Loss Of Proliferative Capacity Research Articles

Timing the spinal cord development with neural progenitor cells losing their proliferative capacity: a theoretical analysis

In the developing neural tube in chicken and mammals, neural stem cells proliferate and differentiate according to a stereotyped spatiotemporal pattern. Several actors have been identified in the control of this process, from tissue-scale morphogens patterning to intrinsic determinants in neural progenitor cells. In a previous study (Bonnet et al. eLife 7, 2018), we have shown that the CDC25B phosphatase promotes the transition from proliferation to differentiation by stimulating neurogenic divisions, suggesting that it acts as a maturating factor for neural progenitors. In this previous study, we set up a mathematical model linking fixed progenitor modes of division to the dynamics of progenitors and differentiated populations. Here, we extend this model over time to propose a complete dynamical picture of this process. We start from the standard paradigm that progenitors are homogeneous and can perform any type of divisions (proliferative division yielding two progenitors, asymmetric neurogenic divisions yielding one progenitor and one neuron, and terminal symmetric divisions yielding two neurons). We calibrate this model using data published by Saade et al. (Cell Reports 4, 2013) about mode of divisions and population dynamics of progenitors/neurons at different developmental stages. Next, we explore the scenarios in which the progenitor population is actually split into two different pools, one of which is composed of cells that have lost the capacity to perform proliferative divisions. The scenario in which asymmetric neurogenic division would induce such a loss of proliferative capacity appears very relevant.

Evaluation of radiation-induced genotoxicity on hu-man melanoma cells (SK-MEL-37) by flow cytometry

Micronucleus assay is a test used to evaluate genotoxic damage in cells, which can be caused by various factors, like ionizing radiation. Interactions between radiation energies and DNA can cause breakage, leading to use chromosomal mutations or loss of genetic material, important events that could be induced in solid tumors to mitigate its expansion within human body. Melanoma has been described as a tumor with increased radio resistance. This work evaluated micronuclei percentages (%MN) in human melanoma cells (SK-MEL-37), irradiated by gamma radiation, with doses between 0 and 16Gy. Cell suspensions were irradiated in PBS by a 60Co source in doses between 0 and 16Gy, and incubated by 48h. Then cell membranes were lysed in the presence of SYTOX Green and EMA dyes, preserving nuclear membranes. Using this method, EMA-stained nuclei could be discriminated as those derived from dead cells, and SYTOX nuclei and micronuclei could be quantified. Micronuclei percentages were found to be proportional to dose, (R2 = 0.997). Only the highest dose (16Gy) could induce statistically significant increase of MN (p<0.0001), although cultures irradiated by 4, 8 and 16Gy showed significant increase of dead cell fractions. Calculation of the nuclei-to-beads ratio showed that 8 and 16Gy could reduce melanoma cell proliferation. Results showed that although cell death and loss of proliferative capacity could be observed on cultures irradiated at lower doses, genotoxic damage could be induced only on a higher dose. Resistance to radiation-induced genotoxicity could explain a relatively high radio resistance of melanoma tumors.

Elevated Local Senescence in Diabetic Wound Healing Is Linked to Pathological Repair via CXCR2

Cellular senescence can be broadly defined as a stable, but essentially irreversible, loss of proliferative capacity. Historically, senescence has been described as a negative outcome of advanced cellular age. It is now clear, however, that senescence represents a dynamic autonomous stress response, integral to long-term tumor suppression. Transient induction of a senescent phenotype has actually been suggested to promote regeneration in both liver and skin. Here, we explored the role of senescence in pathological aged and diabetic murine wound healing. Aged and diabetic wounds had greater numbers of senescent cells, and diabetic macrophages maintained altered retention of polarization and produced a CXCR2-enriched senescence-associated secretory phenotype (i.e., SASP). Of translational relevance, targeted expression of CXCR2 in primary human dermal fibroblasts led to paracrine induction of nuclear p21. Furthermore, a selective agonist to CXCR2 was able to reverse delayed healing in diabetic mice and accelerate exvivo human skin wound healing. Collectively, these data suggest a hitherto unappreciated role for CXCR2 in mediating cellular senescence in pathological wound repair.

