MiRNA-146a/AKT/ß-Catenin Activation Regulates Cancer Stem Cell Phenotype in Oral Squamous Cell Carcinoma by Targeting CD24.

CD44highCD24low population has been previously reported as cancer stem cells (CSCs) in Oral Squamous Cell Carcinoma (OSCC). Increasing evidence suggests potential involvement of microRNA (miRNA) network in modulation of CSC properties. MiRNAs have thus emerged as crucial players in tumor development and maintenance. However, their role in maintenance of OSCC stem cells remains unclear. Here we report an elevated expression of miR-146a in the CD44highCD24low population within OSCC cells and primary HNSCC tumors. Moreover, over-expression of miR-146a results in enhanced stemness phenotype by augmenting the CD44highCD24low population. We demonstrate that miR-146a stabilizes ß-catenin with concomitant loss of E-cadherin and CD24. Interestingly, CD24 is identified as a novel functional target of miR-146a and ectopic expression of CD24 abrogates miR-146a driven potential CSC phenotype. Mechanistic analysis reveals that higher CD24 levels inhibit AKT phosphorylation leading to ß-catenin degradation. Using stably expressing miR-146a/CD24 OSCC cell lines, we also validate that the miR-146a/CD24/AKT loop significantly alters tumorigenic ability in vivo. Furthermore, we confirmed that ß-catenin trans-activates miR-146a, thereby forming a positive feedback loop contributing to stem cell maintenance. Collectively, our study demonstrates that miR-146a regulates CSCs in OSCC through CD24-AKT-ß-catenin axis.

MicroRNA-324-5p-CUEDC2 Axis Mediates Gain-of-Function Mutant p53-Driven Cancer Stemness.

Regulation of cancer stemness has recently emerged as a new gain-of-function (GOF) property of mutant p53. In this study, we identify miR-324-5p as a critical epigenetic regulator of cancer stemness and demonstrate its role in mediating GOF-mutant p53-driven stemness phenotypes. We report that miR-324-5p is upregulated in human cancer cell lines and non-small cell lung carcinoma (NSCLC) tumors carrying TP53 GOF mutations. Mechanistically, we show that GOF mutant p53 upregulates miR-324-5p expression via c-Myc, an oncogenic transcription factor in cancer cells. Our experimental results suggest that miR-324-5p-induced CSC phenotypes stem from the downregulation of CUEDC2, a downstream target gene of miR-324-5p. Accordingly, CUEDC2 complementation diminishes elevated CSC marker expression in miR-324-5p-overexpressing cancer cells. We further demonstrate that mutant p53 cancer cells maintain a low level of CUEDC2 that is rescued upon miR-324-5p inhibition. Importantly, we identify CUEDC2 downregulation as a novel characteristic feature of TP53-mutated human cancers. We show that activation of NF-κB due to downregulation of CUEDC2 by miR-324-5p imparts stemness in GOF mutant p53 cancer cells. Finally, we provide evidence that TP53 mutations coupled with high miR-324-5p expression predict poor prognosis in patients with lung adenocarcinoma. Thus, our study delineates an altered miR-324-5p-CUEDC2-NF-κB pathway as a novel regulator of GOF mutant p53-driven cancer stemness. IMPLICATIONS: Our findings implicate miRNA-324-5p as a novel epigenetic modifier of human cancer stemness.

Cancer Stemness: p53 at the Wheel.

The tumor suppressor p53 maintains an equilibrium between self-renewal and differentiation to sustain a limited repertoire of stem cells for proper development and maintenance of tissue homeostasis. Inactivation of p53 disrupts this balance and promotes pluripotency and somatic cell reprogramming. A few reports in recent years have indicated that prevalent TP53 oncogenic gain-of-function (GOF) mutations further boosts the stemness properties of cancer cells. In this review, we discuss the role of wild type p53 in regulating pluripotency of normal stem cells and various mechanisms that control the balance between self-renewal and differentiation in embryonic and adult stem cells. We also highlight how inactivating and GOF mutations in p53 stimulate stemness in cancer cells. Further, we have explored the various mechanisms of mutant p53-driven cancer stemness, particularly emphasizing on the non-coding RNA mediated epigenetic regulation. We have also analyzed the association of cancer stemness with other crucial gain-of-function properties of mutant p53 such as epithelial to mesenchymal transition phenotypes and chemoresistance to understand how activation of one affects the other. Given the critical role of cancer stem-like cells in tumor maintenance, cancer progression, and therapy resistance of mutant p53 tumors, targeting them might improve therapeutic efficacy in human cancers with TP53 mutations.

