Downregulation of miR-506-3p Facilitates EGFR-TKI Resistance through Induction of Sonic Hedgehog Signaling in Non-Small-Cell Lung Cancer Cell Lines

Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation eventually develop resistance to EGFR-targeted tyrosine kinase inhibitors (TKIs). Treatment resistance remains the primary obstacle to the successful treatment of NSCLC. Although drug resistance mechanisms have been studied extensively in NSCLC, the regulation of these mechanisms has not been completely understood. Recently, increasing numbers of microRNAs (miRNAs) are implicated in EGFR-TKI resistance, indicating that miRNAs may serve as novel targets and may hold promise as predictive biomarkers for anti-EGFR therapy. MicroRNA-506 (miR-506) has been identified as a tumor suppressor in many cancers, including lung cancer; however, the role of miR-506 in lung cancer chemoresistance has not yet been addressed. Here we report that miR-506-3p expression was markedly reduced in erlotinib-resistant (ER) cells. We identified Sonic Hedgehog (SHH) as a novel target of miR-506-3p, aberrantly activated in ER cells. The ectopic overexpression of miR-506-3p in ER cells downregulates SHH signaling, increases E-cadherin expression, and inhibits the expression of vimentin, thus counteracting the epithelial–mesenchymal transition (EMT)-mediated chemoresistance. Our results advanced our understanding of the molecular mechanisms underlying EGFR-TKI resistance and indicated that the miR-506/SHH axis might represent a novel therapeutic target for future EGFR mutated lung cancer treatment.     

A Regulatory Loop Involving miR-200c and NF-κB Modulates Mortalin Expression and Increases Cisplatin Sensitivity in an Ovarian Cancer Cell Line Model

Ovarian cancer is currently the most lethal gynecological cancer. At present, primary debulking surgery combined with platinum-based chemotherapy is the standard treatment strategy for ovarian cancer. Although cisplatin-based chemotherapy has greatly improved the prognosis of patients, the subsequent primary or acquired drug resistance of cancer cells has become an obstacle to a favorable prognosis. Mortalin is a chaperone that plays an important role in multiple cellular and biological processes. Our previous studies have found that mortalin is associated with the proliferation and migration of ovarian cancer cells and their resistance to cisplatin-based chemotherapy. In this study, microRNA (miR)-200b/c downregulated mortalin expression and inhibited the proliferation and migration of the paired cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) epithelial ovarian cancer cell lines. Moreover, miR-200c increased the sensitivity of ovarian cancer cells to cisplatin treatment by regulating mortalin levels. Nuclear factor (NF)-κB directly regulated mortalin and miR-200b/c expression levels, while NF-κB and miR-200b/c jointly regulated the expression of mortalin. The combination of cisplatin and miR-200c significantly enhanced the therapeutic effects on ovarian cancer in vivo, suggesting that miR-200c may serve as a potential therapeutic agent for ovarian cancer.     

Doxorubicin-Resistant TNBC Cells Exhibit Rapid Growth with Cancer Stem Cell-like Properties and EMT Phenotype,Which Can Be Transferred to Parental Cells through Autocrine Signaling

Emerging evidence suggests that breast cancer stem cells (BCSCs), and epithelial–mesenchymal transition (EMT) may be involved in resistance to doxorubicin. However, it is unlear whether the doxorubicin-induced EMT and expansion of BCSCs is related to cancer dormancy, or outgrowing cancer cells with maintaining resistance to doxorubicin, or whether the phenotypes can be transferred to other doxorubicin-sensitive cells. Here, we characterized the phenotype of doxorubicin-resistant TNBC cells while monitoring the EMT process and expansion of CSCs during the establishment of doxorubicin-resistant MDA-MB-231 human breast cancer cells (DRM cells). In addition, we assessed the potential signaling associated with the EMT process and expansion of CSCs in doxorubicin-resistance of DRM cells. DRM cells exhibited morphological changes from spindle-shaped MDA-MB-231 cells into round-shaped giant cells. They exhibited highly proliferative, EMT, adhesive, and invasive phenotypes. Molecularly, they showed up-regulation of Cyclin D1, mesenchymal markers (β-catenin, and N-cadherin), MMP-2, MMP-9, ICAM-1 and down-regulation of E-cadherin. As the molecular mechanisms responsible for the resistance to doxorubicin, up-regulation of EGFR and its downstream signaling, were suggested. AKT and ERK1/2 expression were also increased in DRM cells with the advancement of resistance to doxorubicin. Furthermore, doxorubicin resistance of DRM cells can be transferred by autocrine signaling. In conclusion, DRM cells harbored EMT features with CSC properties possessing increased proliferation, invasion, migration, and adhesion ability. The doxorubicin resistance, and doxorubicin-induced EMT and CSC properties of DRM cells, can be transferred to parental cells through autocrine signaling. Lastly, this feature of DRM cells might be associated with the up-regulation of EGFR.     