Differential effects of protein kinase C-eta on apoptosis versus senescence

Protein kinase C-eta (PKCη) is considered an anti-apoptotic kinase, which promotes cell survival and chemoresistance in several cancers, including breast cancer. We have recently shown that PKCη positively regulates the anti-apoptotic protein Mcl-1 in breast cancer cells, and depletion of PKCη induced proteasomal degradation of Mcl-1. We therefore examined if depletion of PKCη would enhance cellular sensitivity to chemotherapeutic agents. Silencing of PKCη by siRNA attenuated apoptosis induced by doxorubicin and paclitaxel in both MCF-7 and T47D breast cancer cells. While silencing of Mcl-1 caused a substantial increase in apoptosis induced by doxorubicin, the combined knockdown of PKCη and Mcl-1 was less effective. Depletion of PKCη also caused an increase in the abundance of the cell cycle inhibitor p27 and a decrease in the clonogenic survival of MCF-7 and T47D cells. PKCη knockdown was associated with an increase in senescence-associated β-galactosidase (SA-β-gal) activity but this increase was attenuated by knockdown of p27. The suppression of doxorubicin-induced apoptosis by PKCη knockdown was partially relieved when p27 was depleted. Since loss of proliferative capacity during senescence could cause resistance to chemotherapeutic drugs, our results suggest that PKCη knockdown inhibits apoptosis by inducing p27-mediated senescence.

Abstract 464: Cell death and senescence: Role of protein kinase C in promoting senescence

Abstract Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. Senescent cells undergo massive genome modulation including induction of the senescence-associated secretory phenotype (SAPS). Components of SASP can implement cell cycle arrest or recruit the immune system to clear senescent cells, thus contributing to tumor suppression. However, some secreted pro-inflammation molecules, act as pro-tumorigenic agents contributing to tumor progression, suggesting that elimination of senescent cells could be beneficial. Very few studies showed a role for PKC in senescence. Here we show that PKCeta, an epithelial specific and anti-apoptotic kinase (epithelia is the origin of about 90% of human tumors), promotes senescence induced by oxidative stress and DNA damage. Using PKCeta-knockdown breast adenocarcinoma MCF-7 cells we show that PKCeta promotes senescence induced by oxidative stress and DNA damage via its ability to upregulate the expression of the cell cycle inhibitors p21Cip1 and p27Kip1 and to modulate transcription of major components of SASP such as IL-6 and IL-8. Moreover, we demonstrate that PKCeta creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor. Thus, the expression of PKCeta modulates major components of SASP. Furthermore, our studies demonstrate that PKCeta interferes with γH2AX phosphorylation, which mark DNA double-strand breaks for repair. The phosphorylation of γH2AX may reflect the extent of DNA breaks but also the cellular response to DNA damage. Our experiments suggest that the response to DNA damage (repair processes) was more efficient in PKCeta-knockdown cells. The phosphorylation on ATM and Chk2 was also lower in PKCeta expressing cells. Revealing the molecular regulators of senescence will improve our ability to develop new therapeutic strategies for clearing tumor cells. Our studies showing a role for PKCeta in senescence could identify PKCeta and/or its activated signaling cascades as targets for intervention senescence induction. Since PKCeta is highly expressed in epithelia that are the origin of about 90% of human tumors, this may provide critical inhibitors as therapeutic adjuvants for different types of carcinomas. Citation Format: Etta Livneh, Assaf Ben Ari, Udi Zurgil. Cell death and senescence: Role of protein kinase C in promoting senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 464.

Abstract 42: MYC through HIF-2α regulates the altruistic stemness program in human leukemia stem cells

