Studies on Δ40p53 mediated regulation of specific coding and non-coding RNAs: Consequences on cellular processes

Abstract

p53 is a crucial transcriptional regulator which influences almost every possible pathway that impinges on the life of a cell. As expected of such an instrumental protein, the molecular network of p53 is as diverse as it is intense. Our laboratory has been working on the only known translational isoform of p53, Δ40p53. Δ40p53 isoform controls the folding, oligomerization and post-translational modifications of p53 complexes, thereby regulating the transcriptional and growth suppressive effects of p53. p53 has been intricately integrated into the non-coding RNA network of the cell through rigorous research over the last decade. Several non-coding RNAs that p53 regulates have been identified over the past few years of extensive studies. However, the role of Δ40p53 in regulating non-coding RNAs either via co-regulation through hetero-tetramerization with full-length p53 (FLp53) or via direct regulatory mechanisms has remained largely unexplored. p53, in consistence with its effect as the guardian angel of cells, is inhibitory to the development and survival of cancer stem cells. Δ40p53 has proven to be different from its "big brother," FLp53. It has been implicated in anti-aging, maintenance of hematopoietic stem cells and developmental programs. Against this anomalous functional behavior of Δ40p53, a pertinent question is whether this isoform plays a role in developing and maintaining cancer stem cells or stem-like cells.

To explore the direct regulatory mechanisms of ∆40p53, we have studied its role in regulating non-coding RNAs and its possible consequences on cellular processes. Moreover, we have also studied the atypical function of Δ40p53 in the biogenesis of stem-like cells. Therefore, the specific objectives are as follows: 1. To study the mechanism and implications of lncRNA regulation by ∆40p53. 2. To elucidate the specific role of ∆40p53 in modulating the expression of miRNAs and their downstream mRNA targets. 3. To investigate the definite effect of ∆40p53 on the biogenesis of cancer stem-like cells mediated by regulation of cellular mRNAs

Objective 1: To study the mechanism and implications of lncRNA regulation by ∆40p53

To study the lncRNAs that are influenced differentially by ∆40p53 and FLp53, an RNA microarray was earlier performed in our laboratory by overexpression of ∆40p53 and FLp53 in H1299 cells (p53 null adenocarcinoma cell line). From the same data set, we validated the lncRNAs, of which many were uncharacterized in the literature. Among the lncRNAs, we selected LINC00176 for further detailed investigation due to its known function of miRNA regulation and differential effect on cellular processes. Upon validation, LINC00176 levels were affected predominantly by the overexpression and knockdown of ∆40p53. Further, under DNA damage, ER stress, and glucose deprivation, LINC00176 was upregulated in HCT116 p53-/- cells (harboring only ∆40p53) compared to HCT116 p53+/+ cells. To understand the mechanism of regulation of the lncRNA, ChIP, RNA Stability and UV-Crosslinking Assays were performed. It revealed that Δ40p53 (and not FLp53) could transactivate LINC00176 transcriptionally and regulate its stability. Results suggest that the increased abundance of LINC00176 in the presence of ∆40p53 compared to p53 could be due to its dual regulation at the genome and transcriptome level. LINC00176 was ectopically overexpressed and partially silenced in HCT116-/- cells to determine the direct cellular effect. Results indicate that LINC00176 increases epithelial cell markers while reducing cell viability and proliferation. Interestingly, RNA pulldown also revealed that LINC00176 sequesters several putative miRNA targets (miR-761, miR-184, miR-508, miR-138, miR-15b-5p) to regulate the cellular processes. Results provide essential insights into the pivotal role of ∆40p53 in regulating the novel LINC00176 RNA-microRNA-mRNA axis independent of FLp53 and in maintaining cellular homeostasis.

Objective 2: To elucidate the specific role of ∆40p53 in modulating the expression of miRNAs and their downstream mRNA targets.