MicroRNA Expression Profiles in Superficial Esophageal Squamous Cell Carcinoma before Endoscopic Submucosal Dissection: A Pilot Study

Esophageal squamous cell carcinoma (ESCC) has a poor prognosis when diagnosed at an advanced stage, and early detection and treatment are essential to improve survival. However, intraobserver and interobserver variation make the diagnosis of superficial ESCC difficult, and suitable biomarkers are urgently needed. Here, we compared the microRNA (miRNA) expression profiles of superficial ESCC tissues and adjacent normal tissues obtained immediately before esophageal endoscopic submucosal dissection. We found that ESCC and normal tissues differed in their miRNA expression profiles. In particular, miR-21-5p and miR-146b-5p were significantly upregulated and miR-210-3p was significantly downregulated in tumor tissues compared with normal tissues. We also detected significant associations between miRNA expression and ESCC invasion depth and lymphovascular invasion. The same differential expression of miR-21-5p, miR-146b-5p, and miR-210-3p was detected in ESCC cell lines compared with normal esophageal epithelial cells in vitro. However, transfection of ESCC cells with miR-210-3p and miR-21-5p mimics or inhibitors had partial effects on cell proliferation and invasion in vitro. These results indicate that miRNA expression is significantly deregulated in superficial ESCC, and suggest that the potential contribution of differentially expressed miRNAs to the malignant phenotype should be further investigated.     

BMAL1 Knockdown Leans Epithelial–Mesenchymal Balance toward Epithelial Properties and Decreases the Chemoresistance of Colon Carcinoma Cells

The circadian clock coordinates biological and physiological functions to day/night cycles. The perturbation of the circadian clock increases cancer risk and affects cancer progression. Here, we studied how BMAL1 knockdown (BMAL1-KD) by shRNA affects the epithelial–mesenchymal transition (EMT), a critical early event in the invasion and metastasis of colorectal carcinoma (CRC). In corresponding to a gene set enrichment analysis, which showed a significant enrichment of EMT and invasive signatures in BMAL1_high CRC patients as compared to BMAL1_low CRC patients, our results revealed that BMAL1 is implicated in keeping the epithelial–mesenchymal equilibrium of CRC cells and influences their capacity of adhesion, migration, invasion, and chemoresistance. Firstly, BMAL1-KD increased the expression of epithelial markers (E-cadherin, CK-20, and EpCAM) but decreased the expression of Twist and mesenchymal markers (N-cadherin and vimentin) in CRC cell lines. Finally, the molecular alterations after BMAL1-KD promoted mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (i.e., HCT116 and SW480) compared to the metastatic cell line SW620. As a consequence, migration/invasion and drug resistance capacities decreased in HCT116 and SW480 BMAL1-KD cells. Together, BMAL1-KD alerts the delicate equilibrium between epithelial and mesenchymal properties of CRC cell lines, which revealed the crucial role of BMAL1 in EMT-related CRC metastasis and chemoresistance.     

Glomerular Endothelial Cell-Derived miR-200c Impairs Glomerular Homeostasis by Targeting Podocyte VEGF-A

Deciphering the pathophysiological mechanisms of primary podocytopathies that can lead to end-stage renal disease and increased mortality is an unmet need. Studying how microRNAs (miRs) interfere with various signaling pathways enables identification of pathomechanisms, novel biomarkers and potential therapeutic options. We investigated the expression of miR-200c in urine from patients with different renal diseases as a potential candidate involved in podocytopathies. The role of miR-200c for the glomerulus and its potential targets were studied in cultured human podocytes, human glomerular endothelial cells and in the zebrafish model. miR-200c was upregulated in urine from patients with minimal change disease, membranous glomerulonephritis and focal segmental glomerulosclerosis and also in transforming growth factor beta (TGF-β) stressed glomerular endothelial cells, but not in podocytes. In zebrafish, miR-200c overexpression caused proteinuria, edema, podocyte foot process effacement and glomerular endotheliosis. Although zinc finger E-Box binding homeobox 1/2 (ZEB1/2), important in epithelial to mesenchymal transition (EMT), are prominent targets of miR-200c, their downregulation did not explain our zebrafish phenotype. We detected decreased vegfaa/bb in zebrafish overexpressing miR-200c and could further prove that miR-200c decreased VEGF-A expression and secretion in cultured human podocytes. We hypothesize that miR-200c is released from glomerular endothelial cells during cell stress and acts in a paracrine, autocrine, as well as context-dependent manner in the glomerulus. MiR-200c can cause glomerular damage most likely due to the reduction of podocyte VEGF-A. In contrast, miR-200c might also influence ZEB expression and therefore EMT, which might be important in other conditions. Therefore, we propose that miR-200c-mediated effects in the glomerulus are context-sensitive.     