Abstract Leukemia is hierarchically organized into two distinct population of cancer cells, cancer stem cells (CSCs) and non-stem cancer cells (NSCC). Numerous studies suggest that CSCs may express embryonic stem (ES) cells related stemness genes including Nanog, Sox-2, and Oct-4. However, potential cell surface markers that could specifically enrich the leukemia stem cell (LSC) subfraction based on the expression of these stemness genes have not yet been identified. Previously, we reported that ABCG2, a drug efflux pump, could enrich a subpopulation of cells exhibiting very high level of Nanog, Sox-2, and Oct-4 in ES cells. Additionally, these ABCG2+ ES cells also exhibited high HIF-2α, a transcription factor, which in cooperation with MYC-regulated both Nanog and Sox-2. In addition to these features, ABCG2+ cells demonstrated highly cytoprotective altruistic behavior by secreting high levels of glutathione. In the present study, we evaluated whether ABCG2+ subfraction of human LSCs exhibits high level of Nanog, Sox2, and Oct4. We also investigated the altruistic behavior of the LSCs, a potential novel mechanism of drug resistance and disease relapse. Methods: ABCG2+ cells from cervical lymph node and peripheral blood of T- cell acute lymphoblastic lymphoma/leukemia (T-ALL) and chronic myeloid leukemia (CML) patients (n=12) were enriched using immunomagnetic sorting. These cells were then expanded and used for different experiments including flow cytometry, ChiP on ChiP assay, in vivo transplantation assay, and siRNA inhibition treatment, to demonstrate the role of HIF-2α and MYC in regulating the altruistic stemness program in human LSCs. Results: We were able to enrich ABCG2+ cell subfraction from human T-ALL (n=5) and CML (n=7) patients exhibiting high levels of stemness genes such as Nanog, Sox-2, and Oct-4 in addition to MYC and HIF-2α. Higher engraftment potential was observed in ABCG2+ cells in comparison to ABCG2- cells as indicated by in vivo transplantation assay in NOD/SCID mice. The inhibition of stemness genes Nanog and Sox2 by siRNA gene silencing led to the loss of expression of MYC and HIF-2α, indicating that these genes are regulated MYC and HIF-2α. We then investigated the altruistic stemness phenotype of the ABCG2+ cells by studying the distinct molecular signature of MYC binding to HIF-2α in these cells. siRNA HIF-2α treatment led to ABCG2+ cell loss of proliferative capacity and reduced GSH levels, suggesting reduction of cytoprotective altruistic behavior in vitro. Additionally, siRNA HIF-2α treated ABCG2+ cells, when injected in mice, exhibited increased survival. Importantly, treatment of ABCG2+ cell harboring mice with FM19G11, a HIF inhibitor, led to marked loss in the secondary engraftment in NOD/SCID mice, thus indicating loss of ABCG2+ cell self-renewal capacity. Then we found that siRNA MYC inhibition led to loss of HIF-2α expression, suggesting that MYC might be regulating HIF-2α mediated altruistic stemness program in the ABCG2+ cells. Conclusion: To summarize, we found that ABCG2 is an excellent marker to enrich the LSC subfraction having high expression of stemness genes. The ABCG2+ LSCs exhibit altruistic stemness phenotype that includes high secretion of GSH and the distinct binding of MYC to HIF-2a, Sox2, and Nanog. Thus, we now report that a subfraction of LSC could also be enriched such that it exhibits altruistic stemness phenotype which was previously reported by us in ES cells. Our findings may open up new understanding of the LSC-mediated cancer relapse and drug resistance. Citation Format: Bidisha Pal, Anupam Sarma, Joyeeta Talukdar, Seema Bhuyan, Sora Sandhya, Sukanya Gayan, Gayatri Gogoi, Debabrat Baishya, Amal Chandra Kataki, Dean W. Felsher, Bikul Das. MYC through HIF-2α regulates the altruistic stemness program in human leukemia stem cells [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 42.

Efficacy of gamma-irradiated adipose-derived stem cells for treatment of thin endometrium in rats

Transplantation of adipose-derived stem cells (ADSCs) is associated with potential risks of late complications including tumorigenesis due to the active proliferation of the cells. We aimed to test the effect of transplantation of ADSCs with suppressed proliferation by gamma irradiation in the treatment of thin endometrium in rats. ADSCs were isolated from female SD rats and identified by detecting the surface antigens with flow cytometry. After exposure to gamma irradiation at 0, 5 Gy and 10 Gy, the cells were examined for changes in colony-forming ability. Twenty-four female rats with chemically induced thin endometrium were randomized into 4 equal groups and at 6-8 h after modeling, the rats received intrauterine injection of non-irradiated ADSCs (group I), 5 Gy irradiated ADSCs (group II), 10 Gy irradiated ADSCs (group III), or PBS only (group IV). Endometrial pathology was analyzed with HE staining in these rats in the third estrus phase following the cell transplantation. The ADSCs showed a complete loss of proliferative capacity after exposure to 10 Gy irradiation. After the cell transplantation, the endometrium thickness was thicker in group I and II than in group IV (P<0.01), but there was no significant difference between groups III and IV. Gamma irradiation impairs the proliferative capacity of ADSCs in vitro. Exposure to 10 Gy irradiation causes a total loss of proliferation capacity of the ADSCs, which have no therapeutic potential; 5 Gy irradiation causes partial loss of proliferation capacity of the cells, which still retain the activity to promote endometrial cell regeneration.

