A xCT role in tumour-associated ferroptosis shed light on novel therapeutic options.

Solute carrier family 7 member 11 (SLC7A11; also known as xCT), a key component of the cystine/glutamate antiporter, is essential for the maintenance of cellular redox status and the regulation of tumor-associated ferroptosis. Accumulating evidence has demonstrated that xCT overexpression, resulting from different oncogenic and tumor suppressor signaling, promotes tumor progression and multidrug resistance partially via suppressing ferroptosis. In addition, recent studies have highlighted the role of xCT in regulating the metabolic flexibility in cancer cells. In this review, the xCT activities in intracellular redox balance and in ferroptotic cell death have been summarized. Moreover, the role of xCT in promoting tumor development, drug resistance, and nutrient dependency in cancer cells has been explored. Finally, different therapeutic strategies, xCT-based, for anti-cancer treatments have been discussed.

The disruption of the CCDC6 - PP4 axis induces a BRCAness like phenotype and sensitivity to PARP inhibitors in high-grade serous ovarian carcinoma.

BACKGROUND: Treatment with PARP inhibitors (PARPi) is primarily effective against high-grade serous ovarian cancers (HGSOC) with BRCA1/2 mutations or other deficiencies in homologous recombination (HR) repair mechanisms. However, resistance to PARPi frequently develops, mostly as a result of BRCA1/2 reversion mutations. The tumour suppressor CCDC6 is involved in HR repair by regulating the PP4c phosphatase activity on γH2AX. In this work, we reported that in ovarian cancer cells, a physical or functional loss of CCDC6 results synthetic lethal with the PARP-inhibitors drugs, by affecting the HR repair. We also unravelled a role for CCDC6 as predictive marker of PARPi sensitivity in ovarian cancer, and the impact of CCDC6 downregulation in overcoming PARPi resistance in these tumours. METHODS: A panel of HGSOC cell lines (either BRCA-wild type or mutant) were treated with PARPi after CCDC6 was attenuated by silencing or by inhibiting USP7, a CCDC6-deubiquitinating enzyme, and the effects on cell survival were assessed. At the cellular and molecular levels, the processes underlying the CCDC6-dependent modification of drugs' sensitivity were examined. Patient-derived xenografts (PDXs) were immunostained for CCDC6, and the expression of the protein was analysed statistically after digital or visual means. RESULTS: HGSOC cells acquired PARPi sensitivity after CCDC6 depletion. Notably, CCDC6 downregulation restored the PARPi sensitivity in newly generated or spontaneously resistant cells containing either wild type- or mutant-BRCA2. When in an un-phosphorylated state, the CCDC6 residue threonine 427 is crucial for effective CCDC6-PP4 complex formation and PP4 sequestration, which maintains high γH2AX levels and effective HR. Remarkably, the PP4-dependent control of HR repair is influenced by the CCDC6 constitutively phosphorylated mutant T427D or by the CCDC6 loss, favouring PARPi sensitivity. As a result, the PP4 regulatory component PP4R3α showed to be essential for both the activity of the PP4 complex and the CCDC6 dependent PARPi sensitivity. It's interesting to note that immunohistochemistry revealed an intense CCDC6 protein staining in olaparib-resistant HGSOC cells and PDXs. CONCLUSIONS: Our findings suggest that the physical loss or the functional impairment of CCDC6 enhances the PP4c complex activity, which causes BRCAness and PARPi sensitivity in HGSOC cells. Moreover, CCDC6 downregulation might overcome PARPi resistance in HGSOCs, thus supporting the potential of targeting CCDC6 by USP7 inhibitors to tackle PARPi resistance.

Altered brain regional homogeneity is associated with depressive symptoms in COVID-19.

