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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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

CCDC6 gene product is a pro-apoptotic protein substrate of ATM, whose loss or inactivation enhances tumour progression. In primary tumours, the impaired function of CCDC6 protein has been ascribed to CCDC6 rearrangements and to somatic mutations in several neoplasia. Recently, low levels of CCDC6 protein, in NSCLC, have been correlated with tumor prognosis. However, the mechanisms responsible for the variable levels of CCDC6 in primary tumors have not been described yet.We show that CCDC6 turnover is regulated in a cell cycle dependent manner. CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis. The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl. The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response.Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.

Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC., (© 2014 UICC.)

Background: DNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation. CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners. In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors., Methods: To achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses., Results: The analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H₂O₂, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased., Conclusions: Therefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.

Background: Recent studies have reported nuclear delocalization of plakoglobin in acantholytic pemphigus vulgaris cells. The objective of this study was to evaluate the role of plakoglobin in the pathogenesis of acantholysis in pemphigus vulgaris (PV) and its relation with the urokinase-type plasminogen activator receptor (uPAR) expression., Materials and Methods: Plakoglobin and uPAR expressions were evaluated by immunohistochemistry in 22 cases of PV at various stages of the disease, and as controls in 18 specimens of skin/oral mucosa from healthy patients., Results: Healthy skin/normal oral mucosa showed strong plakoglobin expression in the basal and spinous layers with prevalent cellular membrane distribution; the intensity of staining progressively decreased toward the superficial layers of the epithelium. In PV patients, a progressive displacement of the plakoglobin signal toward the nucleus was found in 18/22 of the cases. Healthy skin/normal oral mucosa showed low uPAR expression with prevalent cellular membrane distribution. In the PV patients, strong uPAR expression was present in the acantholytic cells in 16/22 of the cases. There was direct correlation (p < 0.05) between the uPAR expression and nuclear plakoglobin., Conclusions: The uPAR overexpression in acantholytic PV may be considered a direct consequence of plakoglobin abnormal distribution. Nuclear delocalization of plakoglobin, a direct consequence of plakoglobin-Dsg-3 dissociation induced by PV IgG, probably induces uPAR overexpression. This evidence suggests a central role for plakoglobin in PV pathogenesis because of its delocalization toward the nucleus, which is the probable cause of the uPAR gene expression.

Background: The WNT gene family is a group of developmental genes involved in cell growth regulation, differentiation and organogenesis in both vertebrates and invertebrates. These genes are also involved in oncogenesis: beta-catenin, a component of the WNT pathway, has been reported to be involved in the genesis of numerous human cancers. WNT-1 pathway signaling is mediated via interactions between beta-catenin, a multifunctional protein playing an important role in cell-to-cell adhesion and gene expression, and members of the LEF-1/TCF family of transcription factors. The WNT signal stabilizes beta-catenin protein and determines its accumulation in the cytoplasm and nucleus., Materials and Methods: In order to evaluate the role of WNT-1 in the neoplastic progression of basal cell carcinoma (BCC), an immunohistochemical and confocal study of its expression and its correlation with beta-catenin distribution was performed in 46 selected cases of BCCs of the head and neck region., Results: While normal skin showed a WNT-1-positive staining only of the cutaneous annexa and a few cells in the basal/parabasal layers, the areas of de-differentiated BCCs showed a high granular positive staining (50-80% of cells). On the other hand, normal skin was characterized by an intense membranous staining for beta-catenin, with a progressive displacement of the signal toward the periphery of the cells. In BCC the absence of membrane localization and cytosolic staining for beta-catenin were detected in de-differentiated cases. A significant correlation (by Pearson's analysis) between overexpression of WNT-1 and free pools of beta-catenins was observed in these tumors., Conclusion: According to these data, the potential role of the WNT-1 gene in BCC seems to correlate with its ability to induce elevated cytoplasmic beta-catenin levels, suggesting that the WNT-1 gene can activate an intracellular signaling pathway involved in the process of cell transformation.