Forkhead Box Protein 1 (FoxO1) Inhibits Accelerated β Cell Aging in Pancreas-specific SMAD7 Mutant Mice

The mechanisms underlying the effects of exocrine dysfunction on the development of diabetes remain largely unknown. Here we show that pancreatic depletion of SMAD7 resulted in age-dependent increases in β cell dysfunction with accelerated glucose intolerance, followed by overt diabetes. The accelerated β cell dysfunction and loss of proliferation capacity, two features of β cell aging, appeared to be non-cell-autonomous, secondary to the adjacent exocrine failure as a "bystander effect." Increased Forkhead box protein 1 (FoxO1) acetylation and nuclear retention was followed by progressive FoxO1 loss in β cells that marked the onset of diabetes. Moreover, forced FoxO1 expression in β cells prevented β cell dysfunction and loss in this model. Thus, we present a model of accelerated β cell aging that may be useful for studying the mechanisms underlying β cell failure in diabetes. Moreover, we provide evidence highlighting a critical role of FoxO1 in maintaining β cell identity in the context of SMAD7 failure.

Proteasome activation enhances stemness and lifespan of human mesenchymal stem cells

The age-associated decline of adult stem cell function contributes to the physiological failure of homeostasis during aging. The proteasome plays a key role in the maintenance of proteostasis and its failure is associated with various biological phenomena including senescence and aging. Although stem cell biology has attracted intense attention, the role of proteasome in stemness and its age-dependent deterioration remains largely unclear. By employing both Wharton's-Jelly- and Adipose-derived human adult mesenchymal stem cells (hMSCs), we reveal a significant age-related decline in proteasome content and peptidase activities, accompanied by alterations of proteasomal complexes. Additionally, we show that senescence and the concomitant failure of proteostasis negatively affects stemness. Remarkably, the loss of proliferative capacity and stemness of hMSCs can be counteracted through proteasome activation. At the mechanistic level, we demonstrate for the first time that Oct4 binds at the promoter region of β2 and β5 proteasome subunits and thus possibly regulates their expression. A firm understanding of the mechanisms regulating proteostasis in stem cells will pave the way to innovative stem cell-based interventions to improve healthspan and lifespan.

GDF11 improves tubular regeneration after acute kidney injury in elderly mice.

The GDF11 expression pattern and its effect on organ regeneration after acute injury in the elderly population are highly controversial topics. In our study, GDF11/8 expression increased after kidney ischemia–reperfusion injury (IRI), and the relatively lower level of GDF11/8 in the kidneys of aged mice was associated with a loss of proliferative capacity and a decline in renal repair, compared to young mice. In vivo, GDF11 supplementation in aged mice increased vimentin and Pax2 expression in the kidneys as well as the percentage of 5-ethynyl-2′-deoxyuridine (EdU)-positive proximal tubular epithelial cells. GDF11 improved the renal repair, recovery of renal function, and survival of elderly mice at 72 h after IRI. Moreover, the addition of recombinant GDF11 to primary renal epithelial cells increased proliferation, migration, and dedifferentiation by upregulating the ERK1/2 pathway in vitro. Our study indicates that GDF11/8 in the kidney decreases with age and that GDF11 can increase tubular cell dedifferentiation and proliferation as well as improve tubular regeneration after acute kidney injury (AKI) in old mice.

Cytostatic Effect of Repeated Exposure to Simvastatin: A Mechanism for Chronic Myotoxicity Revealed by the Use of Mesodermal Progenitors Derived from Human Pluripotent Stem Cells.

