P53-induced Cell Cycle Arrest Research Articles

Genome-Wide Analysis of p53 Targets Reveals SCN2A as a Novel Player in p53-Induced Cell Arrest in HPV-Positive Cells.

The host transcription factor p53 is a critical tumor suppressor in HPV-induced carcinogenesis, regulating target genes involved in cell cycle arrest and apoptosis. However, the p53 targets have not been thoroughly analyzed in HPV-infected cells. In this study, p53 signaling in HPV16 and HPV18 cells was activated by depleting the viral oncoprotein E6. Subsequently, p53-regulated genes were identified by comparing them with genes altered in p53-silenced cells. True p53 targets were defined as genes with at least one overlapping p53 binding site and ChIP peak near their locus. Our analysis revealed that while some p53 targets were common to both the HPV16 and HPV18 cells, the majority of the targets differed between these two types, potentially contributing to the varying prevalence of HPV16 and HPV18 in cervical cancer. Additionally, we identified SCN2A as a novel p53 target involved in p53-induced cell cycle arrest in HPV-related carcinogenesis. This study provides new insights into the mechanisms by which p53 inhibits HPV-induced carcinogenesis.

Disulfiram Upgrades the Radiosensitivity of Osteosarcoma by Enhancing Apoptosis and P53-Induced Cell Cycle Arrest.

The prognosis of osteosarcoma has not been improved for decades. As radioresistance is one of the major reasons, effective radiotherapy sensitization drugs need to be discovered. HOS and K7M2 osteosarcoma cell lines were treated with disulfiram (DSF) and radiation to assess cell viability, proliferation, migration ability, apoptosis level, ROS and Ca2+ level, and cell cycle in vitro. A HOS-derived subcutaneous tumor mouse model was constructed to evaluate tumor growth after DSF combined with radiation, and the Tunel assay and immunohistochemistry of Ki67 were conducted. Western blot was used to evaluate the protein expression level. The IC50 and working concentration of DSF in osteosarcoma cell lines were ascertained. When combined with radiation, DSF effectively suppressed cell viability, proliferation, and migration, while enhancing apoptosis in osteosarcoma cells. The cell cycle postirradiation exhibited a downward shift in the G1 phase, but the addition of DSF counteracted this trend. The combination of DSF and radiation exhibited inhibitory effects on tumor growth invivo, which was corroborated by Ki67 staining and Tunel assay. Western blot analysis revealed that DSF upregulated the expression of P53, P21, CDKN2C, BAX, and cleaved Caspase-3 while downregulating BCL2, CDK4/6, and CyclinD1 after irradiation. Our results document that DSF exerts its radiosensitization effects invivo and in vitro, and is a valuable radiosensitizing drug option for osteosarcoma. The radiosensitization effect is mainly achieved by activating the apoptotic pathway and promoting cell cycle arrest induced by P53/P21 and CDKN2C after irradiation.

Abstract B033: Histone H3.3 K27M and G34R/V glioma driver mutations inhibit mitotic phosphorylation of H3.3 S31, driving tumor formation by inducing chromosomal instability