BACKGROUND: COVID-19 is an infectious disease that has spread worldwide in 2020, causing a severe pandemic. In addition to respiratory symptoms, neuropsychiatric manifestations are commonly observed, including chronic fatigue, depression, and anxiety. The neural correlates of neuropsychiatric symptoms in COVID-19 are still largely unknown. METHODS: A total of 79 patients with COVID-19 (COV) and 17 healthy controls (HC) underwent 3 T functional magnetic resonance imaging at rest, as well as structural imaging. Regional homogeneity (ReHo) was calculated. We also measured depressive symptoms with the Patient Health Questionnaire (PHQ-9), anxiety using the General Anxiety Disorder 7-item scale, and fatigue with the Multidimension Fatigue Inventory. RESULTS: In comparison with HC, COV showed significantly higher depressive scores. Moreover, COV presented reduced ReHo in the left angular gyrus, the right superior/middle temporal gyrus and the left inferior temporal gyrus, and higher ReHo in the right hippocampus. No differences in gray matter were detected in these areas. Furthermore, we observed a negative correlation between ReHo in the left angular gyrus and PHQ-9 scores and a trend toward a positive correlation between ReHo in the right hippocampus and PHQ-9 scores. LIMITATIONS: Heterogeneity in the clinical presentation in COV, the different timing from the first positive molecular swab test to the MRI, and the cross-sectional design of the study limit the generalizability of our findings. CONCLUSIONS: Our results suggest that COVID-19 infection may contribute to depressive symptoms via a modulation of local functional connectivity in cortico-limbic circuits.

18F-FDG-PET/MRI texture analysis in rectal cancer after neoadjuvant chemoradiotherapy.

OBJECTIVE: Reliable markers to predict the response to neoadjuvant chemoradiotherapy (nCRT) in locally advanced rectal cancer (LARC) are lacking. We aimed to assess the ability of 18F-FDG PET/MRI to predict response to nCRT among patients undergoing curative-intent surgery. METHODS: Patients with histological-confirmed LARC who underwent curative-intent surgery following nCRT and restaging with 18F-FDG PET/MRI were included. Statistical correlation between radiomic features extracted in PET, apparent diffusion coefficient (ADC) and T2w images and patients' histopathologic response to chemoradiotherapy using a multivariable logistic regression model ROC-analysis. RESULTS: Overall, 50 patients were included in the study. A pathological complete response was achieved in 28.0% of patients. Considering second-order textural features, nine parameters showed a statistically significant difference between the two groups in ADC images, six parameters in PET images and four parameters in T2w images. Combining all the features selected for the three techniques in the same multivariate ROC curve analysis, we obtained an area under ROC curve of 0.863 (95% CI, 0.760-0.966), showing a sensitivity, specificity and accuracy at the Youden's index of 100% (14/14), 64% (23/36) and 74% (37/50), respectively. CONCLUSION: PET/MRI texture analysis seems to represent a valuable tool in the identification of rectal cancer patients with a complete pathological response to nCRT.

Thyroid hormone and androgen signals mutually interplay and enhance inflammation and tumorigenic activation of tumor microenvironment in prostate cancer.

Prostate Cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in males and the fifth leading cause of death worldwide. The majority of PCas are androgen-sensitive, with a significant up-regulation of Androgen Receptor (AR) that causes a stimulatory effect on growth and progression of cancer cells. For this reason, the first-line therapy for PCa is androgen ablation, even if it ultimately fails due to the onset of hormone-refractory state, in which the malignant cells do not sense the androgen signal anymore. Besides androgens, a growing number of evidence suggests that Thyroid Hormones (THs) mediate tumor-promoting effects in a variety of human cancers, as Epithelial-to-Mesenchymal Transition (EMT), invasion and metastasis and also stimulation of angiogenesis and tumor metabolism. Moreover, epidemiological studies demonstrated an increased risk for PCa in patients with lower levels of Thyreotropin (TSH). Here, we investigated if intracellular TH metabolism affects Benign Prostatic Hyperplasia (BPH) and PCa formation and progression. We found that the intracellular TH metabolism is a crucial determinant of PCa behavior. We observed that a dynamic stage-specific expression of the THs modulating enzymes, the deiodinases, is required for the progression of BPH to PCa malignancy. By acting simultaneously on epithelial cancer cells and fibroblasts, THs exert a proliferative and pro-inflammatory effect cooperating with androgens. These findings suggest that androgens and THs may interplay and mediate a coordinate effect on human PCa formation and progression. In light of our results, future perspective could be to explore the potential benefits of THs intracellular modulators aimed to counteract PCa progression.

