Gustavo Baldassarre

Quality of radiation shapes survival, invasiveness, and migration in ovarian cancer cell lines with different molecular profiles and varying alpha/beta ratios: an in vitro study on behalf of the Multicenter Italian Trials in Ovarian Cancer (MITO) group

The results of radiotherapy (RT) in oligometastatic ovarian cancers (OCs) lead to the query whether it is possible to stratify patients based on tumor hallmarks to ensure the best-personalized RT treatment. To address this question, we designed a preclinical study to evaluate the effects of high and low linear energy transfer (LET) radiation while considering molecular features and alpha/beta ratios of different OC cell lines. Exponentially growing human OVSAHO, OVCAR8, COV362, and OVCAR3 cells cultured in T-25 and T-75 flasks were exposed to different single physical doses of photons, protons, and carbon ion (CIRT) irradiation. We assessed ovarian cells' in vitro response using clonogenic survival (fitted using LQ model), migration by Boyden chamber assay, and invasion through BioCoat Matrigel invasion assay. Following photon irradiation, OVCAR3 was the most radioresistant and OVCAR8 the most radiosensitive cell line. OC cell migration decreased in a dose-dependent manner after irradiation, with CIRT showing the strongest effect, evident by the α/β ratio. The number of invading cells decreased following irradiation with all types. However, the greatest reduction was seen in CIRT, particularly at higher α/β ratios. Proton irradiation demonstrated similar potential to photons but did not match the effects of carbon ions in terms of survival, migration, and invasion

Serum- and glucocorticoid- inducible kinase 2, SGK2, is a novel autophagy regulator and modulates platinum drugs response in cancer cells

AbstractFor many tumor types chemotherapy still represents the therapy of choice and many standard treatments are based on the use of platinum (PT) drugs. However, de novo or acquired resistance to platinum is frequent and leads to disease progression. In Epithelial Ovarian Cancer (EOC) patients, PT-resistant recurrences are very common and improving the response to treatment still represents an unmet clinical need. To identify new modulators of PT-sensitivity, we performed a loss-of-function screening targeting 680 genes potentially involved in the response of EOC cells to platinum. We found that SGK2 (Serum-and Glucocorticoid-inducible kinase 2) plays a key role in PT-response. We show here that EOC cells relay on the induction of autophagy to escape PT-induced death and that SGK2 inhibition increases PT sensitivity inducing a block in the autophagy cascade due to the impairment of lysosomal acidification. Mechanistically we demonstrate that SGK2 controls autophagy in a kinase-dependent manner by binding and inhibiting the V-ATPase proton pump. Accordingly, SGK2 phosphorylates the subunit V1H (ATP6V1H) of V-ATPase and silencing or chemical inhibition of SGK2, affects the normal autophagic flux and sensitizes EOC cells to platinum. Hence, we identified a new pathway that links autophagy to the survival of cancer cells under platinum treatment in which the druggable kinase SGK2 plays a central role. Our data suggest that blocking autophagy via SGK2 inhibition could represent a novel therapeutic strategy to improve patients’ response to platinum.

Splicing factor proline- and glutamine-rich (SFPQ) protein regulates platinum response in ovarian cancer-modulating SRSF2 activity

AbstractIn epithelial ovarian cancer (EOC), response to platinum (PT)-based chemotherapy dictates subsequent treatments and predicts patients’ prognosis. Alternative splicing is often deregulated in human cancers and can be altered by chemotherapy. Whether and how changes in alternative splicing regulation could impact on the response of EOC to PT-based chemotherapy is still not clarified. We identified the splicing factor proline and glutamine rich (SFPQ) as a critical mediator of response to PT in an unbiased functional genomic screening in EOC cells and, using a large cohort of primary and recurrent EOC samples, we observed that it is frequently overexpressed in recurrent PT-treated samples and that its overexpression correlates with PT resistance. At mechanistic level, we show that, under PT treatment, SFPQ, in complex with p54nrb, binds and regulates the activity of the splicing factor SRSF2. SFPQ/p54nrb complex decreases SRSF2 binding to caspase-9 RNA, favoring the expression of its alternative spliced antiapoptotic form. As a consequence, SFPQ/p54nrb protects cells from PT-induced death, eventually contributing to chemoresistance. Overall, our work unveils a previously unreported SFPQ/p54nrb/SRSF2 pathway that in EOC cells plays a central role in regulating alternative splicing and PT-induced apoptosis and that could result in the design of new possible ways of intervention to overcome PT resistance.

The future of gynecologic oncologic surgery: a narrative review of current surgical trials

Recent advances in gynecologic oncology have driven a paradigm shift toward less invasive, more personalized surgical approaches. This narrative review critically examines key ongoing international trials investigating innovative surgical strategies across vulvar, cervical, ovarian, and endometrial cancers, with a focus on improving oncologic outcomes while minimizing morbidity. In vulvar cancer, trials are exploring the use of neoadjuvant chemotherapy and the replacement of inguinofemoral lymphadenectomy with chemoradiation in selected patients. For cervical cancer, large multicenter randomized trials are evaluating the oncologic safety of minimally invasive hysterectomy, surgical staging for para-aortic disease, and robotic-assisted surgery. In the contest of ovarian cancer, randomized trials are assessing the role of lymphadenectomy in early-stage disease, the optimal timing of cytoreductive surgery (primary versus interval), and the potential benefits of hyperthermic intraperitoneal chemotherapy, even in cases of platinum-resistant recurrence. For endometrial cancer, both observational and interventional studies are investigating sentinel lymph nodes mapping and robotic-assisted hysterectomy as alternatives to traditional staging procedures. Collectively, these trials underscore the growing importance of individualized treatment strategies guided by disease stage, histologic subtype, response to neoadjuvant therapy, and patient-specific factors. While minimally invasive techniques and surgical de-escalation appear promising for selected patient populations, critical questions remain regarding long-term oncologic safety, cost-effectiveness, and the consistency of practice across institutions. This narrative review synthesizes current evidence and outlines how the outcomes of these pivotal studies are expected to influence future guidelines in gynecologic cancer surgery.