Abstract Diffuse gliomas are pediatric high-grade gliomas that frequently harbor heterozygous histone H3.3 K27M, G34R, or G34V mutations. These mutations alter histone methylations, although the precise mechanism(s) of gliomagenesis remains uncertain. H3.3 contains a unique Serine at position 31 that is phosphorylated in prophase and dephosphorylated in anaphase during normal cell division. Previously we have reported that chromosome missegregation triggers H3.3 S31 phosphorylation along chromosome arms in ana/telophase leading to p53-induced cell cycle arrest. Here we show, in vitro and in vivo, that H3.3 K27M, G34R, and G34V suppress S31 phosphorylation by its mitotic kinase, Chk1. In diploid cells, overexpression of H3.3 K27M, G34R, G34V, or non-phosphorylatable S31A significantly increases the frequency of chromosome missegregation, while H3.3 K36M did not. This missegregation phenotype could be rescued by overexpression of K27M/S31E or G34R/S31E double mutants. Next, we used CRISPR/Cas9 gene editing to revert K27M or G34V patient-derived DMG cells to WT or replace the mutant allele with S31A. Reversion to WT increased metaphase S31 phosphorylation and reduced chromosome missegregations, but inserting an S31A mutation caused more mitotic defects. Following a mitotic error, newly formed aneuploid cells overexpressing K27M, G34R, or G34V suppress p53 levels and continue to divide, while WT overexpressing cells always arrest. To test the effects of diminished S31 phosphorylation on gliomagenesis, we utilized a novel mouse model of glioma. Expression of PDGFß linked to H3.3K27M, H3.3G34R, and H3.3S31A in neural stem cells in vivo promoted the development of high-grade gliomas in under 100 days, whereas PDFß-H3.3WT did not. Notably, the S31A tumors had histologically normal levels of K27me3 and K36me3. This work shows that a single glioma-associated point mutation in H3.3 can generate chromosomal instability, and which is a major contributing factor to H3.3 K27M and G34-altered glioma formation. Citation Format: Charles A. Day, Florina Grigore, Alyssa Langfald, Faruck Hakkim, David Daniels, Kevin Vaughan, James Robinson, Edward Hinchcliffe. Histone H3.3 K27M and G34R/V glioma driver mutations inhibit mitotic phosphorylation of H3.3 S31, driving tumor formation by inducing chromosomal instability [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B033.

Extra centrosomes induce PIDD1-mediated inflammation and immunosurveillance.

Unscheduled increases in ploidy underlie defects in tissue function, premature aging, and malignancy. A concomitant event to polyploidization is the amplification of centrosomes, the main microtubule organization centers in animal cells. Supernumerary centrosomes are frequent in tumors, correlating with higher aggressiveness and poor prognosis. However, extra centrosomes initially also exert an onco-protective effect by activating p53-induced cell cycle arrest. If additional signaling events initiated by centrosomes help prevent pathology is unknown. Here, we report that extra centrosomes, arising during unscheduled polyploidization or aberrant centriole biogenesis, induce activation of NF-κB signaling and sterile inflammation. This signaling requires the NEMO-PIDDosome, a multi-protein complex composed of PIDD1, RIPK1, and NEMO/IKKγ. Remarkably, the presence of supernumerary centrosomes suffices to induce a paracrine chemokine and cytokine profile, able to polarize macrophages into a pro-inflammatory phenotype. Furthermore, extra centrosomes increase the immunogenicity of cancer cells and render them more susceptible to NK-cell attack. Hence, the PIDDosome acts as a dual effector, able to engage not only the p53 network for cell cycle control but also NF-κB signaling to instruct innate immunity.

Genome-wide CRISPR screening uncovers potential targets and mechanisms of vincristine resistance in DLBCL.

In this issue, Rovsing et al. employ unbiased genome-wide CRISPR screening and functional cellular assays to investigate the cellular response to vincristine, an important component of the front-line DLBCL treatment R-CHOP. Their findings reveal intriguing targets and mechanisms that hold promise for enhancing DLBCL treatment and provide a foundation for the development of future drug regimens. This research prompts further exploration of the translational potential to advance more effective and individualized approaches in the clinical management of DLBCL. Commentary on: Rovsing et al. Resistance to vincristine in DLBCL by disruption of p53-induced cell cycle arrest and apoptosis mediated by KIF18B and USP28. Br J Haematol 2023;202:825-839.

Resistance to vincristine in DLBCL by disruption of p53-induced cell cycle arrest and apoptosis mediated by KIF18B and USP28.

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous Phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. The inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 of which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine-induced p53 response suggesting that resistance to vincristine has origin in the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens.

CCRG-02. THE DIFFUSE MIDLINE GLIOMA MUTATIONS, H3.3 K27M AND G34R/V, INHIBIT MITOTIC PHOSPHORYLATION OF SERINE 31, LEADING TO TUMOR FORMATION THROUGH INCREASED CHROMOSOMAL INSTABILITY

