Induction Of Cellular Senescence Research Articles

Induction and Validation of Cellular Senescence in Primary Human Cells

Induction and Validation of Cellular Senescence in Primary Human Cells

Lung cellular senescence is independent of aging in a mouse model of COPD/emphysema

Cigarette smoke (CS) induces lung cellular senescence that plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). How aging influences cellular senescence and other molecular hallmarks, and increases the risk of CS-induced damage remains unknown. We hypothesized that aging-associated changes in lungs worsen the COPD/emphysema by CS exposure. Younger and older groups of C57BL/6J mice were exposed to chronic CS for 6 months with respective age-matched air-exposed controls. CS caused a decline in lung function and affected the lung structure of both groups of mice. No alterations were observed in the induction of inflammatory mediators between the air-exposed younger and older controls, but aging increased the severity of CS-induced lung inflammation. Aging per se increased lung cellular senescence and significant changes in damage-associated molecular patterns marker S100A8. Gene transcript analysis using the nanoString nCounter showed a significant upregulation of key pro-senescence targets by CS (Mmp12, Ccl2, Cdkn2a, Tert, Wrn, and Bub1b). Aging independently influenced lung function and structure, as well as increased susceptibility to CS-induced inflammation in emphysema, but had a negligible effect on cellular senescence. Thus, aging solely does not contribute to the induction of cellular senescence by CS in a mouse model of COPD/emphysema.

The efficacy of brusatol as a chemotherapeutic agent in pancreatic cancer and its relevance to potential chemoresistance conferred by Nrf2

While several studies link a state of hypothyroidism to extended lifespan of humans and mice, the role of thyroid hormone in cancer is more controversial since tumor-promoting as well as tumor-suppressive effects are known. In general, aberrant thyroid hormone levels are associated with increased cancer incidence. For prostate cancer (PCa) a prospective cohort study indicates that lower thyrotropin (TSH) and higher thyroxin (T4) levels are associated with an increased risk of PCa. However, triiodothyronine (T3) can attenuate PCa progression.Here we show that T3 treatment of human PCa cells reduces cell proliferation, by induction of cellular senescence. Interestingly, we could neither detect an increased expression of p16INK4A nor p21CIP1 cell cycle inhibitors, which are mediators of the two major pathways for senescence induction. This suggests that the T3-induced cellular senescence of PCa cells is driven by an alternative pathway. We show that T3-mediated cellular senescence is associated with increase of DEC1 expression encoded by the BHLHE40 gene and p15INK4B encoded by CDKN2B. Each DEC1/BHLHE40 and p15INK4B/CDKN2B knockdown reduced significantly the level of T3-mediated cellular senescence. The data suggest that DEC1 and p15INK4B are crucial for the T3-induced cellular senescence. In line with a protective role of cellular senescence in cancer, public databases provide evidence linking low DEC1 expression to poor survival of PCa patients. Further we show that the BHLHE40 promoter is responsive to T3 suggesting BHLHE40 being a target gene for the thyroid hormone receptor (TR). Taken together, the data suggest that T3 mediates cellular senescence in PCa cells through induction of DEC1- and p15INK4B -dependent pathway.

SRT1720, a potential sensitizer for radiotherapy and cytotoxicity effects of NVB-BEZ235 in metastatic breast cancer cells

BackgroundChemo-radio therapy (CRT) resistance is a main barrier in treating the triple negative breast cancer (TNBC). The success of conventional treatment may be ameliorated by elevating the responsiveness of the cancer cells to CRT. NVP-BEZ235 as a PI3K/AKT/mTOR dual inhibitor has been shown promising results in treating breast cancer cells. However, potential radiation-sensitizing effect of NVP-BEZ235 in TNBC remained unclear. In addition, SIRT-1 activation state and environmental cytokine were identified as being responsible for cancer cells responses to CRT. Herein, we investigate the role of interleukin 6 (IL-6) as a tumor environmental cytokine and SIRT1 in the effectiveness of NVP-BEZ235 plus radiotherapy. Material and methodsTNBC cells were pre-treated with/without IL-6 and were exposed to single and combination of SRT1720 (SIRT1 activator)/EX-527 (SIRT1 inhibitor) and/or NVP-BEZ235 and/or gamma radiation. The effect of our treatments on cellular growth was determined by MTT and the cellular death and CSCs percentage were determined by Flow cytometry. Senescence detection kit was used to assay the effect of our treatments on cellular senescence induction. ResultsActivation of SIRT1 via SRT1720 increased the efficacy of CRT in TNBC cells, especially when IL-6 exists in tumor microenvironment. Additionally, IL-6 pre-treatment followed by exposure to SRT1720 and NVP-BEZ235 significantly increased sensitivity of the cancer stem cells to radiation (p < 0.05). ConclusionOur result shows that combination of NVP-BEZ235 and SRT1720 may effectively improve late stage breast cancer cells therapeutics approach. Activation of SIRT1 and STAT3 in resistance breast cancer cells improves the in-vitro therapeutic efficacy of CRT.

