Abstract
BackgroundEarlier, proteomic profiling of a Serous Ovarian Carcinoma (SeOvCa) progression model in our lab had identified significantly enriched expression of three double-strand break (DSB) -repair proteins viz. RAD50, NPM1, and XRCC5 in transformed cells over pre-transformed, non-tumorigenic cells. Analysis of the functional relevance of enhanced levels of these proteins was explored in transformed ovarian cancer cells.MethodsExpression profiling, validation and quantitation of the DSB-repair proteins at the transcriptional and protein levels were carried out. Further analyses included identification of their localization, distribution and modulation on exposure to Estradiol (E2) and cisplatin. Effects on silencing of each of these under conditions of genomic-stress were studied with respect to apoptosis, alterations in nuclear morphology and DNA fragmentation; besides profiling known mitotic and spindle check-point markers in DSB-repair.ResultsWe identified that levels of these DSB-repair proteins were elevated not only in our model, but generally in cancer and are specifically triggered in response to genotoxic stress. Silencing of their expression led to aberrant DSB repair and consequently, p53/p21 mediated apoptosis. Further compromised functionality generated genomic instability.ConclusionsPresent study elucidates a functional relevance of NPM1, RAD50 and XRCC5 DSB-repair proteins towards ensuring survival and evasion of apoptosis during ovarian transformation, emphasizing their contribution and association with disease progression in high-grade SeOvCa.
Highlights
Earlier, proteomic profiling of a Serous Ovarian Carcinoma (SeOvCa) progression model in our lab had identified significantly enriched expression of three double-strand break (DSB) -repair proteins viz. Human homolog of Saccharomyces cerevisiae Rad50 (RAD50), NPM1, and X-ray repair cross-complementing 5 (XRCC5) in transformed cells over pre-transformed, non-tumorigenic cells
In exploring the functional relevance associated with disease progression, we focused on these three LEx proteins of DSB-repair pathways
Transformation-associated DSB Repair proteins are enriched in ovarian cancer An earlier study on proteomic profiling of the SeOvCa progression in our lab had categorized differentially identified proteins into two groups, where Group I comprised EEx and LEx sub-groups consisting 10 and 34 proteins respectively, while Group II comprised 31 and 48 differentially upregulated proteins identified in A4-P and A4-T cells respectively (Figure 1A)
Summary
Proteomic profiling of a Serous Ovarian Carcinoma (SeOvCa) progression model in our lab had identified significantly enriched expression of three double-strand break (DSB) -repair proteins viz. RAD50, NPM1, and XRCC5 in transformed cells over pre-transformed, non-tumorigenic cells. DNA double-strand break (DSB) repair, homologous recombination (HR) mediated repair pathway is reported to be frequently disrupted in solid high-grade serous ovarian adenocarcinoma (SeOvCa) tumors [3]. Overexpression of such DSB-repair associated proteins is assumed to be associated with endogenous replication stress and high frequency of DNA breaks in transformed cells [4,5], though functional relevance of these markers remained largely uncharacterized during the disease progression. Nineteen of these clones underwent spontaneous immortalization One of these immortal clones viz. A4 with slow-cycling and non-tumorigenic properties, got transformed (passage ~20-25) into an aggressively proliferating clone that exhibited tumorigenic and metastatic capabilities in in vitro assays. Categorization of proteins into functional networks provided a clear insight of cellular functionality and major pathways regulating ovarian cell transformation
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