Statin treatment of hypercholesterolemia can lead to chronic myotoxicity which is, in most cases, alleviated by drug withdrawal. Cellular and molecular mechanisms of this adverse effect have been elusive, in particular because of the lack of in vitro models suitable for long-term exposures. We have taken advantage of the properties of human pluripotent stem cell-derived mesodermal precursors, that can be maintained unaltered in vitro for a long period of time, to develop a model of repeated exposures to simvastatin during more than 2 weeks. This approach unveiled major differences, both in functional and molecular terms, in response to single versus repeated-dose exposures to simvastatin. The main functional effect of the in vitro simvastatin-induced long-term toxicity was a loss of proliferative capacity in the absence of concomitant cell death, revealing that cytostatic effect could be a major contributor to statin-induced myotoxicity. Comparative analysis of molecular modifications induced by simvastatin short-term versus prolonged exposures demonstrated powerful adaptive cell responses, as illustrated by the dramatic decrease in the number of differentially expressed genes, distinct biological pathway enrichments, and distinct patterns of nutrient transporters expressed at the cell surface. This study underlines the potential of derivatives of human pluripotent stem cells for developing new approaches in toxicology, in particular for chronic toxicity testing.

Abstract 1263: Cellular senescence in Hodgkin's lymphoma

Abstract Hodgkin's lymphoma (HL), a B cell originated malignancy of the immune system, is one of the most frequent lymphomas in the Western world. Although the rate of cure is high, about 20-35% of patients relapse, and about half of them eventually die of the disease or treatment-related late toxicities and secondary malignancies. Conceptually novel treatment strategies are thus needed, particularly for this category of patients. The malignant cells, called Hodgkin and Reed-Sternberg (HRS) cells, only make up 1-2% of the total tumor cellularity; the remaining mass comprising a mixed infiltrate population is thought to be recruited to the lymph node by HRS-driven pro-inflammatory signals. The origin of the pro-inflammatory signals remains elusive, and represents a critical gap in our knowledge of the pathogenesis of HL. We propose that a sub-population of HRS cells, which we call herein large RS cells, have characteristics of senescent cells, and, thus produce large amounts of inflammatory mediators (the so-called “senescence-associated secretory phenotype”, or SASP). Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. We show that the senescence markers the cell cycle inhibitors p16INK4a and p21Cip1 are expressed in large RS cells in all HL biopsies examined. Moreover, the large RS cells are negative for Ki-67 staining, demonstrating that these cells have ceased to proliferate. We also show that large, ‘RS-like’ cells in a HL-derived line, L428, stained for the characteristic senescence marker β-galactosidase (β-gal). Oxidative stress and chemotherapy increased the proportion of β-gal positive large RS cells. Furthermore, we show that the large RS cells secrete high levels of cytokines. We suggest that these senescent RS cells may be responsible, at least in part, for creating a pro-inflammatory microenvironment, promoting HL pathogenesis, and mediating chemo-resistance in relapsed disease. Understanding the pathways important for the establishment of senescence in HRS cells, as well gaining insight into targetable mechanisms for the eradication of these cells, will provide new therapeutic approaches for HL patients with recurrent or chemo-resistant disease. Citation Format: Jacob Gopas, Udi Zurgil, Estee Zeev, Daniel Benharoch, Jodi Ezratty, Giora Shubinsky, Etta Livneh. Cellular senescence in Hodgkin's lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1263. doi:10.1158/1538-7445.AM2015-1263

Quantification of the chromosomal radiation damage induced by labelling of leukocytes with [18F]FDG