Abstract Diffuse midline gliomas (DMG) are high-grade pediatric gliomas that frequently harbor heterozygous histone H3.3 K27M, G34R, or G34V mutations. These mutations alter histone methylations, although the precise mechanism(s) of gliomagenesis remains uncertain. H3.3 contains a unique Serine at position 31 that is phosphorylated in prophase and dephosphorylated in anaphase during normal cell division. In an aberrant division, chromosome missegregation triggers S31 phosphorylation along chromosome arms in anaphase leading to p53-induced cell cycle arrest. Here we show in vitro and in vivo, that H3.3 K27M, G34R, and G34V significantly suppress S31 phosphorylation by Chk1 (the mitotic S31 kinase). In diploid cells, overexpression of H3.3 K27M, G34R, G34V, or non-phosphorylatable S31A significantly increases the frequency of chromosome missegregation, while H3.3 K36M did not. This missegregation phenotype could be rescued by overexpression of K27M/S31E or G34R/S31E double mutants. Next, we used genome editing to revert K27M or G34V patient-derived DMG cells to WT or replace the mutant allele with S31A. Reversion to WT increased metaphase S31 phosphorylation and reduced chromosome missegregations, but inserting an S31A mutation caused more mitotic defects. Following a mitotic error, newly formed aneuploid cells overexpressing K27M, G34R, or G34V suppress p53 levels and continue to divide, while WT overexpressing cells always arrest. To test the effects of diminished S31 phosphorylation on gliomagenesis, we utilized a novel mouse model of DMG. Expression of PDGFβ linked to H3.3K27M, H3.3G34R, and H3.3S31A in neural stem cells in vivo promoted the development of high-grade gliomas in under 100 days, whereas PDGFβ-H3.3WT did not. Notably, the S31A tumors had histologically normal levels of K27me3 and K36me3. This work shows that a single DMG-associated point mutation in H3.3 can generate chromosomal instability and suggests that chromosomal instability is a major contributing factor to DMG K27-altered and DMG G34-altered tumor formation.

Albendazole exerts an anti-hepatocellular carcinoma effect through a WWOX-dependent pathway

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most common cause of cancer-related deaths. The WW-domain containing oxidoreductase (WWOX) protein suppresses carcinogenesis and its absence is closely related to aggressive HCC phenotypes. In this study, by using SPR analysis, cell viability assay and xenograft mice models, we found that albendazole (ABZ), a safe and effective anthelmintic drug, exhibited the binding affinity with WWOX protein and potential inhibition effect on HCC cells in vitro and in vivo. Overexpression and knockdown of WWOX confirmed that the suppression of HCC by ABZ. Flow cytometric analysis, western blotting analysis and Co-IP were conducted to study the mechanism of ABZ. Our data showed that ABZ regulated the interaction between WWOX and its binding proteins including p53 and C-MYC. Furthermore, ABZ triggered p53-induced intrinsic apoptosis and suppressed EMT-mediated migration by C-MYC/Fibronectin axis. In addition, ∆NP73 expression was significantly inhibited by ABZ, which further sensitized p53-induced intrinsic apoptosis and cell cycle arrest. In summary, ABZ could suppress the proliferation and migration of HCC cells by regulating WWOX-dependent signaling pathway.

CBIO-11. HISTONE H3.3 G34R/V MUTATIONS STIMULATE PEDIATRIC HIGH-GRADE GLIOMA FORMATION THROUGH THE INDUCTION OF CHROMOSOMAL INSTABILITY

Abstract H3.3 G34R/V mutations are drivers of high-grade pediatric glioma (pHGG). H3.3 G34R/V mutations are linked to altered H3.3 K36 trimethylation (H3K36me3); implicating epigenetic gene regulation as a possible contributor to pHGG formation. Here we show that H3.3 G34R/V also induces chromosomal instability (CIN); a hallmark of pHGG. If CIN promotes pHGG formation is unknown. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. And, H3.3 G34R reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Chk1 regulates chromosome segregation through phosphorylation of pericentromeric H3.3 S31 during early mitosis. Overexpression of H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells caused a significant increase in CIN. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of CIN as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in anaphase. However, following chromosome missegregation, phospho-S31 spreads and stimulates p53-induced cell cycle arrest. Here we show that WT p53 cells expressing mutant G34 fail to arrest following chromosome mis-segregation. These studies demonstrate that H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferating CIN cells. To determine if this process contributes to tumorigenesis, we used RCAS Nestin-TVA mice to overexpress H3.3 WT, G34R, or S31A – P2A-linked to PDGFB expression in glial precursor cells of newborn mice. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free. Our work implicates CIN as a driver of H3.3 G34 mutant pHGG formation. Our ongoing studies utilize K36M and double mutants to further define the contributions of S31 phosphorylation (CIN) and H3K36me3 (epigenetic gene regulation) to tumorigenesis.

