PARP inhibitor olaparib induced differential protein expression in cervical cancer cells.

PARP inhibitors are a potential class of chemotherapeutic drugs but PARP inhibitor response has not been explored systematically. We lack a specific understanding of the subset of the proteome preferentially modified in various cancers by PARP inhibitors. Implications of PARP inhibitor and PARP1 in cervical cancer treatment and resistance are not fully elucidated. We conducted a mass spectrometry-based proteomic analysis of cervical cancer Hela cells treated with olaparib. We aimed to identify the alteration in the protein signaling pathway induced by PARP inhibitors beyond the DNA damage response pathway. Our data demonstrate a significant reduction in PARP activity and enhanced cell death after olaparib treatment. We further observed articulated proteomic changes with a significant enrichment of proteins in diverse cellular processes. The differentially expressed proteins were predominantly associated with RNA metabolism, mRNA splicing, processing, and RNA binding. Our data also identified proteins that could probably contribute to survival mechanisms resulting in resistance to PARP inhibitors. Hence, we put forth the overview of proteomic changes induced by PARP inhibitor olaparib in cervical cancer cells. This study highlights the significant proteins modified during PARP inhibition and thus could be a probable target for combination therapies with PARP inhibitors in cervical cancer. SIGNIFICANCE.

HSc70 interactome reveal major role of macroautophagy and minor role of chaperone mediated autophagy in K-Ras G12V cell proliferation and survival.

Constitutively active K-Ras oncogene mutation at G12V changes the proteome of cells and activates macroautophagy for cell advantage. Inhibition of macroautophagy impairs K-Ras mediated tumor progression to a limited extent with increase of spontaneous tumors due to poorly understood mechanisms. Here, we show that inhibition of macroautophagy in K-Ras G12V mouse embryonic fibroblasts (MEFs) hyper activates chaperon mediated autophagy (CMA). Quantitative identification of CMA substrates through co-immunoprecipitation of CMA component heat shock cognate 70 (Hsc70) demonstrates a shift of proteins from macroautophagy to CMA mediated degradation. However, macroautophagy impairment show significant inhibition on proliferation and CMA hyper activation provides a basal support to macroautophagy-inhibited MEFs for survival. On the other hand, K-Ras G12V MEFs impaired of CMA reduces number of Hsc70 clients but activated macroautophagy significantly compensated CMA loss. Nonetheless, co-inhibition of CMA and macroautophagy had a synergistic detrimental effect on both proliferation and survival of MEFs expressing K-Ras G12V mutant. Our results point to K-Ras G12V MEFs dependency on macroautophagy and CMA partly compensates its loss for survival but not hyper-proliferation; implicating that targeting both macroautophagy and CMA as a promising therapeutic target in G12V mutation associated K-Ras cancers. SIGNIFICANCE: The present study provides a framework of Hsc70 interacting proteins, which differentially interact with Hsc70 in response to autophagy alterations. The role of proteins accumulation and induced proteo-toxicity could be underlying factor in macroautophagy and CMA co-inhibited K-Ras G12V MEFs phenotype. Our study provides rational for adaptive mechanisms in K-Ras tumors inhibited with different autophagy pathways and also supports targeting both macroautophagy and CMA simultaneously as therapeutic target. At the same time current study will help in characterizing the underlying cellular processes that may play a role in escaping tutor suppressor role CMA and macroautophagy in cancers harboring K-Ras G12V mutation that may be further utilized to identify molecular targets for K-Ras-driven cancers.

Detection of UV-Induced Thymine Dimers.

Ultraviolet rays induce interstrand and intrastrand DNA cross-links, usually thymine-thymine cyclobutane dimer (T-T) and thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct (T (6-4) T). These DNA cross-links, if left unrepaired, increase the risk of these mutation being incorporated in the genetic material (i.e., DNA). Numerous studies have reported the mutagenic potential of above mentioned DNA adducts in prokaryotes, yeast and mammalian cells. Different techniques have been developed to identify such DNA adducts such as immuno-Southern blotting. This is a routinely used quantitative method to determine especially the amount of thymine dimers formed, following irradiation. In this chapter, the detailed methodology to identify thymine dimers formation is provided, using specific antibody against these adducts.

ATM and ATR signaling at a glance.

ATM and ATR signaling pathways are well conserved throughout evolution and are central to the maintenance of genome integrity. Although the role of both ATM and ATR in DNA repair, cell cycle regulation and apoptosis have been well studied, both still remain in the focus of current research activities owing to their role in cancer. Recent advances in the field suggest that these proteins have an additional function in maintaining cellular homeostasis under both stressed and non-stressed conditions. In this Cell Science at a Glance article and the accompanying poster, we present an overview of recent advances in ATR and ATM research with emphasis on that into the modes of ATM and ATR activation, the different signaling pathways they participate in - including those that do not involve DNA damage - and highlight their relevance in cancer.