Epithelial-to-mesenchymal transition (EMT) is an important driver of tumor invasion and metastasis, which causes many cancer deaths. Cancer stem cells (CSC) that maintain and initiate tumors have also been implicated in invasion and metastasis, but whether EMT is an important contributor to CSC function is unclear. In this study, we investigated whether a population of CSCs that have undergone EMT (EMT CSCs) exists in squamous cell carcinoma (SCC). We also determined whether a separate population of CSCs that retain epithelial characteristics (non-EMT CSCs) is also present. Our studies revealed that self-renewing CSCs in SCC include two biologically-distinct phenotypes. One phenotype, termed CD44 high ESA high , was proliferative and retained epithelial characteristics (non-EMT CSCs), whereas the other phenotype, termed CD44 high ESA low , was migratory and had mesenchymal traits characteristic of EMT CSCs. We found that non-EMT and EMT CSCs could switch their epithelial or mesenchymal traits to reconstitute the cellular heterogeneity which was characteristic of CSCs. However, the ability of EMT CSCs to switch to non-EMT character was restricted to cells that were also ALDH1 þ , implying that only ALDH1 þ EMT cells had the ability to seed a new epithelial tumor. Taken together, our findings highlight the identification of two distinct CSC phenotypes and suggest a need to define therapeutic targets that can eradicate both of these variants to achieve effective SCC treatment. Cancer Res; 71(15); 5317-26. Ó2011 AACR.

Cancer stem cells (CSCs) drive tumour spread and therapeutic resistance, and can undergo epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) to switch between epithelial and post-EMT sub-populations. Examining oral squamous cell carcinoma (OSCC), we now show that increased phenotypic plasticity, the ability to undergo EMT/MET, underlies increased CSC therapeutic resistance within both the epithelial and post-EMT sub-populations. The post-EMT CSCs that possess plasticity exhibit particularly enhanced therapeutic resistance and are defined by a CD44highEpCAMlow/− CD24+ cell surface marker profile. Treatment with TGFβ and retinoic acid (RA) enabled enrichment of this sub-population for therapeutic testing, through which the endoplasmic reticulum (ER) stressor and autophagy inhibitor Thapsigargin was shown to selectively target these cells. Demonstration of the link between phenotypic plasticity and therapeutic resistance, and development of an in vitro method for enrichment of a highly resistant CSC sub-population, provides an opportunity for the development of improved chemotherapeutic agents that can eliminate CSCs.

Autophagy, as an important non-selective degradation mechanism, could promote tumor initiation and progression by maintaining cellular homeostasis and the cell metabolism as well as cell viability. CircCDR1as has been shown to function as an oncogene in cancer progression, however, it remains largely unknown as to how autophagy is regulated by circCDR1as in oral squamous cell carcinoma (OSCC). In this study, we validated the functional roles of circCDR1as in regulation of autophagy in OSCC cells and further investigated how circCDR1as contributed to cell survival via up-regulating autophagy under a hypoxic microenvironment by using combination of human tissue model, in vitro cell experiments and in vivo mice model. We found that hypoxia promoted the expression level of circCDR1as in OSCC cells and elevated autophagy. In addition, circCDR1as further increased hypoxia-mediated autophagy by targeting multiple key regulators of autophagy. We revealed that circCDR1as enhanced autophagy in OSCC cells via inhibition of rapamycin (mTOR) activity and upregulation of AKT and ERK½ pathways. Overexpression of circCDR1as enhanced OSCC cells viability, endoplasmic reticulum (ER) stress, and inhibited cell apoptosis under a hypoxic microenvironment. Moreover, circCDR1as promoted autophagy in OSCC cells by sponging miR-671-5p. Collectively, these results revealed that high expression of circCDR1as enhanced the viability of OSCC cells under a hypoxic microenvironment by promoting autophagy, suggesting a novel treatment strategy involving circCDR1as and the inhibition of autophagy in OSCC cells.

Mutations in POLG disrupt mtDNA replication and cause devastating diseases often with neurological phenotypes. Defining disease mechanisms has been hampered by limited access to human tissues, particularly neurons. Using patient cells carrying POLG mutations, we generated iPSCs and then neural stem cells. These neural precursors manifested a phenotype that faithfully replicated the molecular and biochemical changes found in patient post-mortem brain tissue. We confirmed the same loss of mtDNA and complex I in dopaminergic neurons generated from the same stem cells. POLG-driven mitochondrial dysfunction led to neuronal ROS overproduction and increased cellular senescence. Loss of complex I was associated with disturbed NAD + metabolism with increased UCP2 expression and reduced phosphorylated SirT1. In cells with compound heterozygous POLG mutations, we also found activated mitophagy via the BNIP3 pathway. Our studies are the first that show it is possible to recapitulate the neuronal molecular and biochemical defects associated with POLG mutation in a human stem cell model. Further, our data provide insight into how mitochondrial dysfunction and mtDNA alterations influence cellular fate determining processes.