title: USP7 inhibitors, downregulating CCDC6, sensitize lung neuroendocrine cancer cells to PARP-inhibitor drugs.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Umberto
authors.initials: U
authors.lastname: Malapelle
authors.affiliation: Institute for the Experimental Endocrinology and Oncology, Research National Council, Naples, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Morra
authors.affiliation: Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy.
authors.forename: Gennaro
authors.initials: G
authors.lastname: Ilardi
authors.affiliation: Institute for the Experimental Endocrinology and Oncology, Research National Council, Naples, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Visconti
authors.affiliation: Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Merolla
authors.affiliation: Institute for the Experimental Endocrinology and Oncology, Research National Council, Naples, Italy.
authors.forename: Aniello
authors.initials: A
authors.lastname: Cerrato
authors.affiliation: Institute for the Experimental Endocrinology and Oncology, Research National Council, Naples, Italy.
authors.forename: Virginia
authors.initials: V
authors.lastname: Napolitano
authors.affiliation: Pathology Unit, A. Cardarelli Hospital, Naples, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Monaco
authors.affiliation: Thoracic Surgery Unit, A. Cardarelli Hospital, Naples, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Guggino
authors.affiliation: Thoracic Surgery Unit, A. Cardarelli Hospital, Naples, Italy.
authors.forename: Guglielmo
authors.initials: G
authors.lastname: Monaco
authors.affiliation: Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Staibano
authors.affiliation: Department of Public Health, University of Naples Federico II, Naples, Italy.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Troncone
authors.affiliation: Institute for the Experimental Endocrinology and Oncology, Research National Council, Naples, Italy. Electronic address: celetti@unina.it.
authors.forename: Angela
authors.initials: A
authors.lastname: Celetti
mesh_terms: D000091162:AMP-Activated Protein Kinase Kinases; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000627:therapeutic use; D018278:Carcinoma, Neuroendocrine / Q000235:genetics* / Q000473:pathology; D002945:Cisplatin / Q000627:therapeutic use; D003598:Cytoskeletal Proteins / Q000187:drug effects* / Q000235:genetics; D015536:Down-Regulation; D005260:Female; D016147:Genes, Tumor Suppressor; D016158:Genes, p53 / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D018358:Neuroendocrine Tumors / Q000378:metabolism* / Q000473:pathology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000627:therapeutic use*; D020641:Polymorphism, Single Nucleotide; D011237:Predictive Value of Tests; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D013876:Thiophenes; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics; D043222:Ubiquitin Thiolesterase / Q000378:metabolism; D000076084:Ubiquitin-Specific Peptidase 7 / Q000037:antagonists & inhibitors*
chemical_list: D000970:Antineoplastic Agents; C088832:CCDC6 protein, human; D003598:Cytoskeletal Proteins; C576408:P5091; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C495901:TP53 protein, human; D013876:Thiophenes; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D017346:Protein Serine-Threonine Kinases; C109790:STK11 protein, human; D000091162:AMP-Activated Protein Kinase Kinases; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7; D002945:Cisplatin
keywords: CCDC6; L-NET; NGS; P5091; PARP-Inhibitors; USP7