WW Domain‐containing Oxidoreductase Induces Cellular Senescence in Response to Replication Stress

The induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for the maintenance of cellular genome integrity during DNA replication. Under replication stress, cellular senescence is a DDR mechanism that prevents the proliferation of cells with DNA damage to avoid mitotic anomalies and inheritance of the damage over cell generations. A reduction in the ability of cells to trigger senescence has been suggested to be associated with cancer initiation and progression. Human WWOX gene resides within a common chromosomal fragile site FRA16D and encodes a tumor suppressor WW domain‐containing oxidoreductase. Downregulation of WWOX protein expression has been frequently found in various human cancers. Increased genome instability has been shown in the cells null for WWOX. However, the role of WWOX in DDR remains largely unclear. In this study, we demonstrated that Wwox gene knockout (Wwox−/−) mouse embryonic fibroblasts (MEFs) failed to undergo replication‐induced cellular senescence after multiple passages in culture. Strikingly, accelerated cell cycle progression and increased apoptosis were detected in late‐passage Wwox−/− MEFs. Increased γH2AX‐positive cells were observed in late‐passage Wwox−/− MEFs, indicating massive accumulation of DNA damages in these cells. The p16INK4A/Rb‐ and p53/p21cip1/waf1‐mediated signaling pathways have been suggested to control cellular senescence. We determined significantly reduced expression of p16INK4A and p21cip1/waf1 proteins in late‐passage Wwox−/− MEFs. Treatment of Wwox−/− MEFs with DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine reversed p16INK4A and p21cip1/waf1 expression, and ectopic expression of p16INK4A and p21cip1/waf1 increased cellular senescence in late‐passage Wwox−/− MEFs. These results suggest that hypermethylation of p16INK4A and p21cip1/waf1 promoters may lead to the failure of senescence induction in Wwox−/− MEFs. Moreover, inhibition of ROS production by N‐acetyl‐L‐cysteine during cell replication in vitro increased protein expression of p16INK4A and p21cip1/waf1 and the induction of cellular senescence in Wwox−/− MEFs. In conclusion, our results support a crucial role for WWOX in DDR for maintaining genome integrity via the induction of cellular senescence in response to replication stress.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines.

TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA)-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK) inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment.

Timosaponin A-III inhibits oncogenic phenotype via regulation of PcG protein BMI1 in breast cancer cells.

Polycomb group (PcG) protein BMI1 is an important regulator of oncogenic phenotype and is often overexpressed in several human malignancies including breast cancer. Aberrant expression of BMI1 is associated with metastasis and poor prognosis in cancer patients. At present, therapy reagents that can efficiently inhibit the expression of BMI1 are not very well known. Here, we report that Timosaponin A-III (TA-III), a steroidal saponin obtained from the rhizomes of an herb, Anemarrhena asphodeloides, strongly inhibits expression of BMI1 in breast cancer cells. Treatment of breast cancer cells with TA-III resulted in inhibition of oncogenic phenotypes such as proliferation, migration and invasion, and induction of cellular senescence. Inhibition of these oncogenic phenotypes was accompanied by downregulation of BMI1 expression and histone posttranslational modification activity of PRC1. The mechanistic analysis of TA-III-induced inhibition of oncogenic activity and BMI1 expression suggests that downregulation of c-Myc mediates TA-III effect on BMI1. We further show that exogenous BMI1 overexpression can overcome TA-III-induced inhibition of oncogenic phenotypes. We also show that TA-III induces expression of tumor suppressive miR-200c and miR-141, which are negatively regulated by BMI1. In summary, our data suggest that TA-III is a potent inhibitor of BMI1 and that it can be successfully used to inhibit the growth of tumors where PcG protein BMI1 and PcG activities are upregulated.

Loss of RNA-binding protein HuR facilitates cellular senescence through posttranscriptional regulation of TIN2 mRNA.