CD44 Is Associated with the Aggressive Phenotype of Nasopharyngeal Carcinoma through Redox Regulation

Recent studies have shown that cancer stem-like cells (CSCs) within a tumor have the capacity for self-renewal and differentiation, and are associated with an aggressive phenotype and therapeutic resistance. Studies have also associated tumor progression with alterations in the levels of intracellular reactive oxygen species (ROS). In this study, we cultured nasopharyngeal carcinoma (NPC) CSCs in conditions that allowed sphere formation. The resulting sphere cells displayed stemness properties, characteristics of the epithelial–mesenchymal transition (EMT), and increased expression of the CSC surface marker CD44. We further evaluated the association between CD44 expression and EMT marker expression, and any correlation with redox status, in these CSCs. We showed that the EMT in sphere cells is associated with the upregulation of CD44 expression and increased ROS generation, which might promote NPC aggressiveness. We also identified the coexpression of CD44 with the EMT marker N-cadherin in sphere cells, and downregulated CD44 expression after the addition of the antioxidant N-acetyl cysteine. Our results indicate that CD44 plays a role in the EMT phenotype of CSCs in NPC, and suggest its involvement in EMT-associated ROS production. These findings might facilitate the development of a novel therapy for the prevention of NPC recurrence and metastasis.     

Circulating miR-200 Family and CTCs in Metastatic Breast Cancer before,during, and after a New Line of Systemic Treatment

The extracellular circulating microRNA (miR)-200 regulates epithelial-mesenchymal transition and, thus, plays an essential role in the metastatic cascade and has shown itself to be a promising prognostic and predictive biomarker in metastatic breast cancer (MBC). Expression levels of the plasma miR-200 family were analyzed in relationship to systemic treatment, circulating tumor cells (CTC) count, progression-free survival (PFS), and overall survival (OS). Expression of miR-200a, miR-200b, miR-200c, miR-141, and miR-429, and CTC status (CTC-positive ≥ 5 CTC/7.5 mL) was assessed in 47 patients at baseline (BL), after the first completed cycle of a new line of systemic therapy (1C), and upon the progression of disease (PD). MiR-200a, miR-200b, and miR-141 expression was reduced at 1C compared to BL. Upon PD, all miR-200s were upregulated compared to 1C. At all timepoints, the levels of miR-200s were elevated in CTC-positive versus CTC-negative patients. Further, heightened miR-200s expression and positive CTC status were associated with poorer OS at BL and 1C. In MBC patients, circulating miR-200 family members decreased after one cycle of a new line of systemic therapy, were elevated during PD, and were indicative of CTC status. Notably, increased levels of miR-200s and elevated CTC count correlated with poorer OS and PFS. As such, both are promising biomarkers for optimizing the clinical management of MBC.     

Dexamethasone Suppresses Palatal Cell Proliferation through miR-130a-3p

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital birth defects. This study aims to identify novel pathogenic microRNAs associated with cleft palate (CP). Through data analyses of miRNA-sequencing for developing palatal shelves of C57BL/6J mice, we found that miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p were significantly upregulated, and that miR-19a-3p, miR-130a-3p, miR-301a-3p, and miR-486b-5p were significantly downregulated, at embryonic day E14.5 compared to E13.5. Among them, overexpression of the miR-449 family (miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p) and miR-486b-5p resulted in reduced cell proliferation in primary mouse embryonic palatal mesenchymal (MEPM) cells and mouse cranial neural crest cell line O9-1. On the other hand, inhibitors of miR-130a-3p and miR-301a-3p significantly reduced cell proliferation in MEPM and O9-1 cells. Notably, we found that treatment with dexamethasone, a glucocorticoid known to induce CP in mice, suppressed miR-130a-3p expression in both MEPM and O9-1 cells. Moreover, a miR-130a-3p mimic could ameliorate the cell proliferation defect induced by dexamethasone through normalization of Slc24a2 expression. Taken together, our results suggest that miR-130-3p plays a crucial role in dexamethasone-induced CP in mice.     