The tumour suppressor CCDC6 is involved in ROS tolerance and neoplastic transformation by evading ferroptosis.

Coiled-coil domain containing 6 (CCDC6) is a tumour suppressor gene involved in apoptosis and DNA damage response. CCDC6 is known to be functionally impaired upon gene fusions, somatic mutations, and altered protein turnover in several tumours. Testicular germ cell tumours are among the most common malignancies in young males. Despite the high cure rate, achieved through chemotherapy and/or surgery, drug resistance can still occur. In a human cellular model of testis Embryonal Carcinoma, the deficiency of CCDC6 was associated with defects in DNA repair via homologous recombination and sensitivity to PARP1/2 inhibitors. Same data were obtained in a panel of murine testicular cell lines, including Sertoli, Spermatogonia and Spermatocytes. In these cells, upon oxidative damage exposure, the absence of CCDC6 conferred tolerance to reactive oxygen species affecting regulated cell death pathways by apoptosis and ferroptosis. At molecular level, the loss of CCDC6 was associated with an enhancement of the xCT/SLC7A11 cystine antiporter expression which, by promoting the accumulation of ROS, interfered with the activation of ferroptosis pathway. In conclusion, our data suggest that the CCDC6 downregulation could aid the testis germ cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants contributing to testicular neoplastic growth. Novel therapeutic options will be discussed.

Neuropilin-1 Expression Associates with Poor Prognosis in HNSCC and Elicits EGFR Activation upon CDDP-Induced Cytotoxic Stress.

Head and neck squamous cell carcinoma (HNSCC) includes a group of aggressive malignancies characterized by the overexpression of the epidermal growth factor receptor (EGFR) in 90% of cases. Neuropilin-1 (NRP-1) acts as an EGFR co-receptor, enhancing, upon ligand stimulation, EGFR signaling in several cellular models. However, NRP-1 remains poorly characterized in HNSCC. By utilizing in vitro cellular models of HNSCC, we report that NRP-1 is involved in the regulation of EGFR signaling. In fact, NRP-1 can lead to cisplatin-induced EGFR phosphorylation, an escape mechanism activated by cancer cells upon cytotoxic stress. Furthermore, we evaluated Neuropilin-1 staining in tissue samples of an HNSCC case series (n = 218), unraveling a prognostic value for the Neuropilin-1 tissue expression. These data suggest a potential role for NRP-1 in HNSCC cancer progression, expanding the repertoire of signaling in which NRP-1 is involved and eliciting the need for further investigations on NRP-1 as a suitable target for HNSCC novel therapeutic approaches.

Toll-Like Receptor 7 Mediates Inflammation Resolution and Inhibition of Angiogenesis in Non-Small Cell Lung Cancer.

Pattern recognition receptors (PRR) promote inflammation but also its resolution. We demonstrated that a specific PRR-formyl peptide receptor 1 (FPR1)-sustains an inflammation resolution response with anti-angiogenic and antitumor potential in gastric cancer. Since toll-like receptor 7 (TLR7) is crucial in the physiologic resolution of airway inflammation, we asked whether it could be responsible for pro-resolving and anti-angiogenic responses in non-small cell lung cancer (NSCLC). TLR7 correlated directly with pro-resolving and inversely with angiogenic mediators in NSCLC patients, as revealed by a publicly available RNAseq analysis. In NSCLC cells, depletion of TLR7 caused an upregulation of angiogenic mediators and a stronger vasculogenic response of endothelial cells compared to controls, assessed by qPCR, ELISA, protein array, and endothelial cell responses. TLR7 activation induced the opposite effects. TLR7 silencing reduced, while its activation increased, the pro-resolving potential of NSCLC cells, evaluated by qPCR, flow cytometry, and EIA. The increased angiogenic potential of TLR7-silenced NSCLC cells is due to the lack of pro-resolving mediators. MAPK and STAT3 signaling are responsible for these activities, as demonstrated through Western blotting and inhibitors. Our data indicate that TLR7 sustains a pro-resolving signaling in lung cancer that inhibits angiogenesis. This opens new possibilities to be exploited for cancer treatment.

