Myelin protein zero-like 3 (MPZL3) is an Immunoglobulin-containing transmembrane protein with predicted cell adhesion molecule function. Loss of 11q23, where the
ABSTRACT High grade serous ovarian cancer (HGSOC) is the most lethal gynecological cancer. Platinumbased therapies such as cisplatin are standard-of-care for HGSOC patients; however, the majority of HGSOCs initially treated with cisplatin will recur with widespread disseminated disease. Cisplatin induces cellular senescence, a stable cell cycle arrest. Although they are non-proliferative, senescent cells secrete a complex mix of cytokines and small molecules, named the senescence associated secretory phenotype (SASP), that have been shown to have pro-tumorigenic effects. To investigate how the SASP contributes to HGSOC progression, we used conditioned media from cisplatin therapy-induced senescent cells to culture naïve HGSOC spheroids. We report that while the SASP does not affect spheroid formation, the adhesion of cells within spheroids is altered, leading to cell detachment from spheroids. Interestingly, our data indicate that this occurs in an MMP-independent manner. Analysis of RNA-Seq samples indicates many adhesion-related genes and adhesion factors are transcriptionally downregulated by the SASP, particularly fibronectin and integrins, which was validated by immunofluorescence in spheroids. These data reveal that senescent cells contribute to a transcriptional program in nearby cancer cells in a paracrine fashion that decreases their adhesion, which may contribute to tumor dissemination.
This study aims to decipher crucial biomarkers regulated by p73 for the early detection of colorectal cancer (CRC) by employing a combination of integrative bioinformatics and expression profiling techniques. The transcriptome profile of HCT116 cell line p53
Abstract p73 is a member of the p53 tumor suppressor family, which transactivates p53-responsive genes and mediates DNA damage response. Recent evidences suggest that p73 exerts its tumor suppressor functions by suppressing metastasis, but the exact mechanism remains unknown. It is increasingly evident that long non-coding RNAs (lncRNAs) play a significant role in tumor suppression. However, we still have a limited knowledge of the clinical significance of lncRNAs in colorectal cancer. The present study is aimed to identify novel lncRNAs that play a role in p73-mediated suppression of metastasis in colorectal cancer cells. p73 was knocked down in HCT116p53−/−p73+/+ cells and transcriptome analysis was performed to detect the differentially expressed lncRNAs in presence and absence of p73. The top two up-regulated and down-regulated lncRNAs were selected for further analysis based on the false discovery rate (FDR), fold-change (FC) and P-values. Out of these, FER1L4 lncRNA was found to be significantly induced by DNA damage in a p73-dependent manner. Through bioinformatics analysis, we identified two p73-binding sites in FER1L4 promoter. Consistent with this, p73 binding to FER1L4 promoter was confirmed through luciferase reporter assays and Chromatin Immunoprecipitation (ChIP) assays. Site-directed mutagenesis of both the binding sites totally abrogated p73 responsiveness, indicating that both the sites are equally responsible and essential for p73 binding. In addition, Real-time quantitative PCR demonstrated a rapid increase in endogenous FER1L4 mRNA upon induction of p73. Furthermore, knockdown of FER1L4 and p73 significantly increased the invasion and migration rate of colorectal cancer cells as confirmed by wound-healing assay. Also, knockdown of FER1L4 decreased the expression of E-cadherin, a cancer metastasis suppressor, and increased the expression of other prominent EMT markers such as N-cadherin, Snail, Vimentin and Fibronectin. Cell functional assays further revealed that FER1L4 causes a G2/M cell cycle arrest in a p73-dependent manner in HCT116p53−/−p73+/+ colon cancer cells and upon FER1L4kd, normal cell cycle progression was observed. Annexin V/PI and TUNEL apoptosis assays revealed that FER1L4 induced apoptosis in HCT116p53−/−p73+/+ colon cancer cells with increase in time-dependent treatment of etoposide and FER1L4kd inhibited apoptosis even in the presence of p73. The protein expression level of several pro-apoptotic genes such as Bad, Bax, Bik, Bim, BID, Bak and PUMA decreased upon FER1L4kd and p73kd, confirming that FER1L4 plays a role in p73-mediated apoptosis and cell cycle arrest. Taken together, we provide evidence that FER1L4 is a direct transcriptional target of p73 and p73 exerts its anti-metastatic function by inducing the expression of FER1L4 in response to genotoxic stress. Citation Format: Apoorva Uboveja, Yatendra Kumar Satija, Chanchal Bareja, Daman Saluja. Long noncoding RNA FER1L4 is a novel p73 transcriptional target and inhibits colon cancer cell migration and invasion in a p73-dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4707.