Developmental Acquisition of p53 Functions.

Remarkably, the p53 transcription factor, referred to as “the guardian of the genome”, is not essential for mammalian development. Moreover, efforts to identify p53-dependent developmental events have produced contradictory conclusions. Given the importance of pluripotent stem cells as models of mammalian development, and their applications in regenerative medicine and disease, resolving these conflicts is essential. Here we attempt to reconcile disparate data into justifiable conclusions predicated on reports that p53-dependent transcription is first detected in late mouse blastocysts, that p53 activity first becomes potentially lethal during gastrulation, and that apoptosis does not depend on p53. Furthermore, p53 does not regulate expression of genes required for pluripotency in embryonic stem cells (ESCs); it contributes to ESC genomic stability and differentiation. Depending on conditions, p53 accelerates initiation of apoptosis in ESCs in response to DNA damage, but cell cycle arrest as well as the rate and extent of apoptosis in ESCs are p53-independent. In embryonic fibroblasts, p53 induces cell cycle arrest to allow repair of DNA damage, and cell senescence to prevent proliferation of cells with extensive damage.

Circulating MIR-34 is associated with the development of knee osteoarthritis in community-dwelling older men

Purpose: MicroRNAs (miRNAs) are involved in various biological processes, including cell apoptosis, differentiation, development, proliferation, and metabolism. MicroRNA-34 (miR-34) is essential as a tumor suppressor gene in downregulating its target genes via SIRT/p53 signaling pathway. MiR-34 is also involved in p53-induced cell cycle arrest, cell senescence, apoptosis, and other biological behaviors. In a previous study, silencing miR-34 by intra-articular injection of lentivirus may attenuate disease progression in a rat model of OA.

Phenanthroline impairs βAPP processing and expression, increases p53 protein levels and induces cell cycle arrest in human neuroblastoma cells

Mis-functional βAPP processing is deemed to be the major phenomenon resulting in increased neuronal cell death, impaired neurogenesis and the loss of synapses, which eventually manifest as the complex symptoms of Alzheimer’s disease. Despite of several milestones having been achieved in the field of drug development, the stigma of the disorder as an incurable disease still remains. Some ADAM proteases mediate the physiological non-amyloidogenic α-secretase processing of βAPP that generates neuroprotective sAPPα production. Earlier studies have also pointed out the role of p53 in Alzheimer’s disease neuropathology, although a direct link with metalloprotease activities remains to be established. In this study, we explored the consequences of α-secretase inhibition on p53 status in cultured human neuroblastoma SH-SY5Y cells by means of specific inhibitors of ADAM10 and ADAM17 and the metal chelator and general metalloprotease inhibitor phenanthroline. We establish that, beyond the ability of all inhibitors to affect sAPPα production to varying degrees, phenanthroline specifically and dose-dependently lessened βAPP expression, a phenomenon that correlated with a strong increase in p53 protein levels and a concomitant decrease of the p53-degrading calpain protease. Furthermore, treatment of cells at concentrations of phenanthroline similar to those inducing increased levels of p53 induced cell cycle arrest leading to apoptosis. Altogether, our results identify new roles of phenanthroline in perturbing βAPP, p53 and calpain biology, and suggest that the use of this compound and its derivatives as antimicrobial and anti-cancer therapies might trigger Alzheimer’s disease pathogenesis.