MicroRNA-130b Promotes Cell Aggressiveness by Inhibiting Peroxisome Proliferator-Activated Receptor Gamma in Human Hepatocellular Carcinoma

MircroRNA-130b (miR-130b) is proposed as a novel tumor-related miRNA and has been found to be significantly dysregulated in tumors. In this study, the expression level of miR-130b was found to be obviously higher in hepatocellular carcinoma (HCC) tissues than that in nontumor tissues. Further, miR-130b was expressed at significantly higher levels in aggressive and recurrent tumor tissues. Clinical analysis indicated that high-expression of miR-130b was prominently correlated with venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage in HCC. Elevated miR-130b expression was observed in all HCC cell lines (HepG2, SMMC-7721, Huh7, Hep3B and MHCC97H) as compared with that in a nontransformed hepatic cell line (LO2). Furthermore, an inverse correlation between miR-130b and E-cadherin and a positive correlation between miR-130b and Vimentin were observed in HCC tissues. Down-regulation of miR-130b expression reduced invasion and migration in both Hep3B and MHCC97H cells. Peroxisome proliferator-activated receptor gamma (PPAR-γ) was inversely correlated with miR-130b expression in HCC tissues. In addition, down-regulation of miR-130b restored PPAR-γ expression and subsequently suppressed epithelial-mesenchymal transition (EMT) in HCC cells. We identified PPARγ as a direct target of miR-130b in HCC in vitro. Notably, PPAR-γ knockdown abolished down-regulation of miR-130b-inhibited EMT in MHCC97H cells. In conclusion, miR-130b may promote HCC cell migration and invasion by inhibiting PPAR-γ and subsequently inducing EMT.     

Cisplatin Plus Cetuximab Inhibits Cisplatin-Resistant Human Oral Squamous Cell Carcinoma Cell Migration and Proliferation but Does Not Enhance Apoptosis

Cisplatin is among the most widely used anticancer drugs used in the treatment of several malignancies, including oral cancer. However, cisplatin treatment often promotes chemical resistance, subsequently causing treatment failure. Several studies have shown that epidermal growth factor receptors (EGFRs) play a variety of roles in cancer progression and overcoming cisplatin resistance. Therefore, this study focused on EGFR inhibitors used in novel targeted therapies as a method to overcome this resistance. We herein aimed to determine whether the combined effects of cisplatin and cetuximab could enhance cisplatin sensitivity by inhibiting the epithelial-to-mesenchymal transition (EMT) process in cisplatin-resistant cells. In vitro analyses of three cisplatin-resistant oral squamous cell carcinoma cells, which included cell proliferation assay, combination index calculation, cell cytotoxicity assay, live/dead cell count assay, Western blot assay, propidium iodide staining assay, scratch assay, and qRT-PCR assay were then conducted. Our results showed that a cisplatin/cetuximab combination treatment inhibited cell proliferation, cell motility, and N-cadherin protein expression but induced E-cadherin and claudin-1 protein expression. Although the combination of cisplatin and cetuximab did not induce apoptosis of cisplatin-resistant cells, it may be useful in treating oral cancer patients with cisplatin resistance given that it controls cell motility and EMT-related proteins.     

miR-200c-3p Regulates Epitelial-to-Mesenchymal Transition in Epicardial Mesothelial Cells by Targeting Epicardial Follistatin-Related Protein 1

Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, by using an in vitro model of epicardial EMT, we investigated the role of miRNAs as regulators of this process and their potential targets. EMT was induced in murine epicardial-mesothelial cells (EMCs) through TGF β1 treatment for 48, 72, and 96 h as indicated by the expression of EMT-related genes by qRT-PCR, WB, and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted as the most strongly suppressed. Interestingly, we also found a significant upregulation of Follistatin-related protein 1 (FSTL1), a miR-200c predicted target already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. Dual-luciferase reporter assay demonstrated that miR-200c-3p directly targeted the 3′-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-3p significantly increased FSTL1 expression, whereas overexpression of miR-200c-3p counteracted TGF β1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction by TGF β1, and attenuated EMT-associated traits, including migration and stemness. In conclusion, epicardial FSTL1, an important cardiogenic factor in its secreted form, induces EMT, stemness, and migration of EMCs in a miR-200c-3p dependent pathway.     