<div>Abstract<p>p16 is a tumor suppressor encoded by the <i>CDKN2A</i> gene whose expression is lost in approximately 50% of all human cancers. In its canonical role, p16 inhibits the G<sub>1</sub>–S-phase cell cycle progression through suppression of cyclin-dependent kinases. Interestingly, p16 also has roles in metabolic reprogramming, and we previously published that loss of p16 promotes nucleotide synthesis via the pentose phosphate pathway. However, the broader impact of p16/<i>CDKN2A</i> loss on other nucleotide metabolic pathways and potential therapeutic targets remains unexplored. Using CRISPR knockout libraries in isogenic human and mouse melanoma cell lines, we determined several nucleotide metabolism genes essential for the survival of cells with loss of p16/<i>CDKN2A</i>. Consistently, many of these genes are upregulated in melanoma cells with p16 knockdown or endogenously low <i>CDKN2A</i> expression. We determined that cells with low p16/<i>CDKN2A</i> expression are sensitive to multiple inhibitors of <i>de novo</i> purine synthesis, including antifolates. Finally, tumors with p16 knockdown were more sensitive to the antifolate methotrexate <i>in vivo</i> than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/<i>CDKN2A</i><sup>low</sup> tumors as loss of p16/<i>CDKN2A</i> may provide a therapeutic window for these agents.</p>Significance:<p>Antimetabolites were the first chemotherapies, yet many have failed in the clinic due to toxicity and poor patient selection. Our data suggest that p16 loss provides a therapeutic window to kill cancer cells with widely-used antifolates with relatively little toxicity.</p></div>
Abstract Introduction: p73 transcription factor belongs to the p53 tumor suppressor family. Recent studies revealed that p73 wields its tumor suppressor properties by inhibiting metastasis. Although the literature on the p73 transcriptional circuit has chiefly concentrated on protein-coding genes, it has been progressively pointed out that p73 is also able to transcriptionally modulate non-coding RNA (ncRNA) members. These involve microRNAs (miRNAs) and many p53-regulated long non-coding RNAs (lncRNAs). Methods: Identification of p73 binding sites in the FER1L4 promoter region was made by JASPER and TF BIND which was further confirmed by chromatin immunoprecipitation (ChIP) and site-directed mutagenesis experiments. Effect of FER1L4 in p73 mediated cell cycle arrest and apoptosis was checked by cell cycle analysis, Annexin-V/PI, and TUNEL apoptosis assays. Depletion of FER1L4 enhanced cell proliferation, migration, and invasion in a p73-dependent manner. Furthermore, RNA-In situ hybridization (RNA-ISH) analysis of non-metastatic and metastatic human colon cancer tissue samples was carried out to compare the levels of FER1L4 and p73 in metastatic and non-metastatic tumor tissue samples. We also checked the expression of different miRNA including miR1273g-3p and its effect on PTEN expression. Results: We have identified lncRNA FER1L4 as a novel p73 transcriptional target that gets induced as a result of genotoxic stress. The binding of p73 to FER1L4 promoter was established by bioinformatics analysis, luciferase reporter, and ChIP assays. Both FER1L4 and p73 knockdown enhanced the migration and invasion rate of colorectal cancer cells. FER1L4 knockdown reduced E-cadherin expression and enhanced the expression of N-cadherin, Vimentin, Snail, and Fibronectin. Cell cycle assays revealed that FER1L4 induces a G2/M cell cycle arrest in a p73-dependent manner. Annexin V/PI and TUNEL assays revealed FER1L4 induced apoptosis in HCT116p53-/-p73+/+ colon cancer cells under genotoxic stress and FER1L4 knockdown inhibited apoptosis even in the presence of p73. The expression of pro-apoptotic markers such as Bad, Bax, Bik, Bim, Bid, Bak and PUMA decreased upon FER1L4 and p73 knockdown. FER1L4 sponges the expression of miR-1273g-3p, which in turn increases PTEN expression leading to cell cycle arrest. RNA In-situ hybridization revealed the down-regulation of both p73 and FER1L4 expression in metastatic colon cancer tissue as compared to non-metastatic tissue. Conclusion: We provide conclusive proof that p73 exerts its anti-metastatic properties by inducing lncRNA FER1L4 in response to genotoxic stress which in turn sponges the expression of miR1273g-3p, a regulator of PTEN. Citation Format: Daman Saluja, Apoorva Uboveja, Yatendra kumar Satija, Fouzia Siraj. Deciphering the mechanism of action of Long non-coding RNA fer1l4 in the suppression of invasion, migration and metastasis of colon cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1552.
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