Tareq Saleh

Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells

Radiotherapy continues to be a primary modality in the treatment of cancer. In addition to promoting apoptosis, radiation-induced DNA damage can promote autophagy and senescence, both of which can theoretically function to prolong tumor survival. In this work, we tested the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiosensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair-proficient HCT116 colon carcinoma cells and a repair-deficient ligase IV–/– isogenic cell line. Exposure to radiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines, however, inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Exposure to radiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the ligase IV–/– cells, however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors, olaparib and niraparib, increased the extent of persistent DNA damage induced by radiation exposure as well as the extent of both autophagy and senescence. Neither cell line underwent significant apoptosis by radiation exposure alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiosensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10–20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence.

Proteomics Insights into Autophagy

Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS‐based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno‐capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy.

Autophagy Inhibition and Chemosensitization in Cancer Therapy

Abstract Autophagy is a cellular degradation mechanism involving protein turnover as well as the recycling of excessive and dysfunctional organelles. Autophagy is generally considered to occur at basal levels in cells, but may be induced to higher levels under conditions of stress, such as starvation, hypoxia, ionizing radiation treatment, or chemotherapy. Aberrations in the autophagic machinery may represent either cause or effect in various diseases. In cancer, autophagy has been shown to be one of the mechanisms affecting the response to chemotherapy and/or radiation treatment. In this chapter, the different types and functional forms of autophagy, the autophagy signaling pathway, as well as the role of autophagy in transformation and tumor suppression are discussed. Modulation of autophagy for improving tumor cell chemosensitivity and radiosensitivity is currently being explored in clinical trials; the potential impact and limitations of these studies are discussed. We also briefly review the intersection between autophagy and the immune system with regard to the effectiveness of chemotherapy. The chapter concludes with future directions in cancer treatment in relation to novel therapies and potential drug targets for autophagy modulation.

Differential Radiation Sensitivity in p53 Wild-Type and p53-Deficient Tumor Cells Associated with Senescence but not Apoptosis or (Nonprotective) Autophagy

Studies of radiation interaction with tumor cells often focus on apoptosis as an end point; however, clinically relevant doses of radiation also promote autophagy and senescence. Moreover, functional p53 has frequently been implicated in contributing to radiation sensitivity through the facilitation of apoptosis. To address the involvement of apoptosis, autophagy, senescence and p53 status in the response to radiation, the current studies utilized isogenic H460 non-small cell lung cancer cells that were either p53-wild type (H460wt) or null (H460crp53). As anticipated, radiosensitivity was higher in the H460wt cells than in the H460crp53 cell line; however, this differential radiation sensitivity did not appear to be a consequence of apoptosis. Furthermore, radiosensitivity did not appear to be reduced in association with the promotion of autophagy, as autophagy was markedly higher in the H460wt cells. Despite radiosensitization by chloroquine in the H460wt cells, the radiation-induced autophagy proved to be essentially nonprotective, as inhibition of autophagy via 3-methyl adenine (3-MA), bafilomycin A1 or ATG5 silencing failed to alter radiation sensitivity or promote apoptosis in either the H460wt or H460crp53 cells. Radiosensitivity appeared to be most closely associated with senescence, which occurred earlier and to a greater extent in the H460wt cells. This finding is consistent with the in-depth proteomics analysis on the secretomes from the H460wt and H460crp53 cells (with or without radiation exposure) that showed no significant association with radioresistance-related proteins, whereas several senescence-associated secretory phenotype (SASP) factors were upregulated in H460wt cells relative to H460crp53 cells. Taken together, these findings indicate that senescence, rather than apoptosis, plays a central role in determination of radiosensitivity; furthermore, autophagy is likely to have minimal influence on radiosensitivity under conditions where autophagy takes the nonprotective form.

Non-Cell Autonomous Effects of the Senescence-Associated Secretory Phenotype in Cancer Therapy

In addition to promoting various forms of cell death, most conventional anti-tumor therapies also promote senescence. There is now extensive evidence that therapy-induced senescence (TIS) might be transient, raising the concern that TIS could represent an undesirable outcome of therapy by providing a mechanism for tumor dormancy and eventual disease recurrence. The senescence-associated secretory phenotype (SASP) is a hallmark of TIS and may contribute to aberrant effects of cancer therapy. Here, we propose that the SASP may also serve as a major driver of escape from senescence and the re-emergence of proliferating tumor cells, wherein factors secreted from the senescent cells contribute to the restoration of tumor growth in a non-cell autonomous fashion. Accordingly, anti-SASP therapies might serve to mitigate the deleterious outcomes of TIS. In addition to providing an overview of the putative actions of the SASP, we discuss recent efforts to identify and eliminate senescent tumor cells.