Cellular senescence can be induced by high levels of reactive oxygen species (ROS) produced by mitochondria. However, the mechanism by which elevated mitochondrial ROS levels are produced during replicative senescence is not yet fully understood. Here, we report that loss of the RNA-binding protein, human antigen R (HuR), during replicative senescence leads to an increase in ROS levels through enhanced mitochondrial localization of the telomeric protein TIN2. HuR binds to the 3′ untranslated region of TIN2 mRNA. This association decreases TIN2 protein levels by both destabilizing TIN2 mRNA and reducing its translation. Conversely, depletion of HuR levels enhances TIN2 expression, leading to increased mitochondrial targeting of TIN2. Mitochondrial localization of TIN2 increases ROS levels, which contributes to induction and maintenance of cellular senescence. Our findings provide compelling evidence for a novel role of HuR in controlling the process of cellular senescence by regulating TIN2-mediated mitochondrial ROS production, and for a useful therapeutic route for modulating intracellular ROS levels in treating both aging-related complications and cancer.

Transcriptional repression of DNA repair genes is a hallmark and a cause of cellular senescence

Cellular senescence response is (i) activated by numerous stresses, (ii) is characterized by a stable proliferation arrest, and (iii) by a set of specific features. Timely regulated senescence is thought to be beneficial, whereas chronic senescence such as during normal or premature aging is deleterious as it favors most, if not all, age-related diseases. In this study, using in-house or publicly available microarray analyses of transcriptomes of senescent cells, as well as analyses of the level of expression of several DNA repair genes by RT-qPCR and immunoblot, we show that repression of DNA repair gene expression is associated with cellular senescence. This repression is mediated by the RB/E2F pathway and it may play a causal role in senescence induction, as single DNA repair gene repression by siRNA induced features of premature senescence. Importantly, activating RB independently of direct DNA damage also results in repression of DNA repair genes and in the subsequent induction of DNA damage and senescence. The dogma is that DNA damage observed during cellular senescence is directly provoked by DNA lesions following genotoxic attack (UV, IR, and ROS) or by induction of replicative stress upon oncogenic activation. Our in vitro results support a largely unsuspected contribution of the loss of DNA repair gene expression in the induction and the accumulation of the DNA damage observed in most, if not all, kinds of cellular senescence, and thus in the induction of cellular senescence. Further demonstration using in vivo models will help to generalize our findings.

Baicalin induces cellular senescence in human colon cancer cells via upregulation of DEPP and the activation of Ras/Raf/MEK/ERK signaling

Baicalin is a natural flavonoid glycoside which has potent anti-tumor and antioxidant activity in cancer cells. In the present study, we found that baicalin treatment significantly induced senescence in colon cancer cells. Furthermore, baicalin upregulated the expression of decidual protein induced by progesterone (DEPP) in HCT116 colon cancer cells, which accompanied with the activation of Ras/Raf/MEK/ERK and p16INK4A/Rb signaling pathways. Meanwhile, these phenomena also appeared under the anti-oxidation effect exerted by baicalin. In addition, ectopic expression of DEPP in HCT116 cells significantly induced the activity of senescence-associated β-galactosidase (SA-β-Gal) in tumor cells regulated by Ras/Raf/MEK/ERK signaling pathway. Knockdown of DEPP by RNA interference efficiently counteracted the baicalin-mediated growth inhibition, senescence and cell cycle arrest in cancer cells. Importantly, in a xenograft mouse model of human colon cancer, we further confirmed that baicalin treatment dramatically inhibited tumor growth, which was due to the induction of tumor cellular senescence via the upregulation of DEPP and the activation of Ras/Raf/MEK/ERK signaling in vivo. In addition to baicalin treatment, we found that the hypoxia-response protein DEPP functions as a positive regulator involving the regulations of Ras/Raf/MEK/ERK signaling pathway and inhibition of human colon cancer by other anti-oxidative drugs, such as curcumin and sulforaphane, resulting in tumor cellular senescence. These results collectively suggest that baicalin upregulates the expression of DEPP and activates its downstream Ras/Raf/MEK/ERK and p16INK4A/Rb pathways by acting as an antioxidant, leading to senescence in colon cancer cells.

