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Long non-coding RNA GAS5 acts as proliferation "brakes" in CD133+ cells responsible for tumor recurrence.

ABSTRACT: Presence of quiescent, therapy evasive population often described as cancer stem cells (CSC) or tumor initiating cells (TIC) is often attributed to extreme metastasis and tumor recurrence. This population is typically enriched in a tumor as a result of microenvironment or chemotherapy induced stress. The TIC population adapts to this stress by turning on cell cycle arrest programs that is a "fail-safe" mechanism to prevent expansion of malignant cells to prevent further injury. Upon removal of the "stress" conditions, these cells restart their cell cycle and regain their proliferative nature thereby resulting in tumor relapse. Growth Arrest Specific 5 (GAS5) is a long-non-coding RNA that plays a vital role in this process. In pancreatic cancer, CD133+ population is a typical representation of the TIC population that is responsible for tumor relapse. In this study, we show for the first time that emergence of CD133+ population coincides with upregulation of GAS5, that reprograms the cell cycle to slow proliferation by inhibiting GR mediated cell cycle control. The CD133+ population further routed metabolites like glucose to shunt pathways like pentose phosphate pathway, that were predominantly biosynthetic in spite of being quiescent in nature but did not use it immediately for nucleic acid synthesis. Upon inhibiting GAS5, these cells were released from their growth arrest and restarted the nucleic acid synthesis and proliferation. Our study thus showed that GAS5 acts as a molecular switch for regulating quiescence and growth arrest in CD133+ population, that is responsible for aggressive biology of pancreatic tumors.

SUBMITTER: Sharma NS

PROVIDER: S-EPMC6861230 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Long non-coding RNA GAS5 acts as proliferation "brakes" in CD133+ cells responsible for tumor recurrence.

Sharma Nikita S NS Gnamlin Prisca P Durden Brittany B Gupta Vineet K VK Kesh Kousik K Garrido Vanessa T VT Dudeja Vikas V Saluja Ashok A Banerjee Sulagna S

Oncogenesis 20191118 12

Presence of quiescent, therapy evasive population often described as cancer stem cells (CSC) or tumor initiating cells (TIC) is often attributed to extreme metastasis and tumor recurrence. This population is typically enriched in a tumor as a result of microenvironment or chemotherapy induced stress. The TIC population adapts to this stress by turning on cell cycle arrest programs that is a "fail-safe" mechanism to prevent expansion of malignant cells to prevent further injury. Upon removal of t ...[more]

PMID: 31740660

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Project description:BackgroundMalignant pleural mesothelioma (MPM) is an aggressive cancer with short overall survival. Long non-coding RNAs (lncRNA) are a class of RNAs more than 200 nucleotides long that do not code for protein and are part of the 90% of the human genome that is transcribed. Earlier experimental studies in mice showed GAS5 (growth arrest specific transcript 5) gene deletion in asbestos driven mesothelioma. GAS5 encodes for a lncRNA whose function is not well known, but it has been shown to act as glucocorticoid receptor decoy and microRNA "sponge". Our aim was to investigate the possible role of the GAS5 in the growth of MPM.MethodsPrimary MPM cultures grown in serum-free condition in 3% oxygen or MPM cell lines grown in serum-containing medium were used to investigate the modulation of GAS5 by growth arrest after inhibition of Hedgehog or PI3K/mTOR signalling. Cell cycle length was determined by EdU incorporation assay in doxycycline inducible short hairpinGAS5 clones generated from ZL55SPT cells. Gene expression was quantified by quantitative PCR. To investigate the GAS5 promoter, a 0.77 kb sequence was inserted into a pGL3 reporter vector and luciferase activity was determined after transfection into MPM cells. Localization of GAS5 lncRNA was identified by in situ hybridization. To characterize cells expressing GAS5, expression of podoplanin and Ki-67 was assessed by immunohistochemistry.ResultsGAS5 expression was lower in MPM cell lines compared to normal mesothelial cells. GAS5 was upregulated upon growth arrest induced by inhibition of Hedgehog and PI3K/mTOR signalling in in vitro MPM models. The increase in GAS5 lncRNA was accompanied by increased promoter activity. Silencing of GAS5 increased the expression of glucocorticoid responsive genes glucocorticoid inducible leucine-zipper and serum/glucocorticoid-regulated kinase-1 and shortened the length of the cell cycle. Drug induced growth arrest was associated with GAS5 accumulation in the nuclei. GAS5 was abundant in tumoral quiescent cells and it was correlated to podoplanin expression.ConclusionsThe observations that GAS5 levels modify cell proliferation in vitro, and that GAS5 expression in MPM tissue is associated with cell quiescence and podoplanin expression support a role of GAS5 in MPM biology.