NSCLC Mutated Isoforms of CCDC6 Affect the Intracellular Distribution of the Wild Type Protein Promoting Cisplatinum Resistance and PARP Inhibitors Sensitivity in Lung Cancer Cells.

CCDC6 is implicated in cell cycle checkpoints and DNA damage repair by homologous recombination (HR). In NSCLC, CCDC6 is barely expressed in about 30% of patients and CCDC6 gene rearrangements with RET and ROS kinases are detected in about 1% of patients. Recently, CCDC6 point-mutations naming E227K, S351Y, N394Y, and T462A have been identified in primary NSCLC. In this work, we analyze the effects exerted by the CCDC6 mutated isoforms on lung cancer cells. By pull-down experiments and immunofluorescence, we evaluated the biochemical and morphological effects of CCDC6 lung-mutants on the CCDC6 wild type protein. By using two HR-reporter assays, we analyzed the effect of CCDC6 lung-mutants in perturbing CCDC6 physiology in the HR process. Finally, by cell-titer assay, we evaluated the response to the treatment with different drugs in lung cancer cells expressing CCDC6 mutants. This work shows that the CCDC6 mutated and truncated isoforms, identified so far in NSCLC, affected the intracellular distribution of the wild type protein and impaired the CCDC6 function in the HR process, ultimately inducing cisplatinum resistance and PARP-inhibitors sensitivity in lung cancer cells. The identification of selected molecular alterations involving CCDC6 gene product might define predictive biomarkers for personalized treatment in NSCLC.

CAF-1 Subunits Levels Suggest Combined Treatments with PARP-Inhibitors and Ionizing Radiation in Advanced HNSCC.

Oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinomas show high morbidity and mortality rates. We aimed to investigate the role of the "Chromatin Assembly Factor-1" (CAF-1) p60 and p150 subunits, involved in DNA repair and replication, in OSCC and OPSCC progression and in response to Poly(ADP-ribose) polymerase (PARP)-inhibitors and exposure to ionizing radiation (IR). We immunostained tissue microarrays (TMAs), including 112 OSCC and 42 OPSCC, with anti-CAF-1/p60 and anti-CAF-1/p150 specific antibodies, correlating their expression with prognosis. Moreover, we assessed the sensitivity to PARP inhibitors and the double-strand breaks repair proficiency by cell viability and HR reporter assays, respectively, in HPV-positive and HPV-negative cell lines upon CAF-1/p60 and CAF-1/p150 depletion. The immunohistochemical analysis revealed a significant prognostic value of both tissue biomarkers combined expression in OSCC but not in OPSCC. In in vitro studies, the p60/150 CAF-1 subunits' depletion impaired the proficiency of Homologous Recombination DNA damage repair, inducing sensitivity to the PARP-inhibitors, able to sensitize both the cell lines to IR. These results indicate that regardless of the prognostic meaning of p60/p150 tissue expression, the pharmacological depletion of CAF-1 complex's function, combined to PARP-inhibitors and/or IR treatment, could represent a valid therapeutic strategy for squamous cell carcinomas of head and neck region.

Analysis of CCDC6 as a novel biomarker for the clinical use of PARP1 inhibitors in malignant pleural mesothelioma.

OBJECTIVES: CCDC6 (coiled-coil domain containing 6) is a player of the HR response to DNA damage and has been predicted to interact with BAP1, another HR-DNA repair gene highly mutated in Malignant Pleural Mesothelioma (MPM), an aggressive cancer with poor prognosis. CCDC6 levels are modulated by the deubiquitinase USP7, and CCDC6 defects have been reported in several tumors determining PARP-inhibitors sensitivity. Our aim was to investigate the functional role of CCDC6 in MPM carcinogenesis and response to PARP-inhibitors. MATERIALS AND METHODS: The interaction between CCDC6 and BAP1 was confirmed in MPM cells, by co-immunoprecipitation. Upon USP7 inhibition, that induces CCDC6 degradation, the ability to repair the DSBs and the sensitivity to PARP inhibitors, was explored by HR reporter and by cells viability assays, respectively. A TMA including 34 MPM cores was immunostained for CCDC6, USP7 and BAP1 and the results correlated by statistical analysis. RESULTS: MPM cells depleted of CCDC6 showed defects in DSBs repair and sensitivity to PARP inhibitors. The silencing of CCDC6 when combined with the overexpression of BAP1-mutant (Δ221-238) enhanced the HR-DNA repair defects and the PARP inhibitors sensitivity. In the TMA of MPM primary samples, the staining of CCDC6 and of its de-ubiquitinase USP7 showed a significant correlation in the tested primary samples (p = 0.01). CCDC6 was barely detected in 30% of the tumors that also carried BAP1 defects. CONCLUSION: The combination of CCDC6 and BAP1 staining may indicate therapeutic options for DDR targeting, acting in synergism with cisplatinum.