IntroductionThe aim of our work is to quantify the radiation damage in lymphocytes after labelling with [18F]FDG. Comparison with gold standard [99mTc]HMPAO labelling is established. An approach to cellular dosimetry is proposed. MethodsMixed leukocytes were separated from fresh venous blood and labelled with [18F]FDG and [99mTc]HMPAO following published guidelines. Cytokinesis-block micronucleus (CBMN) assay was performed for both sets of experiments. Tests for quality control of labelling described in guidelines were followed. Cellular dosimetry was calculated according to MIRD. ResultsMN scored after labelling with 37MBq of [18F]FDG were 956±172 and 347±26 for [99mTc]HMPAO (p<0.05). Absorbed dose in cell nucleus was of 0.23Gy for [18F]FDG and 0.08Gy for [99mTc]HMPAO labelling. The CBMN assay after labelling with ~290MBq of [18F]FDG showed radiation induced inhibition of proliferation capacity of the lymphocytes, confirmed by proliferation study. Conclusions[18F]FDG labelling of mixed leukocytes causes severe radiation damage to the cell, higher than with [99mTc]HMPAO in accordance with the absorbed dose. Labelling of mixed leukocytes for clinical purpose induces high cytotoxicity reflected in the loss of proliferation capacity in lymphocytes this statement allows us to consider a low oncogenic risk however the association between MN formation in the PBL and subsequent risk of cancer is not well established. Advances in knowledgeThis is the first work about radiation damage with [18F]FDG labelled cells. We focused on [18F]FDG labelling of leukocytes due to the growing number of research and review articles about this technique. Implications for patient careThe possibility of an increased risk of lymphoid malignancies associated with the administration of radiolabelled lymphocytes is a very controversial subject. Studies on radiation damage on new labelling procedures as the one exposed in this work must be considered.

Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii.

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals' toxic effects observed.

Dynamic changes in the cardiac methylome during postnatal development.

Relatively little is known about the epigenetic control mechanisms that guide postnatal organ maturation. The goal of this study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the first 2 wk of mouse heart development, which is an important period for cardiomyocyte maturation, loss of proliferative capacity and loss of regenerative potential. Gene expression profiling (RNA-seq) and genome-wide sequencing of methylated DNA (MBD-seq) identified dynamic changes in the cardiac methylome during postnatal development [2545 differentially methylated regions (DMRs) from P1 to P14 in the mouse]. The vast majority (~80%) of DMRs were hypermethylated between P1 and P14, and these hypermethylated regions were associated with transcriptional shut down of important developmental signaling pathways, including Hedgehog, bone morphogenetic protein, TGF-β, fibroblast growth factor, and Wnt/β-catenin signaling. Postnatal inhibition of DNA methylation with 5-aza-2'-deoxycytidine induced a marked increase (~3-fold) in cardiomyocyte proliferation and ~50% reduction in the percentage of binucleated cardiomyocytes compared with saline-treated controls. This study provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation.

Abstract 2247: Histone H3 cleavage via Cathepsin L drives a cellular senescence program

Abstract Oncogene-induced senescence (OIS) is characterized by irreversible loss of proliferative capacity. It has been proposed that OIS functions as a tumor suppressor mechanism, and several studies have demonstrated induction of senescence markers in pre-malignant lesions. In addition to profound morphological changes, significant alterations in chromatin structure occur during senescence including formation of senescence-associated heterochromatin foci (SAHF). We have used well-characterized senescence models in human primary lung fibroblasts and melanocytes to study how chromatin alterations contribute to the senescence phenotype. We discovered that the N'-terminal tail of histone H3 is proteolytically cleaved by the lysosomal protease Cathepsin L1 (CTSL1). We show that CTSL1 is induced during senescence both at the mRNA and protein levels, and that it is present in the chromatin fraction of senescent cells. Chemical and shRNA-mediated inhibition of CTSL1 activity effectively block H3 tail cleavage and leads to a SAHF formation defect. We identified two distinct cleaved forms of H3 in senescent cells, which are associated with chromatin and incorporated into nucleosomes. Using antibodies directed against specific post-translational modifications (PTMs) of the histone H3 tail, we have mapped the approximate locations of the cleavage sites. Furthermore, overexpression of flag-tagged cleaved versions of H3, but not the full-length histone, trigger robust senescence in IMR90 cells accompanied by a modest induction of SAHFs. Interestingly, we also detect cleaved H3 in chromatin extracted from benign nevi tissue samples, providing evidence of H3 tail cleavage in senescence models in vivo. Collectively, our results suggest that H3 tail cleavage may contribute significantly to the senescence phenotype, potentially through transcriptional regulation of key senescence target genes. This area of investigation is currently ongoing. Citation Format: Luis F. Duarte, Andrew R. Young, Hsan-Au Wu, Taniya Panda, Zichen Wang, Alexandre G. Maia, Dan Hasson, Avnish Kapoor, Masashi Narita, Emily Bernstein. Histone H3 cleavage via Cathepsin L drives a cellular senescence program. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2247. doi:10.1158/1538-7445.AM2014-2247