Age-related oxidative changes in pancreatic islets are predominantly located in the vascular system

Aged tissues usually show a decreased regenerative capacity accompanied by a decline in functionality. During aging pancreatic islets also undergo several morphological and metabolic changes. Besides proliferative and regenerative limitations, endocrine cells lose their secretory capacity, contributing to a decline in functional islet mass and a deregulated glucose homeostasis. This is linked to several features of aging, such as induction of cellular senescence or the formation of modified proteins, such as advanced glycation end products (AGEs) - the latter mainly examined in relation to hyperglycemia and in disease models. However, age-related changes of endocrine islets under normoglycemic and non-pathologic conditions are poorly investigated. Therefore, a characterization of pancreatic tissue sections as wells as plasma samples of wild-type mice (C57BL/6J) at various age groups (2.5, 5, 10, 15, 21 months) was performed. Our findings reveal that mice at older age are able to secret sufficient amounts of insulin to maintain normoglycemia. During aging the pancreatic islet area increased and the islet size doubled in 21 months old mice when compared to 2.5 months old mice, whereas the islet number was unchanged. This was accompanied by an age-dependent decrease in Ki-67 levels and pancreatic duodenal homeobox-1 (PDX-1), indicating a decline in proliferative and regenerative capacity of pancreatic islets with advancing age. In contrast, the number of p16Ink4a-positive nuclei within the islets was elevated starting from 10 months of age. Interestingly, AGEs accumulated exclusively in the islet blood vessels of old mice associated with increased amounts of inflammatory markers, such as the inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine (3-NT). In summary, the age-related increase in islet size and area was associated with the induction of senescence, accompanied by an accumulation of non-enzymatically modified proteins in the islet vascular system.

NADPH oxidase 4 mediates ROS production in radiation-induced senescent cells and promotes migration of inflammatory cells

Excessive DNA damage induced by ionising radiation (IR) to normal tissue cells is known to trigger cellular senescence, a process termed stress-induced premature senescence (SIPS). SIPS is often accompanied by the production of reactive oxygen species (ROS), and this is reported to be important for the initiation and maintenance of SIPS. However, the source of ROS during SIPS after IR and their significance in radiation-induced normal tissue damage remain elusive. In the present study, we tested the hypothesis that the NADPH oxidase (NOX) family of proteins mediates ROS production in SIPS-induced cells after IR and plays a role in SIPS-associated biological events. X-irradiation of primary mouse embryonic fibroblasts (MEFs) resulted in cellular senescence and the concomitant increase of intracellular ROS. Among all six murine NOX isoforms (NOX1–4 and DUOX1/2), only NOX4 was detectable under basal conditions and was upregulated following IR. In addition, radiation-induced ROS production was diminished by genetic or pharmacological inhibition of NOX4. Meanwhile, NOX4 deficiency did not affect the induction of cellular senescence after IR. Furthermore, the migration of human monocytic U937 cells to the culture medium collected from irradiated MEFs was significantly reduced by NOX4 inhibition, suggesting that NOX4 promotes the recruitment of inflammatory cells. Collectively, our findings imply that NOX4 mediates ROS production in radiation-induced senescent cells and contributes to normal tissue damage after IR via the recruitment of inflammatory cells and the exacerbation of tissue inflammation.

P16Ink4a and p21Cip1/Waf1 promote tumour growth by enhancing myeloid-derived suppressor cells chemotaxis

p16Ink4a and p21Cip1/Waf1 act as tumour suppressors through induction of cellular senescence. However, senescence-independent roles of these CDK inhibitors are not well understood. Here, we report an unexpected function of p16Ink4 and p21Cip1/Waf1, namely, tumour promotion through chemotaxis. In monocytic myeloid-derived suppressor cells (Mo-MDSCs), p16Ink4 and p21Cip1/Waf1 are highly expressed and stimulate CX3CR1 chemokine receptor expression by preventing CDK-mediated phosphorylation and inactivation of SMAD3. Thus, deletion of p16Ink4 and p21Cip1/Waf1 reduces CX3CR1 expression, thereby inhibiting Mo-MDSC accumulation in tumours expressing CX3CL1 and suppressing the tumour progression in mice. Notably, blockade of the CX3CL1/CX3CR1 axis suppresses tumour growth, whereas inactivation of CDKs elicits the opposite effect. These findings reveal an unexpected function of p16Ink4a and p21Waf1/Cip1 and indicate that regulation of Mo-MDSCs chemotaxis is a valuable potential strategy for control of tumour development.