Identification of Novel Biomarkers of Homologous Recombination Defect in DNA Repair to Predict Sensitivity of Prostate Cancer Cells to PARP-Inhibitors.

One of the most common malignancies in men is prostate cancer, for which androgen deprivation is the standard therapy. However, prostate cancer cells become insensitive to anti-androgen treatment and proceed to a castration-resistant state with limited therapeutic options. Therefore, besides the androgen deprivation approach, novel biomarkers are urgently required for specific targeting in this deadly disease. Recently, germline or somatic mutations in the homologous recombination (HR) DNA repair genes have been identified in at least 20-25% of metastatic castration-resistant prostate cancers (mCRPC). Defects in genes involved in HR DNA repair can sensitize cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors, a class of drugs already approved by the Food and Drug Administration (FDA) for breast and ovarian cancer carrying germline mutations in BRCA1/2 genes. For advanced prostate cancer carrying Breast cancer1/2 (BRCA1/2) or ataxia telengiectasia mutated (ATM) mutations, preclinical studies and clinical trials support the use of PARP-inhibitors, which received breakthrough therapy designation by the FDA. Based on these assumptions, several trials including DNA damage response and repair (DDR) targeting have been launched and are ongoing for prostate cancer. Here, we review the state-of-the-art potential biomarkers that could be predictive of cancer cell synthetic lethality with PARP inhibitors. The identification of key molecules that are affected in prostate cancer could be assayed in future clinical studies to better stratify prostate cancer patients who might benefit from target therapy.

CCDC6 and USP7 expression levels suggest novel treatment options in high-grade urothelial bladder cancer.

BACKGROUND: The muscle invasive form of urothelial bladder cancer (UBC) is a deadly disease. Currently, the therapeutic approach of UBC is mostly based on surgery and standard chemotherapy. Biomarkers to establish appropriate drugs usage are missing. Deficiency of the tumor suppressor CCDC6 determines PARP-inhibitor sensitivity. The CCDC6 levels are modulated by the deubiquitinase USP7. In this work we scored CCDC6 and USP7 expression levels in primary UBC and we evaluated the expression levels of CCDC6 in correlation with the effects of the PARP-inhibitors combined with the USP7 inhibitor, P5091, in vitro. Since PARP-inhibitors could be enhanced by conventional chemotherapy or DNA damage inducers, we tested the new agent RRx-001, able to induce DNA damage, to prove the benefit of combined treatments in bladder cancer cells. METHODS: The J82, T24, 5637 and KU-19-19 bladder cancer cells were exposed to USP7 inhibitor P5091 in presence of cycloheximide to analyse the CCDC6 stability. Upon the CCDC6 degradation induced by P5091, the cells sensitivity to PARP-inhibitor was evaluated by cell viability assays. The ability of the DNA damage inducer RRx-001 to modulate CCDC6 protein levels and H2AX phosphorylation was detected at immunoblot. The combination of USP7 inhibitor plus RRx-001 enhanced the PARP-inhibitor sensitivity, as evaluated by cell viability assays. The results of the scores and correlation of CCDC6 and USP7 expression levels obtained by UBC primary biopsies staining were used to cluster patients by a K-mean cluster analysis. RESULTS: P5091 determining CCDC6 degradation promoted bladder cancer cells sensitivity to PARP-inhibitor drugs. RRx-001, by inducing DNA damage, enhanced the effects of the combined treatment. The immunohistochemical staining of both CCDC6 and USP7 proteins allowed to cluster the high grade (G3) UBC patients, on the basis of CCDC6 expression levels. CONCLUSIONS: In high grade UBC the identification of two clusters of patients based on CCDC6 and USP7 expession can possibly indicate the use of PARP-inhibitor drugs, in combination with USP7 inhibitor in addition to the DNA damage inducer RRx-001, that also acts as an immunomodulatory agent, offering novel therapeutic strategy for personalized medicine in bladder cancer patients.