Genome-Wide Small RNA Sequencing Identifies MicroRNAs Deregulated in Non-Small Cell Lung Carcinoma Harboring Gain-of-Function Mutant p53.

Mutations in the TP53 gene are one of the most frequent events in cancers. Certain missense mutant p53 proteins gain oncogenic functions (gain-of-functions) and drive tumorigenesis. Apart from the coding genes, a few non-coding microRNAs (miRNAs) are implicated in mediating mutant p53-driven cancer phenotypes. Here, we identified miRNAs in mutant p53R273H bearing non-small cell lung carcinoma (NSCLC) cells while using small RNA deep sequencing. Differentially regulated miRNAs were validated in the TCGA lung adenocarcinoma patients with p53 mutations and, subsequently, we identified specific miRNA signatures that are associated with lymph node metastasis and poor survival of the patients. Pathway analyses with integrated miRNA-mRNA expressions further revealed potential regulatory molecular networks in mutant p53 cancer cells. A possible contribution of putative mutant p53-regulated miRNAs in epithelial-to-mesenchymal transition (EMT) is also predicted. Most importantly, we identified a novel miRNA from the unmapped sequencing reads through a systematic computational approach. The newly identified miRNA promotes proliferation, colony-forming ability, and migration of NSCLC cells. Overall, the present study provides an altered miRNA expression profile that might be useful in biomarker discovery for non-small cell lung cancers with TP53 mutations and discovers a hitherto unknown miRNA with oncogenic potential.

p53 gain-of-function mutations increase Cdc7-dependent replication initiation.

Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations.

CD44(high)CD24(low) molecular signature determines the Cancer Stem Cell and EMT phenotype in Oral Squamous Cell Carcinoma.

Almost all epithelial tumours contain cancer stem-like cells, which possess a unique property of self-renewal and differentiation. In oral cancer, several biomarkers including cell surface molecules have been exploited for the identification of this highly tumorigenic population. Implicit is the role of CD44 in defining CSCs but CD24 is not well-explored. Here we show that CD44(high)CD24(low) cells isolated from the oral cancer cell lines, not only express stem cell related genes but also exhibit Epithelial-to-Mesenchymal transition (EMT) characteristics. This CD44(high)CD24(low) population gives rise to all other cell types upon differentiation. Typical Cancer Stem Cell (CSC) phenotypes like increased colony formation, sphere forming ability, migration and invasion were also confirmed in CD44(high)CD24(low) cells. Drug transporters were found to be over-expressed in CD44(high)CD24(low) sub-population thereby contributing to elevated chemo-resistance. To validate our findings in-vivo, we determined the relative expression of CD44 and CD24 in clinical samples of OSCC patients. CD44 expression was consistently high whereas CD24 showed significantly lower expression in tumour tissues. Further, the gene expression profile of the CSC and non-CSC population unravels the molecular pathways which may contribute to stemness. We conclude that CD44(high)CD24(low) represents cancer stem-like cells in Oral Squamous Cell Carcinoma.

Transcriptional control of mitosis: deregulation and cancer.

Research over the past few decades has well established the molecular functioning of mitosis. Deregulation of these functions has also been attributed to the generation of aneuploidy in different tumor types. Numerous studies have given insight into the regulation of mitosis by cell cycle specific proteins. Optimum abundance of these proteins is pivotal to timely execution of mitosis. Aberrant expressions of these mitotic proteins have been reported in different cancer types. Several post-transcriptional mechanisms and their interplay have subsequently been identified that control the level of mitotic proteins. However, to date, infrequent incidences of cancer-associated mutations have been reported for the genes expressing these proteins. Therefore, altered expression of these mitotic regulators in tumor samples can largely be attributed to transcriptional deregulation. This review discusses the biology of transcriptional control for mitosis and evaluates its role in the generation of aneuploidy and tumorigenesis.