Project description:The present study aimed to investigate the potential role of long non-coding RNA growth arrest-specific transcript 5 (lncRNA GAS5) in the progression of esophageal squamous cell carcinoma (ESCC) and to reveal its possible regulatory mechanism. The expression of lncRNA GAS5 in ESCC tissues and cell lines was analyzed using reverse transcription-quantitative polymerase chain reaction and western blot analysis. The overexpression vector pc-GAS5 and control vector pc-negative control (NC), containing no GAS5 sequence, were transfected into ESCC cells. The effects of lncRNA GAS5 overexpression on cell proliferation, cell cycle distribution, cell migration and invasion were then analyzed. Besides, the expression levels of ATM-CHK2 pathway-associated proteins and epithelial-mesenchymal transition (EMT)-associated proteins were measured. Expression of lncRNA GAS5 was downregulated in the ESCC tissues compared with adjacent normal tissues, and was also downregulated in ESCC Kyse450 cells compared with the human esophageal epithelial HET-1A cell line. Additionally, lncRNA GAS5 was successfully overexpressed in ESCC cells following transfection with pc-GAS5. Overexpression of lncRNA GAS5 significantly inhibited cell proliferation, induced cell cycle arrest at G2/M phase and suppressed cellular migration and invasion. When cells were transfected with pc-GAS5, the levels of phosphorylated (p)-ATM serine/threonine protein kinase, p-checkpoint kinase 2 (CHK2), p-cell division cycle 25C, p-cyclin-dependent kinase 1, N-cadherin, vimentin and Snail were significantly increased, whereas that of E-cadherin were markedly decreased. The results of the present study indicate that overexpression of lncRNA GAS5 may inhibit cell proliferation, migration and invasion in ESCC. lncRNA GAS5 overexpression may induce cell cycle arrest at G2/M stage by activating the ATM-CHK2 pathway. The results of the current study further indicate that lncRNA GAS5 overexpression may suppress cell migration and invasion via EMT-associated proteins. lncRNA GAS5 could therefore serve as a potential target for ESCC therapy.

Project description:BackgroundEpithelial ovarian cancer (EOC) is the malignant tumor of the female reproductive system with the highest fatality rate. Tolerance of chemotherapeutic drugs like cisplatin (DDP) occurring in very early stage is one of the important factors of the poor prognosis of epithelial ovarian cancer. Here we aim to study the dysregulation of a particular long noncoding RNA, lncRNA GAS5, and its role in EOC progression.MethodsThe low expression of lncRNA GAS5 in EOC tissues and OC cell lines was determined by microarray analyses and Real-Time qPCR. Flow cytometer assays were used to detect cell cycle and apoptosis of OC cells. CCK8 assay were performed to investigate the DDP sensitivity of OC cells. Western blot was carried out to detect cell growth markers, apoptotic markers, PARP1, E2F4, MAPK pathway protein expression and other protein expression in OC cell lines. The binding of GAS5 and E2F4 were proved by RNA pull-down and RIP assay. The effect of E2F4 on PARP1 were determined by CHIP-qPCR assay and luciferase reporter assay. The effect of lncRNA GAS5 on OC cells was assessed in vitro and in vivo.ResultsBy microarray (3 EOC tissues νs. 3 normal ovary tissues) and RT- qPCR (53 EOC tissues νs. 10 normal ovary tissues) we identified lncRNA GAS5 to be dramatically low expressed in EOC samples and correlated with prognosis. Compared with sensitive cell lines, GAS5 was also low expressed in DDP resistant OC cell lines, and over-expression of GAS5 significantly enhanced the sensitivity of OC cells to DDP in vivo and in vitro. Meanwhile the over-expression of GAS5 also caused OC cells G0/G1 arrest and apoptosis increase. Mechanistically, GAS5 might regulate PARP1 expression by recruiting the transcription factor E2F4 to its promoter, and then affect the MAPK pathway activity. Due to the 5'TOP structure, GAS5 could be regulated by transcription inhibitor rapamycin in OC cells.ConclusionHere we explored the specific mechanisms of EOC cisplatin resistance and tumor progress due to lncRNA-GAS5, presented the GAS5-E2F4-PARP1-MAPK axis and its role in OC drug-sensitivity and progression for the first time, and the results may provide experimental basis for clinical application.

Project description:PurposeLong noncoding RNAs (lncRNAs) have been identified as an important class of noncoding RNAs that are deeply involved in multiple biological processes in tumorigenesis. This study is to investigate the critical roles and biological function of lncRNA growth arrest-specific 5 (GAS5) in tumorigenesis of laryngeal squamous cell carcinoma (LSCC).Patients and methodsA total of 59 samples of LSCC and paired adjacent tissue, as well as corresponding clinicopathological information were collected. GAS5 expression in both LSCC tissues and human SUN1076 and SNU899 cell lines were analyzed by Real-time quantitative RT-PCR method. Ectopic expression of GAS5 by vector transfection in LSCC cell lines and followed by in vitro experiments was to investigate the critical roles and function of GAS5 in LSCC. Cell Counting Kit 8 (CCK8) assay and PE/7AAD Annexin V Apoptosis analysis was to evaluate cell proliferation ability and cell apoptosis. Co-transfection of GAS5 and miR-21 was to explore the interaction between GAS5 and miR-21 in LSCC. BAX and CDK6 protein level were analyzed by western blot method.ResultsThis study demonstrated that GAS5 was significantly downregulated in LSCC tissue and human LSCC cell lines. GAS5 levels were correlated with the clinicopathological features of LSCC patients. In addition, the ectopic expression of GAS5 significantly inhibited cell proliferation and promoted apoptosis. Co-expression analyses indicated that GAS5 is negatively correlated with miR-21 in LSCC tissues. Overexpression of miR-21 eliminated GAS5-mediated cell apoptosis and proliferation suppression. Furthermore, GAS5, which upregulated BAX mRNA expression and downregulated CDK6 mRNA expression, was reversed by ectopic expression of miR-21.ConclusionGAS5 suppresses LSCC progression through the negative regulation of miR-21 and its targets involved in cell proliferation and apoptosis, indicating that GAS5 may serve as a biomarker and potential target for LSCC therapy.

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Long non-coding RNA GAS5 acts as proliferation "brakes" in CD133+ cells responsible for tumor recurrence.

Publications

Long non-coding RNA GAS5 acts as proliferation "brakes" in CD133+ cells responsible for tumor recurrence.

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