New combinatorial strategies to improve the PARP inhibitors efficacy in the urothelial bladder Cancer treatment.

BACKGROUND: Novel therapeutic strategies are urgently needed for the treatment of metastatic Urothelial Bladder Cancer. DNA damaging repair (DDR) targeting has been introduced in cinical trials for bladder cancer patients that carry alterations in homologous DNA repair genes, letting to envisage susceptibility to the Poly (adenosine diphosphate [ADP]) ribose polymerase (PARP) inhibitors. MAIN BODY: PARP inhibition, by amplifying the DNA damage, augments the mutational burden and promotes the immune priming of the tumor by increasing the neoantigen exposure and determining upregulation of programmed death ligand 1 (PD-L1) expression. Thus, the combination of PARP-inhibition and the PD/PD-L1 targeting may represent a compelling strategy to treat bladder cancer and has been introduced in recent clinical trials. The targeting of DDR has been also used in combination with epigenetic drugs able to modulate the expression of genes involved in DDR, and also able to act as immunomodulator agents suggesting their use in combination with immune-checkpoint inhibitors. CONCLUSION: In conclusion, it may be envisaged the combination of three classes of drugs to treat bladder cancer, by targeting the DDR process in a tumor context of DDR defect, together with epigenetic agents and immune-checkpoint inhibitors, whose association may amplify the effects and reduce the doses and the toxicity of each single drug.

CCDC6: the identity of a protein known to be partner in fusion.

Coiled Coil Domain Containing 6 gene, CCDC6, was initially isolated as part of a tumorigenic DNA originated by the fusion of CCDC6 with the tyrosine kinase of RET receptor, following a paracentric inversion of chromosome 10. For a long time, CCDC6 has been considered as an accidental partner of the RET protooncogene, providing the promoter and the first 101 aa necessary for the constitutive activation of the oncogenic Tyrosine Kinase (TK) RET in thyroid cells. With the advent of more refined diagnostic tools and bioinformatic algorithms, an exponential growth in fusion genes discoveries has allowed the identification of CCDC6 as partner of genes other than RET in different tumor types. CCDC6 gene product has a proper role in sustaining the DNA damage checkpoints in response to DNA damage. The inactivation of CCDC6 secondary to chromosomal rearrangements or gene mutations could enhance tumor progression by impairing the apoptotic response upon the DNA damage exposure, contributing to the generation of radio- and chemoresistance. Preclinical studies indicate that the attenuation of CCDC6 in cancer, while conferring a resistance to cisplatinum, sensitizes the cancer cells to the small molecule inhibitors of Poly (ADP-ribose) polymerase (PARP1/2) with a synthetic lethal effect. Several CCDC6 mutations and gene rearrangements have been described so far in different types of cancer and CCDC6 may represent a possible predictive biomarker of tumor resistance to the conventional anticancer treatments. Nevertheless, the detection of a CCDC6 impairment in cancer patients may help to select, in future clinical trials, those patients who could benefit of PARP-inhibitors treatment alone or in combination with other treatments.

The between Now and Then of Lung Cancer Chemotherapy and Immunotherapy.

Lung cancer is the most common cancer worldwide. Disappointingly, despite great effort in encouraging screening or, at least, a close surveillance of high-risk individuals, most of lung cancers are diagnosed when already surgically unresectable because of local advancement or metastasis. In these cases, the treatment of choice is chemotherapy, alone or in combination with radiotherapy. Here, we will briefly review the most successful and recent advances in the identification of novel lung cancer genetic lesions and in the development of new drugs specifically targeting them. However, lung cancer is still the leading cause of cancer-related mortality also because, despite impressive initial responses, the patients often develop resistance to novel target therapies after a few months of treatment. Thus, it is literally vital to continue the search for new therapeutic options. So, here, on the basis of our recent findings on the role of the tumor suppressor CCDC6 protein in lung tumorigenesis, we will also discuss novel therapeutic approaches we envision for lung cancer.