393 Myeloid cells alter their effector status in response to the secretome of senescent cancer cells

Cellular senescence plays an important role in tissue development, homeostasis and cancer control. It is characterized by a G0/G1 arrest of the cell cycle, stop in proliferation and induction of senescence associated ß-galactosidase activity thereby promoting aging or tissue degeneration and possibly restraining cancer. We recently found in RIP1-Tag2 mice that the cytokines IFNy and TNF, exogenously delivered by immune cells, can arrest cancer growth through the induction of cellular senescence. Thus, in addition to cancer control through killing, the immune system can arrest a variety of human and mouse cancer cells by inducing senescence. However, senescent cancer cells may also become potentially harmful due to their secretory phenotype. Here we analyzed the effect of the senescence associated secretory phenotype (SASP) of murine ß-cancer cells on the differentiation and function of macrophages. ß-cancer cells driven into senescence via IFNy and TNF show a highly inflammatory SASP. Enhanced secretion of cytokines like CCL2, CCL5, CXCL9 or CXCL10 attracts immune cells and modulates their function. The SASP attracted bone-marrow derived macrophages (BMDM) and altered their polarization status and function. The SASP induced migration and polarization towards a M2-like phenotype in BMDMs. Such M2-like macrophages are characterized by lack of iNOS and concurrent increase in arginase1 protein levels. Simultaneously the BMDMs lost their naïve status and showed a decreased phagocytic ability, supporting the concept that antigen presenting cells transform from naïve phagocytizing to effector cells during maturation. Furthermore we showed that non-senescent tumor cells increased proliferation if challenged with the SASP and SASP-chemokines. These data underline that the removal of senescent cancer cells from the organism may be an important cancer-protective mechanism. Taken together, modulating the secretome of senescent cancer cells may change the tumor microenvironment, trigger different anti-tumor immune responses and directly affect the immune responses to non-senescent cancer cells.

The cell cycle regulator protein P16 and the cellular senescence of dental follicle cells.

Cellular senescence is a restricting factor for regenerative therapies with somatic stem cells. We showed previously that the onset of cellular senescence inhibits the osteogenic differentiation in stem cells of the dental follicle (DFCs), although the mechanism remains elusive. Two different pathways are involved in the induction of the cellular senescence, which are driven either by the cell cycle protein P21 or by the cell cycle protein P16. In this study, we investigated the expression of cell cycle proteins in DFCs after the induction of cellular senescence. The induction of cellular senescence was proved by an increased expression of β-galactosidase and an increased population doubling time after a prolonged cell culture. Cellular senescence regulated the expression of cell cycle proteins. The expression of cell cycle protein P16 was up-regulated, which correlates with the induction of cellular senescence markers in DFCs. However, the expression of cyclin-dependent kinases (CDK)2 and 4 and the expression of the cell cycle protein P21 were successively decreased in DFCs. In conclusion, our data suggest that a P16-dependent pathway drives the induction of cellular senescence in DFCs.

Analysis of individual cells identifies cell-to-cell variability following induction of cellular senescence.

SummarySenescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single‐cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell‐to‐cell variability resulted in a loss of correlation among the expression of several senescence‐associated genes. Many genes encoding senescence‐associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.

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

Abstract LB-335: Induction of Ribosomal Checkpoint Induced Senescence (RCIS) for the treatment of liver cancer

Abstract While ribosomal proteins are generally considered to be essential genes, the existence of ribosomopathies suggests that a therapeutic window for exploiting ribosomal proteins as targets for cancer therapy may exist. We report distinct stress responses of tumor cells and normal cells upon knockdown of Rpl15, a ribosomal protein which was identified in a direct in vivo shRNA screen for new therapeutic targets in liver cancer. While normal cells undergo a reversible cell cycle arrest, we found that in tumor cells, the knockdown of Rpl15 triggers a ribosomal stress response followed by induction of cellular senescence, designated ribosomal checkpoint induced senescence (RCIS). Importantly, Rpl15 suppression triggered RCIS independent of any reduction in global protein translation. Using well established therapy resistant murine HCC models, we show that shRNAs targeting Rpl15 potently suppress tumor development in genetically diverse murine HCCs. Using Rpl15 shRNA transgenic mice, allowing for ubiquitous shRNA mediated suppression of Rpl15, we show that systemic Rpl15 suppression can be tolerated for up to 5 days, thus revealing a therapeutic window for metronomic Rpl15 inhibitory therapies. However, molecular modeling analyses revealed that Rpl15 is not druggable by small molecule inhibitors and we thus set out to explore whether RCIS can be induced via interference with other ribosomal proteins or other factors involved in ribosome biogenesis. We generated and screened a focused shRNA library targeting 41 ribosomal proteins and 19 ribosome biogenesis factors and found that apart from Rpl15, only a small subset of shRNAs scored. From a druggability point of view it was interesting, that shRNAs targeting components of the RNA polymerase I complex had scored, as recently a pharmacological RNA polymerase I inhibitor became available. Both genetic and pharmacological inhibition of RNA polymerase I induced RCIS and mediated an excellent prolongation of survival (far superior to the clinically used standard therapy Sorafenib), however in contrast to targeting Rpl15, targeting RNA polymerase I failed to achieve full long term tumor suppression. Mechanistically we found a less efficient immune mediated clearance of senescent cells as a possible explanation. In line with the idea that distinct secretory profiles underlie differences in immune mediated clearance of RCIS cells, cytokine arrays revealed distinct secretory profiles of RCIS induced by Rpl15 suppression and RCIS induced via shRNA or pharmacological RNA polymerase I inhibition. Citation Format: Katharina Wolter, Marina Pesic, Sabrina Klotz, Nicolas Herranz, Torsten Wuestefeld, Tae-Won Kang, Marco Seehawer, Rishabh Chawla, Stefan Zwirner, Jonathan Cotton, Benyuan Zhou, Marcel Krüger, Frank Klawonn, Thomas Longerich, Bence Sipos, Bernd Pichler, Jesus Gil, Martin Eilers, Prem K. Premsrirut, Antti Poso, Lars Zender. Induction of Ribosomal Checkpoint Induced Senescence (RCIS) for the treatment of liver cancer [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 LB-335. doi:10.1158/1538-7445.AM2017-LB-335