Possible Action of Olaparib for Preventing Invasion of Oral Squamous Cell Carcinoma In Vitro and In Vivo

Despite recent advances in treatment, the prognosis of oral cancer remains poor, and prevention of recurrence and metastasis is critical. Olaparib is a PARP1 inhibitor that blocks polyADP-ribosylation, which is involved in the epithelial–mesenchymal transition (EMT) characteristic of tumor recurrence. We explored the potential of olaparib in inhibiting cancer invasion in oral carcinoma using three oral cancer cell lines, HSC-2, Ca9-22, and SAS. Olaparib treatment markedly reduced their proliferation, migration, invasion, and adhesion. Furthermore, qRT-PCR revealed that olaparib inhibited the mRNA expression of markers associated with tumorigenesis and EMT, notably Ki67, Vimentin, β-catenin, MMP2, MMP9, p53, and integrin α2 and β1, while E-Cadherin was upregulated. In vivo analysis of tumor xenografts generated by injection of HSC-2 cells into the masseter muscles of mice demonstrated significant inhibition of tumorigenesis and bone invasion by olaparib compared with the control. This was associated with reduced expression of proteins involved in osteoclastogenesis, RANK and RANKL. Moreover, SNAIL and PARP1 were downregulated, while E-cadherin was increased, indicating the effect of olaparib on proteins associated with EMT in this model. Taken together, these findings confirm the effects of olaparib on EMT and bone invasion in oral carcinoma and suggest a new therapeutic strategy for this disease.     

Secreted HSP90α-LRP1 Signaling Promotes Tumor Metastasis and Chemoresistance in Pancreatic Cancer

The extracellular heat shock protein 90α (eHSP90α) has been reported to promote cancer cell motility. However, whether pancreatic cancer (PC) cells expressed membrane-bound or secreted HSP90α, as well as its underlying mechanism for PC progression, were still unclear. Our study demonstrated that the amounts of secreted HSP90α proteins were discrepant in multiple PC cells. In addition, highly invasive Capan-2 cells have a higher level of secreted HSP90α compared with those of less invasive PL45 cells. The conditioned medium of Capan-2 cells or recombinant HSP90α treatment stimulated the migration and invasion of PC cells, which could be prevented with a neutralizing anti-HSP90α antibody. Furthermore, secreted HSP90α promoted elements of epithelial–mesenchymal transition in PL45 cells, including increases in vimentin and Snail expressions, decreases in E-cadherin expression, and changes in cell shape towards a mesenchymal phenotype, but these phenomena were reversed by the anti-HSP90α antibody in Capan-2 cells. In addition, high levels of low-density lipoprotein receptor-related protein 1 (LRP1) were associated with worsened patient survival in pancreatic adenocarcinoma. We demonstrated LRP1 as a receptor of eHSP90α for its stimulatory role in metastasis, by activating the AKT pathway. In addition, silencing LRP1 enhanced the chemosensitivity to gemcitabine and doxorubicin in Capan-2 cells. Therefore, our study indicated that blocking secreted HSP90α underlies an aspect of metastasis and chemoresistance in PC.     

The Status of EGFR Modulates the Effect of miRNA-200c on ZEB1 Expression and Cell Migration in Glioblastoma Cells

Migration of glioblastoma cells into surrounding tissue is one of the main features that makes this tumor incurable. We evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns in 30 cases of primary glioblastoma. From the 64 miRNAs that showed differential expression between tumors with a high level of EGFR amplification and tumors without EGFR amplification, 40% were related with cell migration, being miR-200c the most differentially expressed between these two groups. We investigated the effect of miR-200c on ZEB1 expression and cell migration in an in vitro transfection model with a miR-200c mimic, a miR-200c inhibitor and siRNA targeting EGFR in three short-term cultures with different levels of EGFR amplification obtained from resected glioblastomas. The cell culture with the highest EGFR amplification level presented the lowest miR-200c expression and the status of EGFR modulated the effect of miR-200c on ZEB1 expression. Silencing EGFR led to miR-200c upregulation and ZEB1 downregulation in transfected cultures, except in the presence of high levels of EGFR. Likewise, miR-200c upregulation decreased ZEB1 expression and inhibited cell migration, especially when EGFR was not amplified. Our results suggest that modulating miR-200c may serve as a novel therapeutic approach for glioblastoma depending on EGFR status.