The combined effect of USP7 inhibitors and PARP inhibitors in hormone-sensitive and castration-resistant prostate cancer cells.

PURPOSE OF THE STUDY: Reduced levels of the tumor suppressor protein CCDC6 sensitize cancer cells to the treatment with PARP-inhibitors. The turnover of CCDC6 protein is regulated by the de-ubiquitinase USP7, which also controls the androgen receptor (AR) stability. Here, we correlated the expression levels of CCDC6 and USP7 proteins in primary prostate cancers (PC). Moreover, we tested the efficacy of the USP7 inhibitors, in combination with PARP-inhibitors as a novel therapeutic option in advanced prostate cancer.Experimental techniques: PC cells were exposed to USP7 inhibitor, P5091, together with cycloheximide, to investigate the turnover of the USP7 substrates, AR and CCDC6. As outcome of the AR downregulation, transcription targets of AR and its variant V7 were examined by qPCR. As a result of CCDC6 degradation, the induction of PARP inhibitors sensitivity was evaluated by analyzing PC cells viability and foci formation. We scored and correlated CCDC6 and USP7 expression levels in a prostate cancer tissue microarray (TMA). RESULTS: P5091 accelerated the degradation of AR and V7 isoform affecting PSA, UBE2C, CDC20 transcription and PC cells proliferation. Moreover, P5091 accelerated the degradation of CCDC6 sensitizing the cells to PARP-inhibitors, that acted sinergistically with genotoxic agents. The immunohistochemical analysis of both CCDC6 and USP7 proteins exhibited significant correlation for the intensity of staining (p ≤ 0.05).Data interpretation: Thus, CCDC6 and USP7 represent predictive markers for the combined treatment of the USP7-inhibitors and PARP-inhibitors in advanced prostate cancer.

USP7 inhibitors, downregulating CCDC6, sensitize lung neuroendocrine cancer cells to PARP-inhibitor drugs.

OBJECTIVES: CCDC6 gene product is a tumor-suppressor pro-apoptotic protein, substrate of ATM, involved in DNA damage response and repair. Altered levels of CCDC6 expression are dependent on post-translational modifications, being the de-ubiquitinating enzyme USP7 responsible of the fine tuning of the CCDC6 stability. Thus, our aim was to investigate CCDC6 and USP7 expression levels in Lung-Neuroendocrine Tumors (L-NETs) to verify if they correlate and may be exploited as novel predictive therapeutic markers. MATERIALS AND METHODS: Tumor tissues from 29 L-NET patients were investigated on tissue microarrays. CCDC6 levels were scored and correlated with immunoreactivity for USP7. Next generation sequencing (NGS) of a homogenous group of Large Cell Neuroendocrine Carcinoma (LCNEC) (N=8) was performed by Ion AmpliSeq NGS platform and the Ion AmpliSeq Cancer Hotspot Panel v2. The inhibition of USP7, using P5091, was assayed in vitro to accelerate CCDC6 turnover in order to sensitize the neuroendocrine cancer cells to PARP-inhibitors, alone or in association with cisplatinum. RESULTS: The immunostaining of 29 primary L-NETs showed that the intensity of CCDC6 staining correlated with the levels of USP7 expression (p≤0.05). The NGS analysis of 8 LCNEC revealed mutations in the hot spot regions of the p53 gene (in 6 out of 8). Moreover, gene polymorphisms were identified in the druggable STK11, MET and ALK genes. High intensity of p53 immunostaining was reported in the 6 tissues carrying the TP53 mutations. The inhibition of USP7 by P5091 accelerated the degradation of CCDC6 versus control in cycloheximide treated L-NET cells in vitro and sensitized the cells to PARP-inhibitors alone and in combination with cisplatinum. CONCLUSION: Our data suggest that CCDC6 and USP7 have a predictive value for the clinical usage of USP7 inhibitors in combination with the PARP-inhibitors in L-NET in addition to standard therapy.