Abstract 4525: Hypoxia signaling pathway plays a role in ovarian cancer chemoresistance

Abstract Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor that when induced regulates the expression of many genes involved in cytoprotective stimuli, which attenuates apoptosis and improves survival.. Increased expression of HIF-1α gene (HIF1A) has been found in several carcinomas, including ovarian cancer. Ovarian cancers are generally refractory to platinum-based chemotherapy. Despite the large number of studies, molecular events that govern the emergence of aggressive therapy-resistant cells after chemotherapy are poorly defined. Genomic instabilities, such as copy number variation(CNV), may play an important role in chemoresistance and have been implicated in many complex diseases, like cancer.. We analyzed CNV data that is publically available through the Cancer Genome Atlas and others. Of particular interest was the transcription factor HIF1A which plays an integral role in oxidative stress response such as those induced by chemotherapy reagents. The present study provides evidence for the rare escape of tumor cells from drug-induced cell death by entering a non-cycling senescent state. We report the adaptive response of human ovarian surface epithelium cells to CoCl2, a chemical hypoxia-mimicking agent resulting in a senescent-like state of chemoresistant cells. The effect of the treatment was evaluated on CNV of HIF-1α gene expression, cell proliferation, survival, and tumor invasiveness. We show here that CNV duplication events of HIF1α results in an oxidative stress response in cells leading to chemoresistance through the induction of cellular senescence. Understanding the molecular events associated with chemoresistance will ultimately lead to better patient treatment and outcomes. Note: This abstract was not presented at the meeting. Citation Format: Noelle L. Cutter, Tyler Walther, Kimberly H. Doyle, Ryan Frank. Hypoxia signaling pathway plays a role in ovarian cancer chemoresistance [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 4525. doi:10.1158/1538-7445.AM2017-4525

Cellular Senescence, Neurological Function, and Redox State

Cellular senescence, characterized by permanent cell cycle arrest, has been extensively studied in mitotic cells such as fibroblasts. However, senescent cells have also been observed in the brain. Even though it is recognized that cellular energetic metabolism and redox homeostasis are perturbed in the aged brain and neurodegenerative diseases (NDDs), it is still unknown which alterations in the overall physiology can stimulate cellular senescence induction and their relationship with the former events. Recent Advances: Recent findings have shown that during prolonged inflammatory and pathologic events, the blood-brain barrier could be compromised and immune cells might enter the brain; this fact along with the brain's high oxygen dependence might result in oxidative damage to macromolecules and therefore senescence induction. Thus, cellular senescence in different brain cell types is revised here. Most information related to cellular senescence in the brain has been obtained from research in glial cells since it has been assumed that the senescent phenotype is a feature exclusive to mitotic cells. Nevertheless, neurons with senescence hallmarks have been observed in old mouse brains. Therefore, although this is a controversial topic in the field, here we summarize and integrate the observations from several studies and propose that neurons indeed senesce. It is still unknown which alterations in the overall metabolism can stimulate senescence induction in the aged brain, what are the mechanisms and signaling pathways, and what is their relationship to NDD development. The understanding of these processes will expose new targets to intervene age-associated pathologies.-Antioxid. Redox Signal. 28, 1704-1723.