chordoma.

Materials and methods

The levels of LOC554402, miR‐31, EZH2, RNF144B, and epithelial‐mesenchymal transition (EMT) markers were measured in chordoma tissues and the chordoma cell lines via quantitative real‐time PCR (qRT‐PCR) or Western blot. FISH assay demonstrated the LOC554402 expression in chordoma tissues. The chordoma cell lines, U‐CH1 and JHC7, were transfected with siRNA or miRNA mimics and analysed for cell proliferation ability, apoptosis, cell migration, and invasion. RNA pull down, RIP assay, and Luciferase Reporter Assay were used to analyze the interaction between LOC554202 and EZH2. Animal tumour xenografts were generated, and qRT‐PCR was performed to investigate EZH2, miR‐31, and RNB144B expression on tumour growth in vivo.

Results

We found elevated expression of LOC554202 was associated with a decreased level of miR‐31 in cancer tissues. Knockdown of LOC554202 or overexpression of miR‐31 suppressed the proliferation, migration, and invasion of chordoma cells. Unexpectedly, EZH2 as a binding protein of LOC554202, and it was positively regulated by LOC554202, leading to the reduced expression of miR‐31. Furthermore, the impaired function of miR‐31 restored expression of the oncogene RNF144B and maintained the metastasis‐promoting activity in vitro. The results in vivo confirmed the anti‐tumour effects of knockdown of LOC554202, which inhibited EZH2/miR‐31 to activate the oncogene RNF144B.

Conclusion

Our results suggest that LOC554202 may play an important role in the progression of chordoma by the direct upregulation of EZH2 and indirect promotion of RNF144B via miR‐31.

     

Role of Jagged1/STAT3 signalling in platinum‐resistant ovarian cancer

Jagged1, the essential ligand of the Notch signalling pathway, is highly expressed in metastatic prostate cancer, and its high expression in breast cancer is linked to poor survival rates. However, the mechanism of Jagged1′s involvement in platinum‐resistant ovarian cancer has not been thoroughly elucidated to date. The purpose of the present study was to investigate the roles of Jagged1 in the platinum resistance of ovarian cancer and its possible mechanisms. Compared with a platinum responsive group of ovarian epithelial cell carcinomas, we found the positive staining intensity of Notch1, Notch2, Jagged1, STAT3 and Epithelial‐mesenchymal transition (EMT) proteins were lower in a platinum‐resistant group. The DDP‐resistant ovarian cancer cell line (C13K) had a higher IC50 of DDP than its parental cell line (OV2008) (P < 0.05) and acquired an EMT phenotype and invasive characteristics. Inhibiting or knockdown of Jagged1 expression could not only reduce its capacity of migration and invasion but also reverse EMT and down‐regulate the expression of serine 727‐phosphorylated STAT3 (pS727) at the protein level but not total STAT3 or tyrosine 705‐phosphorylated STAT3 (pY705) in C13K cells. Furthermore, it was found that crosstalk between the Jagged1/Notch and JAK/STAT3 signalling pathways were involved in Jagged1‐promoting EMT in C13K cells. Experiments in vivo showed a reduced micrometastatic tumour burden in the lung, liver and spleen of mice implanted with C13K cells with knocked‐down Jagged1 compared with mice implanted with control cells. All of these results demonstrate that Jagged1 can crosstalk with the JAK/STAT3 pathway, and they all cooperate to promote the aberrant occurrence of EMT, further reinforcing the abilities of invasion and migration of platinum‐resistant ovarian cancer in vivo and in vitro.     

Curcumin inhibits the growth of triple‐negative breast cancer cells by silencing EZH2 and restoring DLC1 expression

Enhancer of zeste homolog 2 (EZH2), an oncogene, is a commonly up‐regulated epigenetic factor in human cancer. Hepatocellular carcinoma deletion gene 1 (DLC1) is an antioncogene that is either expressed at low levels or not expressed in many malignant tumours. Curcumin is a promising anticancer drug that has antitumour effects in many tumours, but its mechanism of action is unclear. Our research demonstrated that EZH2 was up‐regulated in breast cancer (BC) tissues and cells, whereas DLC1 was down‐regulated, and the expression of EZH2 and DLC1 was negatively correlated in BC. By analysing the characteristics of clinical cases, we found that positive expression of EZH2 and negative expression of DLC1 may be predictors of poor prognosis in patients with triple‐negative breast cancer (TNBC). Moreover, knockdown of EZH2 expression restored the expression of DLC1 and inhibited the migration, invasion and proliferation, promoted the apoptosis, and blocked the cell cycle of MDA‐MB‐231 cells. Furthermore, we found that curcumin restored the expression of DLC1 by inhibiting EZH2; it also inhibited the migration, invasion and proliferation of MDA‐MB‐231 cells, promoted their apoptosis and blocked the cell cycle. Finally, xenograft tumour models were used to demonstrate that curcumin restored DLC1 expression by inhibiting EZH2 and also inhibited the growth and promoted the apoptosis of TNBC cells. In conclusion, our results suggest that curcumin can inhibit the migration, invasion and proliferation, promote the apoptosis, block the cycle of TNBC cells and restore the expression of DLC1 by inhibiting the expression of EZH2.     

TGF-β1/SH2B3 axis regulates anoikis resistance and EMT of lung cancer cells by modulating JAK2/STAT3 and SHP2/Grb2 signaling pathways

The pathogenesis of lung cancer, the most common cancer, is complex and unclear, leading to limited treatment options and poor prognosis. To provide molecular insights into lung cancer development, we investigated the function and underlying mechanism of SH2B3 in the regulation of lung cancer. We indicated SH2B3 was diminished while TGF-β1 was elevated in lung cancer tissues and cells. Low SH2B3 level was correlated with poor prognosis of lung cancer patients. SH2B3 overexpression suppressed cancer cell anoikis resistance, proliferation, migration, invasion, and EMT, while TGF-β1 promoted those processes via reducing SH2B3. SH2B3 bound to JAK2 and SHP2 to repress JAK2/STAT3 and SHP2/Grb2/PI3K/AKT signaling pathways, respectively, resulting in reduced cancer cell anoikis resistance, proliferation, migration, invasion, and EMT. Overexpression of SH2B3 suppressed lung cancer growth and metastasis in vivo. In conclusion, SH2B3 restrained the development of anoikis resistance and EMT of lung cancer cells via suppressing JAK2/STAT3 and SHP2/Grb2/PI3K/AKT signaling cascades, leading to decreased cancer cell proliferation, migration, and invasion. Subject terms: Cell migration, Lung cancer