Gallotannin Imposes S Phase Arrest in Breast Cancer Cells and Suppresses the Growth of Triple-Negative Tumors In Vivo

Triple-negative breast cancers are associated with poor clinical outcomes and new therapeutic strategies are clearly needed. Gallotannin (Gltn) has been previously demonstrated to have potent anti-tumor properties against cholangiocarcinoma in mice, but little is known regarding its capacity to suppress tumor outgrowth in breast cancer models. We tested Gltn for potential growth inhibitory properties against a variety of breast cancer cell lines in vitro. In particular, triple-negative breast cancer cells display higher levels of sensitivity to Gltn. The loss of proliferative capacity in Gltn exposed cells is associated with slowed cell cycle progression and S phase arrest, dependent on Chk2 phosphorylation and further characterized by changes to proliferation related genes, such as cyclin D1 (CcnD1) as determined by Nanostring technology. Importantly, Gltn administered orally or via intraperitoneal (IP) injections greatly reduced tumor outgrowth of triple-negative breast cells from mammary fat pads without signs of toxicity. In conclusion, these data strongly suggest that Gltn represents a novel approach to treat triple-negative breast carcinomas.

Abstract B36: Premature senescence is enhanced by PKCeta and its polymorphic variant V374I

Abstract Cellular senescence appears now as an important mechanism that could prevent proliferation of potential tumorigenic cells. Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. Senescent cells secrete a complex mixture extracellular proteins and soluble factors, known as the senescence-associated secretory phenotype (SAPS). The senescence secretome exerts diverse effects on the microenviroment and neighboring cells. Components of the senescence secretome reinforce or implement cell cycle arrest and contribute to tumor suppression by signaling and recruiting the immune system. Among these factors, IL-6 was implicated in implementing cell cycle arrest, characteristic of senescence. A limited number of studies suggested a role for PKC in senescence, with opposite effects of different isoforms. Here our aim was to demonstrate that the epithelial specific isoform, PKCeta, plays a role in promoting senescence. Previous studies from our laboratory and other showed a role for PKCeta in the protection from cell death. We suggest that PKCeta expression in epithelial cells could shift the balance between apoptosis and senescence towards senescence thus protecting against cell death. In the present study we have also utilized a polymorphic variant of PKCeta. This polymorphic variation, the nonsynonymous SNP, changing 374V to 374I (V374I), was previously identified as a risk factor for cerebral infarction and rheumatoid arthritis in two independent genetic screens of the Japanese population. As the SNP V374I is located in the functional domain of the kinase- the ATP binding site- we have initially characterized the effect of this polymorphic change on the kinase activity of the protein. Our results show that 374I significantly enhanced the kinase activity of the protein and its phosphorylation on external protein substrates. Overexpression of PKCeta 374V and 374I in the breast adenocarcinoma MCF-7 cells enhanced the secretion of IL-6 and TNFα by LPS, with 374I having a greater effect. Oxidative stress was previously shown to induce senescence phenotype in MCF-7 cells. Oxidative stress resulted also in increased IL-6 secretion by 374I, although the 374V (WT) showed also elevated levels compared to the control treated cells. The role of PKCeta in IL-6 secretion and β-galactosidase staining was also demonstrated using PKCeta knocked-down cells by sh-RNA. Thus, our results reveal that PKCeta itself (WT) is involved in IL-6 secretion, with 374I having a more profound effect. NFkB was shown to be a master regulator of SASP by influencing the expression of NFkB target genes. Notably, our previously published studies reported a role for PKCeta as an upstream regulator of NFkB and its function in cell cycle control. One of the hallmarks of cells undergoing senescence is the involvement of the p16INK4A-Rb and p53 pathways, with p21CIP1 induction also evident. Since our previous studies showed a role for PKCeta in cell cycle regulation at the G1/S transition its effects were also determined. Upon oxidative stress, we show increased expression of p21CIP1 and p16INK4A, under conditions in which senescence is induced. Furthermore, cell expressing 374I exhibited higher levels of p21CIP1 and p16INK4A compared to 374V (WT). Taken together, our present studies reveal a role for PKCeta in establishing the senescence phenotype. There is now considerable interest in therapies that will harness senescence to control tumor promotion. Revealing the molecular regulators of senescence will improve our ability to develop new therapeutic strategies for clearing tumor cells. Citation Format: Udi Zurgil, Assaf Ben Ari, Etta Livneh. Premature senescence is enhanced by PKCeta and its polymorphic variant V374I. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B36.