Background: Pemphigus vulgaris (PV) is an autoimmune blistering disease of the skin and mucosa due to the presence of autoantibodies against the components of desmosomes. To date, less is known about the expression levels of beta- and gamma-catenins in blistering diseases. The objective of this study was to evaluate the role of beta- and gamma-catenins in the pathogenesis of acantholysis in pemphigus vulgaris., Methods: beta- and gamma-catenin expression was evaluated by immunohistochemistry in 30 cases of PV at various stages of the disease and, as controls, in 18 specimens of the skin/oral mucosa of healthy patients., Results: Healthy skin and normal oral mucosa showed a strong beta- and gamma-catenin expression in basal and spinous layers with a prevalent cellular membrane distribution; the intensity of staining progressively decreased toward the superficial layers of epithelium. In PV patients, cytoplasmic expression of gamma-catenin was detected in 28/30 cases, and in 19/30 cases of PV for beta-catenin. Moreover, a progressive displacement of the signal toward the nucleus was found in 14/30 cases for beta-catenin, with dyslocalization toward the nucleus, particularly in areas with intense acantholysis, and in 22/30 cases of PV for gamma-catenin., Conclusions: Abnormal distribution of gamma-catenin, consequent to PV IgG, may be considered a direct consequence of Dg3 dissociation from catenin. gamma-catenin likely plays a direct role in PV pathogenesis through its dyslocalization toward the nucleus or indirectly through the beta-catenin dyslocalization toward the nucleus, which is thought to induce transcription of selected target genes, such as uPAR.

Cell-to-cell adhesion is mediated by cadherins (integral membrane proteins), which form a complex with catenins (cytoplasmatic proteins). While E-cadherin expression has been extensively studied in many human skin diseases, less is known about the expression levels of catenins in oral blistering diseases. The purpose of this study was to evaluate the role of these proteins in the pathogenesis of acantholysis in oral pemphigus vulgaris. We evaluated by immunohistochemistry beta- and gamma-catenin expression in 7 cases of oral pemphigus vulgaris (PV) at various stages of the disease and, as controls, in 18 healthy patients. Healthy cases showed, as reported in the literature, a strong reactivity with both beta- and gamma-catenins, with the intensity of staining progressively decreasing from the spinous to the keratinised layers of epithelium, which had a prevalent cellular membrane expression. In PV patients, we detected a loss of membrane expression of these molecules with a progressive displacement of the signal toward the cytosol and, for gamma-catenin, nuclear dislocation, particularly in areas with intense acantholysis.

Cell-cell adhesion is mediated by cadherins (integral membrane proteins) which form a complex with catenins (cytoplasmatic proteins). Down-regulation of cadherins and more recently of catenins has frequently been detected in many types of human carcinomas, in which it has been associated to tumor progression. While E-cadherin expression has been extensively studied in many forms of human cancers, including oral SCC, less is known about the expression levels of catenins in oral SCCs. The objective of this study was to evaluate the role of these proteins in the carcinogenetic process of the oral cavity. We evaluated by immunohistochemistry beta- and gamma-catenin expression in 30 cases of intraoral squamous cell carcinomas at different degree of cellular differentiation. As already reported for E-cadherin, the beta- and gamma-catenin expression showed an inverse relationship with the degree of differentiation, being the membranous expression of both catenins homogeneously reduced in less differentiated oral squamous cell carcinomas (grade 3). More interestingly, a decreased expression of these molecules was also found at the invasive front of grade 2 and sometimes of grade 1 carcinomas, thus suggesting a more aggressive biological behavior of these cancer cells. An absent staining for both beta- and gamma-catenins could constitute a hallmark of aggressive biological behavior even in tumor still well or moderately differentiated, at least in the peripheral invading front constituted by less differentiated tumor cells.

Background: Catenins belong to a family of proteins that mediate the binding between intracytoplasmic domain of cadherins and cytoskeleton. Few data on distribution of beta and gamma catenins in non-neoplastic tissues are available from current literature. This study aims to evaluate distribution of beta and gamma catenins in oral epithelium., Methods: Nine formalin-fixed and paraffin-embedded samples of oral epithelium were retrieved from files of Department of Oral Pathology of the University of Naples "Federico II". These samples were tested with anti-beta and anti-gamma monoclonal antibodies revealed by standard streptavidin-biotin-peroxidase technique. Sections have been evaluated by two observers by optical microscope using a 40X objective. The number of positive cells has been evaluated using a semi-quantitative method., Results: The results of this study show that beta and gamma catenins were mostly distributed in the upper two-thirds of oral epithelial thickness, except for keratinized areas which appear negative. Basal layer is positive except for the basal side of basal cells. Keratinized layers are negative for beta and gamma catenins., Conclusions: In physiologic conditions staining pattern for beta and gamma catenins is almost exclusively membranous, sometimes cytoplasmic but never nuclear. This staining is well-represented even in conditions of chronic inflammation and leucoplakia.