Abstract 5483: Investigating the role of the retinoblastoma protein in induction and maintenance of oncogene-induced cellular senescence in tumor suppression

Abstract Oncogene-induced cellular senescence is a tumor suppressor response resulting in irreversible cell cycle exit, and is dependent on the p53 and RB pathways. Loss of p53 has recently been shown to allow re-entry of senescent cells into the cell cycle. The role of the RB1 protein in maintenance of the senescent state is less well studied. We investigated whether RB1 is necessary for the induction as well as the maintenance of oncogene-induced senescence, and we also evaluated whether RB1 activation is sufficient to induce and/or maintain senesce in the absence of a functional p53 pathway. Inactivation of RB1 prior to onset of senescence led to failure of senescence induction, with impaired cell cycle exit and failure to induce senescence markers. On the other hand, loss of RB1 after cells had already entered oncogene-induced senescence was able to reverse some features of the senescence phenotype, but did not result in successful cell division and accumulation. Interestingly, in the absence of p53, constitutively active RB1 was sufficient for cells to undergo oncogene-induced senescence, and effectively prevented colony formation. Finally, while p53 inactivation was sufficient for senescent cells to re-enter the cell cycle, constitutive activation of RB1 prevented cell cycle re-entry, even in the absence of p53. Our findings show that RB1 is necessary for both the induction and maintenance of oncogene-induced senescence, and that active RB is sufficient for senescence induction as well as its maintenance, both in the presence and absence of p53. These results have direct implications on targeting RB activation as a senescence-inducing approach in both p53 wild type and p53-defective premalignant and malignant tumors. Note: This abstract was not presented at the meeting. Citation Format: Mohamad Harajly, Hasan Zalzali, Farah Ghamloush, Raya H. Saab. Investigating the role of the retinoblastoma protein in induction and maintenance of oncogene-induced cellular senescence in tumor suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5483. doi:10.1158/1538-7445.AM2017-5483</jats:p

Temporally distinct roles for tumor suppressor pathways in cell cycle arrest and cellular senescence in Cyclin D1-driven tumor

Abstract Background Cellular senescence represents a tumor suppressive response to a variety of aberrant and oncogenic insults. We have previously described a transgenic mouse model of Cyclin D1-driven senescence in pineal cells that opposes tumor progression. We now attempted to define the molecular mechanisms leading to p53 activation in this model, and to identify effectors of Cyclin D1-induced senescence. Results Senescence evolved over a period of weeks, with initial hyperproliferation followed by cell cycle arrest due to ROS production leading to activation of a DNA damage response and the p53 pathway. Interestingly, cell cycle exit was associated with repression of the Cyclin-dependent kinase Cdk2. This was followed days later by formation of heterochromatin foci correlating with RB protein hypophosphorylation. In the absence of the Cdk4-inhibitor p18Ink4c, cell cycle exit was delayed but most cells eventually showed a senescent phenotype. However, tumors later arose from this premalignant, largely senescent lesion. We found that the p53 pathway was intact in tumors arising in a p18Ink4c-/- background, indicating that the two genes represent distinct tumor suppressor pathways. Upon tumor progression, both p18Ink4c-/- and p53-/- tumors showed increased Cdk2 expression. Inhibition of Cdk2 in cultured pre-tumorigenic and tumor cells of both backgrounds resulted in decreased proliferation and evidence of senescence. Conclusion Our findings indicate that the p53 and the RB pathways play temporally distinct roles in senescence induction in Cyclin D1-expressing cells, and that Cdk2 inhibition plays a role in tumor suppression, and may be a useful therapeutic target.</p

CDK2 Transcriptional Repression Is an Essential Effector in p53-Dependent Cellular Senescence—Implications for Therapeutic Intervention

Abstract Cellular senescence, a form of cell-cycle arrest, is a tumor-suppressor mechanism triggered by multiple tumor-promoting insults, including oncogenic stress and DNA damage. The role of cyclin-dependent kinase 2 (CDK2) regulation has been evaluated in models of replicative senescence, but little is known regarding its role in other senescence settings. Using in vitro and in vivo models of DNA damage–and oncogene-induced cellular senescence, it was determined that activation of the tumor-suppressor protein p53 (TP53) resulted in repression of the CDK2 transcript that was dependent on intact RB. Ectopic CDK2 expression was sufficient to bypass p53-dependent senescence, and CDK2-specific inhibition, either pharmacologically (CVT313) or by use of a dominant-negative CDK2, was sufficient to induce early senescence. Pharmacologic inhibition of CDK2 in an in vivo model of pineal tumor decreased proliferation and promoted early senescence, and it also decreased tumor penetrance and prolonged time to tumor formation in animals lacking p53. In conclusion, for both oncogene- and DNA damage–induced cellular senescence, CDK2 transcript and protein are decreased in a p53- and RB-dependent manner, and this repression is necessary for cell-cycle exit during senescence. Implications: These data show that CDK2 inhibition may be useful for cancer prevention in premalignant hyperproliferative lesions, as well as established tumors. Mol Cancer Res; 13(1); 29–40. ©2014 AACR.</jats:p