The pseudo-caspase FLIP(L) regulates cell fate following p53 activation

p53 is the most frequently mutated, well-studied tumor-suppressor gene, yet the molecular basis of the switch from p53-induced cell-cycle arrest to apoptosis remains poorly understood. Using a combination of transcriptomics and functional genomics, we unexpectedly identified a nodal role for the caspase-8 paralog and only human pseudo-caspase, FLIP(L), in regulating this switch. Moreover, we identify FLIP(L) as a direct p53 transcriptional target gene that is rapidly up-regulated in response to Nutlin-3A, an MDM2 inhibitor that potently activates p53. Genetically or pharmacologically inhibiting expression of FLIP(L) using siRNA or entinostat (a clinically relevant class-I HDAC inhibitor) efficiently promoted apoptosis in colorectal cancer cells in response to Nutlin-3A, which otherwise predominantly induced cell-cycle arrest. Enhanced apoptosis was also observed when entinostat was combined with clinically relevant, p53-activating chemotherapy in vitro, and this translated into enhanced in vivo efficacy. Mechanistically, FLIP(L) inhibited p53-induced apoptosis by blocking activation of caspase-8 by the TRAIL-R2/DR5 death receptor; notably, this activation was not dependent on receptor engagement by its ligand, TRAIL. In the absence of caspase-8, another of its paralogs, caspase-10 (also transcriptionally up-regulated by p53), induced apoptosis in Nutlin-3A-treated, FLIP(L)-depleted cells, albeit to a lesser extent than in caspase-8-proficient cells. FLIP(L) depletion also modulated transcription of canonical p53 target genes, suppressing p53-induced expression of the cell-cycle regulator p21 and enhancing p53-induced up-regulation of proapoptotic PUMA. Thus, even in the absence of caspase-8/10, FLIP(L) silencing promoted p53-induced apoptosis by enhancing PUMA expression. Thus, we report unexpected, therapeutically relevant roles for FLIP(L) in determining cell fate following p53 activation.

Voltage‐gated sodium channels β3 subunit promotes tumorigenesis in hepatocellular carcinoma by facilitating p53 degradation

The voltage-gated sodium channels (VGSCs) are aberrantly expressed in a variety of tumors and play an important role in tumor growth and metastasis. Here, we show that VGSCs auxiliary β3 subunit, encoded by the SCN3B gene, promotes proliferation and suppresses apoptosis in HepG2 cells by promoting p53 degradation. β3 significantly increases HepG2 cell proliferation, promotes tumor growth in mouse xenograft models, and suppresses senescence and apoptosis. We found that β3 knockdown stabilizes p53 protein, leading to potentiation of p53-induced cell cycle arrest, senescence, and apoptosis. Mechanistic studies revealed that β3 could bind to p53, promoting p53 ubiquitination and degradation by stabilizing the p53/MDM2 complex. Our results suggest that β3 is a novel negative regulator of p53 and a potential oncogenic factor.

Abstract P2018: PRR Mediates P53 Activation in Aristolochic Acid Induced Renal Proximal Tubule Cell Injury

Aristolochic acid (AA) induces renal proximal tubular injury leading to inflammation, atrophy and fibrosis. These effects of AA were reported to be mediated by p53 induced cell cycle arrest and apoptosis. Recently, our laboratory demonstrated that the (pro)renin receptor (PRR) promotes podocyte inflammation and apoptosis. We hypothesized that PRR mediates AA induced proximal tubular injury via upregulation of p53 activation. Mouse immortalized proximal tubule cells (PTCs) were grown to 70% confluence and transfected with either scramble (Scr) or PRR siRNA followed by treatment with either AA 10ug/mL or culture media alone (control) for 48 hours prior to harvesting for RNA and protein extraction (n=3 for all treatment groups). Compared to control, AA treatment alone significantly increased PRR mRNA expression by 202% (p<0.05). Compared to Scr treatment, PRR siRNA reduced PRR mRNA expression by 49% (p<0.05). Compared to AA treatment, AA+PRR siRNA ameliorated the observed increase in PRR mRNA. Total p53 mRNA expression was not significantly changed by treatment with PRR siRNA or AA. However, compared to the control group, p53 phosphorylation (activity) increased by 167% in the AA treated group (p<0.05). PRR shRNA attenuated (40% reduction) the observed increase in p53 phosphorylation that was induced by AA treatment (p<0.05). We conclude that PRR mediates increased activity of p53 in response to AA in proximal tubule cells.

DREAM and RB cooperate to induce gene repression and cell-cycle arrest in response to p53 activation.