Reversibility of chemotherapy-induced senescence is independent of autophagy and a potential model for tumor dormancy and cancer recurrence.

Autophagy and senescence are both well-established responses to chemotherapy and radiation that often occur in parallel, contributing to growth arrest in tumor cells. However, it has not been established whether this growth arrest is reversible. This question was addressed using non-small cell lung cancer models exposed to the cancer chemotherapeutic drug, etoposide. Senescent cells that were sorted, identified by β-galactosidase staining and alterations in morphology, isolated by flow cytometric cell sorting based on C12FDG staining, and real-time live microscopy were found to be capable of recovering proliferative capacity. Autophagy, monitored by vacuole formation, SQSTM1/p62 degradation, and LC3BII generation did not interfere with either the senescence arrest or proliferative recovery and was nonprotective in function (i.e. autophagy inhibition via both pharmacological and genetic strategies had negligible impact on the response to etoposide). These observations argue against the premise that (chemotherapy-induced) senescence is irreversible and indicate that therapy-induced senescence may ultimately be a transient process in that at least a subpopulation of tumor cells can and will remain metabolically active and recover proliferative capacity independently of autophagic turnover. We therefore propose that dormant tumor cells may be capable of prolonged survival in a state of autophagy/senescence and that disease recurrence may reflect escape from this senescence-arrested state.

Abstract 227: Mass spectrometry-based proteomics analysis of the non-small cell lung cancer secretome

Non-small cell lung cancer (NSCLC) is the more common subtype (~80%) of lung cancer, a leading cause of cancer death worldwide. NSCLC has one of the lowest 5-year relative survival rates due to a combination of late stage diagnosis and treatment relapse. Autophagy, a stress response mechanism in which cancer cells recycle organelles, and proteins to generate the necessary nutrients and metabolic intermediates for survival, is thought to be one of the principal mechanisms of treatment resistance and relapse in cancer. Due to its relatively refractory nature to chemo/radiotherapy, our long-term goal is to identify molecular signatures and pathways of autophagy that can be targeted to improve the sensitivity of NSCLC to radiotherapy. H1299 +/-p53 cells were cultured to ~85% confluency, the media exchanged to serum-free DMEM, and treated with 6Gy of ionizing radiation. After 12 hours, secreted proteomes were collected, processed and analyzed by reverse-phase nanoLC-MS/MS (Q-Exactive, ThermoFisher). A total of 364 secreted proteins were identified, of which 163 were quantified in at least 2 replicates of one condition. 29 unique proteins were identified to be present in all samples except H1299 +p53 cells which were not treated with ionizing radiation. These proteins included HMGA1, LSM8, CAST, CAPZB, CHGB, GSS, and HSPA9 and were associated principally with protein secretion and chaperone activities. Functional enrichment analysis using FunRich revealed that the secretome was enriched for exosomal, lysosomal, cytoplasmic, and cytoskeletal proteins (>2 fold, p-value 2 fold in +p53 cell secretomes. Calnexin, an ER chaperone protein that is associated with senescence, has been identified in previous studies as a sero-marker of lung cancer but not as a marker for distinguishing between the autophagic responses of NSCLC to IR. The novel functional and potential diagnostic value of these differentially secreted proteins as a function of p53 expression, irradiation and functional autophagy status in the context of NSCLC will be discussed. Citation Format: Emmanuel K. Cudjoe, Tareq Saleh, David A. Gewirtz, Adam M. Hawkridge. Mass spectrometry-based proteomics analysis of the non-small cell lung cancer secretome [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 227. doi:10.1158/1538-7445.AM2017-227

Abstract 5470: Reversibility of therapy-induced senescence in non-small cell lung cancer as a model of cancer recurrence