CD147 Promotes Tumorigenesis via Exosome-Mediated Signaling in Rhabdomyosarcoma

International audienceRhabdomyosarcoma (RMS) is an aggressive childhood soft-tissue tumor, with propensity for local invasion and distant metastasis. Exosomes are secreted vesicles that mediate paracrine signaling by delivering functional proteins and miRNA to recipient cells. The transmembrane protein CD147, also known as Basigin or EMMPRIN, is enriched in various tumor cells, as well as in tumor-derived exosomes, and has been correlated with poor prognosis in several types of cancer, but has not been previously investigated in RMS. We investigated the effects of CD147 on RMS cell biology and paracrine signaling, specifically its contribution to invasion and metastatic phenotype. CD147 downregulation diminishes RMS cell invasion and inhibits anchorage-independent growth in vitro. While treatment of normal fibroblasts with RMS-derived exosomes results in a significant increase in proliferation, migration, and invasion, these effects are reversed when using exosomes from CD147-downregulated RMS cells. In human RMS tissue, CD147 was expressed exclusively in metastatic tumors. Altogether, our results demonstrate that CD147 contributes to RMS tumor cell aggressiveness, and is involved in modulating the microenvironment through RMS-secreted exosomes. Targeted inhibition of CD147 reduces its expression levels within the isolated exosomes and reduces the capacity of these exosomes to enhance cellular invasive properties

p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

International audienceThe restoration of p53 has been suggested as a therapeutic approach in tumors. However, the timing of p53 restoration in relation to its efficacy during tumor progression still is unclear. We now show that the restoration of p53 in murine premalignant proliferating pineal lesions resulted in cellular senescence, while p53 restoration in invasive pineal tumors did not. The effectiveness of p53 restoration was not dependent on p19 Arf expression but showed an inverse correlation with Mdm2 expression. In tumor cells, p53 restoration became effective when paired with either DNA-damaging therapy or with nutlin, an inhibitor of p53-Mdm2 interaction. Interestingly, the inactivation of p53 after senescence resulted in reentry into the cell cycle and rapid tumor progression. The evaluation of a panel of human supratentorial primitive neuroectodermal tumors (sPNET) showed low activity of the p53 pathway. Together, these data suggest that the restoration of the p53 pathway has different effects in premalignant versus invasive pineal tumors, and that p53 activation needs to be continually sustained, as reversion from senescence occurs rapidly with aggressive tumor growth when p53 is lost again. Finally, p53 restoration approaches may be worth exploring in sPNET, where the p53 gene is intact but the pathway is inactive in the majority of examined tumors

A temporal in vivo catalog of chromatin accessibility and expression profiles in pineoblastoma reveals a prevalent role for repressor elements

Pediatric pineoblastomas (PBs) are rare and aggressive tumors of grade IV histology. Although some oncogenic drivers are characterized, including germline mutations in RB1 and DICER1, the role of epigenetic deregulation andcis-regulatory regions in PB pathogenesis and progression is largely unknown. Here, we generated genome-wide gene expression, chromatin accessibility, and H3K27ac profiles covering key time points of PB initiation and progression from pineal tissues of a mouse model ofCCND1-driven PB. We identified PB-specific enhancers and super-enhancers, and found that in some cases, the accessible genome dynamics precede transcriptomic changes, a characteristic that is underexplored in tumor progression. During progression of PB, newly acquired open chromatin regions lacking H3K27ac signal become enriched for repressive state elements and harbor motifs of repressor transcription factors like HINFP, GLI2, and YY1. Copy number variant analysis identified deletion events specific to the tumorigenic stage, affecting, among others, the histone gene cluster andGas1, the growth arrest specific gene. Gene set enrichment analysis and gene expression signatures positioned the model used here close to human PB samples, showing the potential of our findings for exploring new avenues in PB management and therapy. Overall, this study reports the first temporal and in vivocis-regulatory, expression, and accessibility maps in PB.</jats:p