Most human cancers acquire mutations causing defects in the p53 signaling pathway. The tumor suppressor p53 becomes activated in response to genotoxic stress and is essential for arresting the cell cycle to facilitate DNA repair or to initiate apoptosis. p53-induced cell cycle-arrest is mediated by expression of the CDK inhibitor p21WAF1/Cip1, which prevents phosphorylation and inactivation of the pocket proteins RB, p130, and p107. In a hypophosphorylated state, pocket proteins bind to E2F factors forming RB-E2F and DREAM transcriptional repressor complexes. Here, we analyze the influence of RB and DREAM on p53-induced gene repression and cell-cycle arrest. We show that abrogation of DREAM function by knockout of the DREAM component LIN37 results in a reduced repression of cell-cycle genes. We identify the genes repressed by the p53-DREAM pathway and describe a set of genes that is downregulated by p53 independent of LIN37/DREAM. Most strikingly, p53-dependent repression of cell-cycle genes is completely abrogated in LIN37−/−;RB−/− cells leading to a loss of the G1/S checkpoint. Taken together, we show that DREAM and RB are key factors in the p53 signaling pathway to downregulate a large number of cell-cycle genes and to arrest the cell cycle at the G1/S transition.

Synthesis and evaluation of modified chalcone based p53 stabilizing agents

Tumor suppressor protein p53 induces cell cycle arrest and apoptotic cell death in response to various cellular stresses thereby preventing cancer development. Activation and stabilization of p53 through small organic molecules is, therefore, an attractive approach for the treatment of cancers retaining wild-type p53. In this context, a series of nineteen chalcones with various substitution patterns of functional groups including chloro, fluoro, methoxy, nitro, benzyloxy, 4-methyl benzyloxy was prepared using Claisen-Schmidt condensation. The compounds were characterized using NMR, HRMS, IR and melting points. Evaluation of synthesized compounds against human colorectal (HCT116) and breast (CAL-51) cancer cell lines revealed potent antiproliferative activities. Nine compounds displayed GI50 values in the low micromolar to submicromolar range; for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (SSE14108) showed GI50 of 0.473±0.043µM against HCT116 cells. Further analysis of these compounds revealed that (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (SSE14105) and (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) caused rapid (4 and 8-h post-treatment) accumulation of p53 in HCT116 cells similar to its induction by positive control, Nutlin-3. Such activities were absent in 3-(4-methoxyphenyl)propiophenone (SSE14106H2) demonstrating the importance of conjugated ketone for antiproliferative and p53 stabilizing activity of the chalcones. We further evaluated p53 levels in the presence of cycloheximide (CHX) and the results showed that the p53 stabilization was regulated at post-translational level through blockage of its degradation. These chalcones can, therefore, act as fragment leads for further structure optimization to obtain more potent p53 stabilizing agents with enhanced anti-proliferative activities.

Abstract A31: Reprimo, a potential tumor suppressor gene TP53-dependent, modulates negatively cell migration and invasion in the MDA-MB-231 breast cancer cell line

Abstract Background: Reprimo, a highly glycosylated protein, is a new downstream effector of p53-induced cell cycle arrest at the G2/M checkpoint, and a putative tumor suppressor gene frequently silenced via methylation of its promoter region in several malignances. The aim of this study was to characterize the epigenetic inactivation and its biological function in BC cell lines. Methods: The correlation between Reprimo methylation and loss of mRNA expression was assessed in six breast cancer cell lines by methylation specific PCR (MSP), 5-Aza-2′-deoxycytidine treatment and qRT-PCR assays. MDA-MB-231 cells were chosen to investigate the phenotypic effect of Reprimo in cell proliferation, cell cycle, cell death, cell migration and invasion. Results: In the cancer methylome system (CMS) (web-based system for visualizing and analyzing genome-wide methylation data of human cancers), the CpG island region of RPRM (1,1Kb) was hypermethylated in breast cancer compared to normal breast tissue. Downregulation of RPRM mRNA by methylation was confirmed in MDA-MB-231 and BT-20 cell lines. In addition, overexpression of RPRM in MDA-MB-231 cells resulted in decreased rates of cell migration, wound healing and invasion in vitro (P < 0.0001). However, RPRM overexpression did not alter cell viability, rate of death cell and G2/M cell cycle transition in this biological model. Conclusion: Taken together, these data suggest that RPRM is involved in decreased cell migration and invasion in vitro, acting as a potential tumor suppressor gene in the MDA-MB-231 cell line. Citation Format: Kurt Buchegger, Tamara Viscarra, Carmen Gloria Ili, Ismael Riquelme, Pablo Letelier, Alejandro Corvalan, Priscilla Brebi, Tim Hui-Ming Huang, Juan Carlos Roa. Reprimo, a potential tumor suppressor gene TP53-dependent, modulates negatively cell migration and invasion in the MDA-MB-231 breast cancer cell line. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A31.