We previously demonstrated that Δ40p53 independently regulates miR-186-5p-YY1 to reduce cell proliferation, implying that it may have broader effects on the expression of other micro RNAs. In this study, we performed small RNA sequencing to identify the pool of miRNAs differentially regulated by ∆40p53 and FLp53. We report that the expression of specific miRNAs is regulated explicitly by ∆40p53. miR-4671-5p, miR-548ae-5p, miR-301b-5p, and miR-34a-5p were shortlisted for further studies based on their significance and differential fold changes obtained in the sequencing. Preliminary results suggested that miR-4671-5p gets differentially regulated, with a significant decrease in its levels upon ∆40p53 overexpression and no change with FLp53 overexpression. miR-4671-5p abundance changed according to the ratios of FLp53 to ∆40p53, indicating the physiological relevance of its regulation by both isoforms. We performed bioinformatic analysis and selected N-sulfoglucosamine sulfohydrolase (SGSH), cyclin-dependent kinases (CDK) 11B and CDK5 regulatory subunit 1 (CDK5R1) as the potential targets of miR-4671-5p, all of which are involved in driving cell cycle progression. To check if miR-4671-5p directly binds to the potential targets, wild-type and mutant target 3'UTRs were cloned. Luciferase assay with the wild type and mutant 3'UTRs in the presence of miR-4671-5p depicted CDK5R1 and SGSH as direct targets of miR-4671-5p. SGSH gene expression levels have potential prognostic relevance on survival that trends in the opposite direction of miR-4671-5p levels associated with the same cancer types, supporting a possible physiological relevance of the interaction. Overexpression of miR-4671-5p directly inhibited SGSH and triggered intra-S-phase cell cycle arrest. ∆40p53-miR-4671-5p-SGSH axis emerges as a novel axis capable of regulating cell cycle progression. These results enhance understanding of ∆40p53 functions mediated by miRNAs that help to maintain metabolic and cellular homeostasis independently of FLp53.

Objective 3: To investigate the role of ∆40p53 in the biogenesis of cancer stem-like cells

Cancer stem cells are a specific subpopulation of cells in a cancerous background, which enjoy the ability of self-renewal and the enhanced capability to generate tumors. p53 is known to inhibit the survival and maintenance of cancer stem cells. However, in this context, Δ40p53 has proven to be different from its "big brother." In a different context, Δ40p53 has been integrated into various development programs and is involved in the maintenance of stemness. It has also been shown to inhibit aging. The molecular factors causing such ∆40p53-mediated consequences are largely unexplored. To characterize the differences in cancer stem-like cell propagation in the presence of p53 and ∆40p53, we have investigated Cancer Stem Cell phenotypic profiles in HCT116-/- and HCT116+/+ cell lines and evaluated their correlation with several CSC functional properties, including spheroid formation ability, side population (SP) phenotype, cell surface marker expression and sensitivity to anticancer compounds.

A higher percentage of cancer stem-like cells (side population cells) was observed in ∆40p53 (in HCT116-/-) than in HCT116+/+. Further, chemosensitivity assays, analysis of stem cell markers, and sphere formation assay with HCT116-/- and HCT116+/+ elucidated new insights into our understanding of the ∆40p53 function. HCT116-/- showed chemoresistance with an increased dosage of 5-FU (5-Fluorouracil) compared to HCT116+/+, which was sensitive under the same drug conditions. We also observed large irregular spheroids for HCT116-/- compared to smaller ones in HCT116+/+. The differential phenotypes observed in the two cell lines indicated that various molecular players could drive them. Therefore, a total RNA sequencing was performed with HCT116-/- and HCT116+/+. We found 206 significantly differentially expressed mRNAs between the two cell lines confirming molecular level changes contributing to the above phenotypes. Further, some of the significant genes were validated, correlating to our above observations and enhancing our understanding of ∆40p53-mediated functions.

Taken together, we have investigated the exclusive role of ∆40p53 on non-coding RNAs (miRNAs and lncRNAs) and their consequences on cellular processes. Parallelly, we have also investigated the atypical role of ∆40p53 (taking p53 as a reference point) in maintaining cancer stem-like cells driving cancer progression.