Probe-based confocal laser endomicroscopy intra-operative evaluation in ovarian cancer: definition of in vivo architectural patterns to determine resection strategies.

Probe-based confocal laser endomicroscopy (pCLE) is a novel real-time imaging technique that is potentially useful for accurately distinguishing between normal and cancerous tissues. The aim of this study was to describe the pCLE patterns of areas suggestive of tumors and evaluate the ability of the method to differentiate between normal and cancerous tissue during cytoreductive surgery for epithelial ovarian cancer. In vivo pCLE images and subsequent biopsies were acquired from various anatomical sites including the parietal and visceral peritoneum, ovaries, and omentum. Each endomicroscopic sequence was analyzed by highly experienced investigators using pCLE imaging for cancer diagnosis. Each pCLE sequence was compared with the histology of the corresponding specimens. We enrolled 18 women with International Federation of Gynecology and Obstetrics stage III/IV high-grade serous epithelial ovarian cancer referred for primary or interval debulking surgery. A total of 112 biopsies were obtained for histologic analysis. The pCLE images of normal tissue showed a regular distribution of stromal fibers and consistent cellular architecture, regardless of the anatomical region, with vascularized areas characterized by regular vessels. Conversely, the extravasation of fluorescein, used as a contrast agent, was a distinguishing feature of malignant nodules, which were easily recognized by leakage and are typical of tumor-associated vessels. The leakage often surrounded the dark clusters of neoplastic cells. A substantial agreement between pCLE and histology emerged (k = 0.66), whereas only a fair concordance between the surgeon's intra-operative assessment and histology was found (k = 0.30). Our results suggest that pCLE is a promising intra-operative technique to assist surgeons in accurately detecting peritoneal metastases in patients with advanced epithelial ovarian cancer, enhancing surgical radicality while avoiding unnecessary resection.

USP1 deubiquitinates PARP1 to regulate its trapping and PARylation activity

PARP inhibitors (PARPi) represent a game-changing treatment for patients with ovarian cancer with tumors deficient for the homologous recombination (HR) pathway treated with platinum (Pt)–based therapy. PARPi exert their cytotoxic effect by both trapping PARP1 on the damaged DNA and by restraining its enzymatic activity (PARylation). How PARP1 is recruited and trapped at the DNA damage sites and how resistance to PARPi could be overcome are still matters of investigation. Here, we described PARP1 as a substrate of the deubiquitinase USP1. At molecular level, USP1 binds PARP1 to remove its K63-linked polyubiquitination and controls PARP1 chromatin trapping and PARylation activity, regulating sensitivity to PARPi. In both Pt/PARPi-sensitive and -resistant cells, USP1/PARP1 combined blockade enhances replicative stress, DNA damage, and cell death. Our work dissected the biological interaction between USP1 and PARP1 and recommended this axis as a promising and powerful therapeutic choice for not only sensitive but also chemoresistant patients with ovarian cancer irrespective of their HR status.

Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

Abstract Platinum (PT)-resistant Epithelial Ovarian Cancer (EOC) grows as a metastatic disease, disseminating in the abdomen and pelvis. Very few options are available for PT-resistant EOC patients, and little is known about how the acquisition of PT-resistance mediates the increased spreading capabilities of EOC. Here, using isogenic PT-resistant cells, genetic and pharmacological approaches, and patient-derived models, we report that Integrin α6 (ITGA6) is overexpressed by PT-resistant cells and is necessary to sustain EOC metastatic ability and adhesion-dependent PT-resistance. Using in vitro approaches, we showed that PT induces a positive loop that, by stimulating ITGA6 transcription and secretion, contributes to the formation of a pre-metastatic niche enabling EOC cells to disseminate. At molecular level, ITGA6 engagement regulates the production and availability of insulin-like growth factors (IGFs), over-stimulating the IGF1R pathway and upregulating Snail expression. In vitro data were recapitulated using in vivo models in which the targeting of ITGA6 prevents PT-resistant EOC dissemination and improves PT-activity, supporting ITGA6 as a promising druggable target for EOC patients.

LINC01605 Is a Novel Target of Mutant p53 in Breast and Ovarian Cancer Cell Lines

TP53 is the most frequently mutated gene in human cancers. Most TP53 genomic alterations are missense mutations, which cause a loss of its tumour suppressor functions while providing mutant p53 (mut_p53) with oncogenic features (gain-of-function). Loss of p53 tumour suppressor functions alters the transcription of both protein-coding and non-protein-coding genes. Gain-of-function of mut_p53 triggers modification in gene expression as well; however, the impact of mut_p53 on the transcription of the non-protein-coding genes and whether these non-protein-coding genes affect oncogenic properties of cancer cell lines are not fully explored. In this study, we suggested that LINC01605 (also known as lincDUSP) is a long non-coding RNA regulated by mut_p53 and proved that mut_p53 directly regulates LINC01605 by binding to an enhancer region downstream of the LINC01605 locus. We also showed that the loss or downregulation of LINC01605 impairs cell migration in a breast cancer cell line. Eventually, by performing a combined analysis of RNA-seq data generated in mut_TP53-silenced and LINC01605 knockout cells, we showed that LINC01605 and mut_p53 share common gene pathways. Overall, our findings underline the importance of ncRNAs in the mut_p53 network in breast and ovarian cancer cell lines and in particular the importance of LINC01605 in mut_p53 pro-migratory pathways.