Abstract 5515: In vivo efficacy of the PARG inhibitor Gallotannin against triple negative breast cancer.

Abstract Breast cancer patients whose tumors fall into the triple negative category have the poorest clinical outcomes. These patients are at high risk for metastatic recurrence and have poor overall survival. Clearly, new therapeutic strategies are needed to combat triple negative tumors both at the time of onset and if necessary, at recurrence. Toward this goal, we initially tested the anti-proliferative properties of Gallotannin (GLTN) against a variety of breast cancer cell lines in vitro. Gallotannin was chosen due to its potent chemotherapeutic effects against cholangiocarcinoma in mice, and the lack of data concerning its potential use as an anti-cancer agent in breast cancer models. We observed potent anti-proliferative properties of GLTN in the low micromolar range in most breast cancer cell lines tested, but not in corresponding non-transformed mammary epithelial cells. Notably, triple negative breast cancer cells displayed the highest level of sensitivity to GLTN. The loss of proliferative capacity in these cells is due to cell cycle arrest in S phase. This corresponded with increased Chk1 phosphorylation, decreased Cyclin D1 protein levels, and changes to growth related genes as determined by Nanostring technology. GLTN may have several cellular targets that could potentially mediate growth arrest. The most thoroughly described target of GLTN is PARG, which is responsible for removing poly(ADP)ribose moieties from target proteins. GLTN potently inhibits PARG activity both in vitro and in vivo. Surprisingly, we found that PARG knockdown results in a dramatic growth inhibition of breast cancer cells, similar to what is observed in cells exposed to GLTN. To our knowledge, this is the first report indicating PARG inhibition may be exploited therapeutically to impair breast cancer growth. Importantly, PARG knockdown cells showed less sensitivity to GLTN, indicating PARG is a primary effector of GLTN-mediated growth inhibition. To test the capacity of GLTN to diminish tumor outgrowth in vivo, we utilized a mouse xenograft model where MDA-MB-468 triple negative breast cancer cells were injected into the mammary fat pad and allowed to grow to a palpable size before drug treatment. GLTN was administered ad libitum orally at 0.05% in drinking water or via intraperitoneal (IP) injections dosed at 10mg/kg daily. Both orally and IP administered GLTN greatly reduced tumor outgrowth by approximately 3 fold within a 25 day period without significant signs of weight loss, morbidity or liver toxicity. Beyond 25 days, orally ingested GLTN continued to prevent tumor growth by 3-fold, whereas tumors exposed to GLTN given by IP exhibited decreased sensitivity. In conclusion, these data strongly suggest that orally administered GLTN represents a novel approach to treat triple negative breast carcinomas and that PARG may represent a new therapeutic target for anti-cancer compounds. Citation Format: Tiejun Zhao, Qiang Sun, Amanda Lovato, Sonia del Rincon, Michael Witcher. In vivo efficacy of the PARG inhibitor Gallotannin against triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5515. doi:10.1158/1538-7445.AM2013-5515

Programmed cell death 1-directed immunotherapy for enhancing T-cell function.

T-cell exhaustion is a unique state that appears during many chronic infections and cancer and is characterized by loss of proliferative capacity and effector function. Complex mechanisms are involved in this T-cell dysfunction but an inhibitory receptor, PD-1, has been identified as a major regulator of T-cell exhaustion. Blockade of the PD-1 pathway can reinvigorate exhausted T cells, resulting in better control of chronic infections and cancer. Notably, recent clinical studies have revealed that PD-1-directed immunotherapy is highly effective in cancer patients, demonstrating that PD-1 is a promising therapeutic target in humans. In this review, we summarize our current understanding of the epigenetic regulation of PD-1 expression in T cells and discuss potential combination therapy with PD-1 blockade toward developing more effective treatment for chronic infections and cancer.