Disease recurrence at either primary or metastatic tumor sites arising from dormant tumors is largely responsible for the morbidity and mortality of many cancers. Although cellular quiescence has been proposed as a mechanism of dormancy, the nature of the growth arrest that allows tumor cells to re-emerge months or years after treatment has never been directly determined. The current work was designed to examine the hypothesis that the senescence induced by cancer chemotherapeutic drugs could be reversible, with implications for tumor dormancy and disease recurrence. Studies were performed using etoposide in models of non-small cell lung cancer (NSCLC). Exposure to etoposide at sublethal concentrations resulted in growth arrest accompanied by the induction of senescence and autophagy. Growth arrest was transient in that proliferative recovery was evident by day 7 post-etoposide exposure. Analysis of senescence based on β-galactosidase activity demonstrated that the restoration of proliferative capacity coincided with a decline in the extent of senescence. Real-time live microscopy demonstrated heterogeneous fates of senescent cells, where the bulk of the cell population remaining in a state of stasis, a subset of cells undergoing apoptosis, and a fraction of the large, flattened, and polyploid cells spontaneously entering into mitosis. Neither genetic nor pharmacological inhibition of autophagy influenced the senescence response to etoposide, the eventual proliferative recovery, or sensitivity to etoposide. These findings indicate that the induction and resolution of senescence is independent of the accompanying autophagy, and that the autophagy induced by etoposide is non-cytoprotective in function. These observations suggest that therapy-induced senescence may ultimately be a transient process in that at least a subpopulation of tumor cells can and will regain proliferative capacity. Consequently, therapy-induced senescence could potentially be studied as a model of tumor dormancy, and the reversibility of senescence as a model of disease recurrence. Citation Format: Tareq Saleh, Emmanuel K. Cudjoe, S Lauren Kyte, Scott C. Henderson, Lynne W. Elmore, David A. Gewirtz. Reversibility of therapy-induced senescence in non-small cell lung cancer as a model of cancer recurrence [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 5470. doi:10.1158/1538-7445.AM2017-5470

Abstract 2848: Autophagy, senescence and proliferative recovery subsequent to DNA damage in radiation sensitization by PARP inhibition

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (-/-) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (-/-) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10-20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence. Citation Format: Moureq R. Alotaibi, Khushboo Sharma, Tareq Saleh, Lawrence Povirk, Eric Hendrickson, David Gewirtz. Autophagy, senescence and proliferative recovery subsequent to DNA damage in radiation sensitization by PARP inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2848.

Abstract 2846: Proliferative recovery after chemotherapy-induced senescence in non-small cell lung cancer (NSCLC) cells as a model of tumor dormancy and disease recurrence

Lung cancer is the leading cause of cancer-related death in both men and women in the United States. Most lung cancer cases are diagnosed in advanced, inoperable stages and are treated with chemoradiation; while chemoradiation is effective in suppressing tumor progression, recurrence following treatment is not infrequent. As there is no well-accepted preclinical model for tumor dormancy and disease recurrence, we hypothesize that the promotion of transient autophagy and senescence could be developed as a model of tumor growth and recovery following treatment. Studies were performed utilizing H460 NSCLC cells as well as a primary NSCLC cell line isolated and grown in our laboratory from a stage IV adenocarcinoma. After treatment with etoposide (1 uM), growth arrest was accompanied by the robust induction of autophagy and senescence. This growth arrest was transient and was followed by proliferative recovery in the course of several days. Genetic and pharmacological inhibition of autophagy failed to interfere with the ability of the cells to regrow, indicating a non-cytoprotective function of autophagy in response to etoposide (in contrast to the cytoprotective function of autophagy exhibited in response to radiation). Quantification of senescence over time based on C12FDG staining and flow cytometry demonstrated that the reversal of growth arrest coincided with a decline in the extent of senescence. To more precisely define the source of the recovered cells, senescent and non-senescent but growth arrested cells were separated by flow cytometry based on their relative β-galactosidase expression and replated. Both cell populations demonstrated the ability to re-emerge from the growth-arrested state and recover proliferative capacity. These observations suggest that senescence is ultimately a transient process in that at least a subpopulation of tumor cells can and will recover proliferative capacity. Furthermore, the reversibility of therapy-induced senescence (TIS) might prove to be a useful model both in cell culture and in vivo to study tumor dormancy and disease recurrence. Studies are currently in progress to determine the impact of senescence inhibition on tumor recovery as well as sensitivity to subsequent therapy given that recurrent disease tends to be relatively refractory to further treatment. Citation Format: Tareq Saleh, Theresa Thekkudan, Moureq R. Alotaibi, David A. Gewirtz. Proliferative recovery after chemotherapy-induced senescence in non-small cell lung cancer (NSCLC) cells as a model of tumor dormancy and disease recurrence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2846.