A novel miR-200c/c-myc negative regulatory feedback loop is essential to the EMT process, CSC biology and drug sensitivity in nasopharyngeal cancer

Abstract

Overexpression of the c-Myc oncogene has been implicated in cancer stem cell - like (CSC) phenotypes and epithelial-to-mesenchymal transition (EMT) in cancer. However, the underlying molecular mechanism by which c-Myc regulates EMT and CSC potential in remains unclear. In the present study, we showed that the expression of c-Myc protein is inversely correlated with microRNA (miR)-200c expression in primary tumor samples from nasopharyngeal cancer (NPC) patients. We further demonstrated that Myc and miR-200c negatively regulate the expression each other in NPC cell lines. c-Myc transcriptionally repressed expression of miR-200c by directly binding to two E-box sites located within a 1 kb segment upstream of TSS of the miR-200c. In addition, miR-200c post-transcriptionally repressed expression of c-Myc by binding to its 3′-untranslated region, suggesting the existence of a negative feedback loop between Myc and miR-200c. Overexpression of c-Myc interfered with this feedback loop and activated the EMT program, induced CSC phenotypes, and enhanced drug sensitivity, whereas miR-200c could counteract these biological effects of c-Myc. Our results provide a novel mechanism governing c-Myc and miR-200c expression and indicate that either targeting c-Myc or restoring miR-200c expression would be a promising approach to overcome oncogenic role of c-Myc in NPC.

Introduction

EMT is the process by which epithelial cell layers lose polarity and cell-cell contacts and undergo the loss of epithelial cell adhesion and cytoskeletal components [1]. A hallmark of EMT is the loss of E-cadherin and overexpression of vimentin, snail and other mesenchymal markers [2]. Increasing evidence indicates that EMT is integral to cancer cell invasion and metastasis. Cancer stem cells (CSCs) with tumor-initiating potential share the properties of normal stem cells regarding their self-renewal ability and the capability to give rise to differentiated or committed progenitors [3]. CSCs are found in many types of solid tumors including breast [4], brain [5], pancreatic [6], and gastric [7] cancers. Metastatic cancer cells that have undergone EMT exhibit a CSC phenotype. Tumor cells that have undergone EMT transition exhibit properties similar to those of CSCs, such as self-renewal and the capacity to form tumor spheroids [8]. Immortalized human mammary epithelial cells (HMLEs) that undergo an EMT change exhibit the characteristics of CSCs, as defined by their CD44-high/CD24-low phenotype [9]. There is also compelling evidence that the EMT transition contributes to a CSC-like phenotype, which may be a prerequisite for cancer cell metastasis, which was related with an initiator (INR) element within the core promoter of target genes [10]. By activating or repressing relevant genes, c-Myc promotes progression through the cell cycle, as well as cell transformation, metabolism, dedifferentiation, genomic instability, and apoptosis [[11], [12], [13]].

MicroRNAs (miRNAs) are small non-coding RNAs that function in the post-transcriptional repression of target mRNAs through sequence-specific binding to the 3′ untranslated region (3′-UTRs) of target genes. miRNAs have been linked to many aspects of cancer cell biology, including EMT and CSC properties [14,15]. MiR-200c was shown to regulate EMT by inhibiting ZEB1/2, which are transcriptional repressors of E-cadherin, and to increase the stem cell and cancer stem cell population through the suppression of BMI1 and Suz12 [16,17]. These results suggest that miR-200c may have an integral role in modulating EMT and CSC phenotypes in cancer.

The proto-oncogene c-Myc has been linked to both EMT and CSC properties in cancer development. c-Myc is a key transcription factor that regulates a variety of biological processes such as cell proliferation, cell cycle progression, metabolism, differentiation, and apoptosis. Overexpression of c-Myc was shown to regulate both EMT and CSC properties in breast cancer [18,19]. Additionally, c-Myc regulates EMT during the nuclear reprogramming of mouse embryonic fibroblasts (MEFs) into iPSCs [20]. Although these studies implicate c-Myc in the regulation of EMT and CSC/stem cell biology, the molecular mechanisms by which c-Myc contributes to these important cellular processes remain unclear.

In the present study, we identified a c-Myc/miR-200c feedback loop involved in the tumorigenesis of nasopharyngeal carcinoma (NPC), one of the most common malignant tumors in Southeast Asia and southern China. Analysis of NPC specimens revealed an inverse correlation between high c-Myc expression and low expression of miR-200c, which predicted a poorer prognosis in patients with this disease. We found that this feedback loop is dysregulated and that the expression of c-Myc and miR-200c is negatively correlated in primary tumor samples from NPC patients. Moreover, we demonstrated that c-Myc transrepresses miR-200c through direct binding to the miR-200c promoter region, and overexpression of c-Myc downregulated the expression of miR-200c in NPC cell lines. On the other hand, miR-200c blocked the expression of c-Myc by binding to the 3′-UTR of c-Myc, suggesting the presence of a negative feedback loop. Overexpression of c-Myc disrupted this feedback loop and activated the EMT program, CSC biology and drug sensitivity. Furthermore, ectopic expression of miR-200c counteracted the effect of c-Myc in the regulation EMT, CSC biology and drug sensitivity. These findings suggest that a negative feedback loop between c-Myc and miR-200c plays an important role in the EMT program, CSC biology and drug sensitivity in NPC.