Reprimo as a modulator of cell migration and invasion in the MDA-MB-231 breast cancer cell line.

BackgroundReprimo (RPRM), a highly glycosylated protein, is a new downstream effector of p53-induced cell cycle arrest at the G2/M checkpoint, and a putative tumor suppressor gene frequently silenced via methylation of its promoter region in several malignances. The aim of this study was to characterize the epigenetic inactivation and its biological function in BC cell lines.MethodsThe correlation between RPRM methylation and loss of mRNA expression was assessed in six breast cancer cell lines by methylation specific PCR (MSP), 5′-Aza-2′-deoxycytidine treatment and RT-PCR assays. MDA-MB-231 cells were chosen to investigate the phenotypic effect of RPRM in cell proliferation, cell cycle, cell death, cell migration and invasion.ResultsIn the cancer methylome system (CMS) (web-based system for visualizing and analyzing genome-wide methylation data of human cancers), the CpG island region of RPRM (1.1 kb) was hypermethylated in breast cancer compared to normal breast tissue; more interesting still was that ERα(+) tumors showed higher methylation intensity than ERα(−). Downregulation of RPRM mRNA by methylation was confirmed in MDA-MB-231 and BT-20 cell lines. In addition, overexpression of RPRM in MDA-MB-231 cells resulted in decreased rates of cell migration, wound healing and invasion in vitro. However, RPRM overexpression did not alter cell viability, phosphatidylserine (PS) translocation or G2/M cell cycle transition.ConclusionTaken together, these data suggest that RPRM is involved in decreased cell migration and invasion in vitro, acting as a potential tumor suppressor gene in the MDA-MB-231 cell line.

Loss of Expression of Reprimo, a p53-induced Cell Cycle Arrest Gene, Correlates with Invasive Stage of Tumor Progression and p73 Expression in Gastric Cancer.

Reprimo (RPRM), a downstream effector of p53-induced cell cycle arrest at G2/M, has been proposed as a putative tumor suppressor gene (TSG) and as a potential biomarker for non-invasive detection of gastric cancer (GC). The aim of this study was to evaluate the epigenetic silencing of RPRM gene by promoter methylation and its tumor suppressor function in GC cell lines. Furthermore, clinical significance of RPRM protein product and its association with p53/p73 tumor suppressor protein family was explored. Epigenetic silencing of RPRM gene by promoter methylation was evaluated in four GC cell lines. Protein expression of RPRM was evaluated in 20 tumor and non-tumor matched cases. The clinical significance of RPRM association with p53/p73 tumor suppressor protein family was assessed in 114 GC cases. Tumor suppressor function was examined through functional assays. RPRM gene expression was negatively correlated with promoter methylation (Spearman rank r = -1; p = 0.042). RPRM overexpression inhibited colony formation and anchorage-independent growth. In clinical samples, RPRM gene protein expression was detected in 75% (15/20) of non-tumor adjacent mucosa, but only in 25% (5/20) of gastric tumor tissues (p = 0.001). Clinicopathological correlations of loss of RPRM expression were significantly associated with invasive stage of GC (stage I to II-IV, p = 0.02) and a positive association between RPRM and p73 gene protein product expression was found (p<0.0001 and kappa value = 0.363). In conclusion, epigenetic silencing of RPRM gene by promoter methylation is associated with loss of RPRM expression. Functional assays suggest that RPRM behaves as a TSG. Loss of expression of RPRM gene protein product is associated with the invasive stage of GC. Positive association between RPRM and p73 expression suggest that other members of the p53 gene family may participate in the regulation of RPRM expression.