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// Renato Franco 1, 2, * , Giosue Scognamiglio 1, * , Elena Valentino 1 , Michela Vitiello 3 , Antonio Luciano 4 , Giuseppe Palma 4 , Claudio Arra 4 , Elvira La Mantia 1 , Luigi Panico 5 , Valentina Tenneriello 5 , Antonello Pinto 6 , Ferdinando Frigeri 6 , Gaetana Capobianco 6 , Gerardo Botti 1 , Laura Cerchia 3 , Annarosaria De Chiara 1 , Monica Fedele 3 1 Surgical Pathology Unit, National Cancer Institute ‘Fondazione Giovanni Pascale’, IRCCS, Naples, Italy 2 Pathology Unit, Second University of Naples, Naples, Italy 3 Institute of Experimental Endocrinology and Oncology (IEOS), National Research Council (CNR), Naples, Italy 4 Animal Facility, National Cancer Institute ‘Fondazione Giovanni Pascale’, IRCCS, Naples, Italy 5 Pathology Unit, Hospital ‘S.G. Moscati’, Avellino, Italy 6 Haematology-Oncology and Stem Cell Transplantation Unit, Department of Hematology, National Cancer Institute, Fondazione ‘G. Pascale’, IRCCS, Naples, Italy * These authors share co-first authorship Correspondence to: Monica Fedele, email: mfedele@unina.it , monica.fedele@cnr.it Renato Franco, email: renato.franco@unina2.it , renfr@yahoo.com Keywords: non-Hodgkin lymphomas, prognostic marker, PATZ1, tissue-microarray, DLBCL Received: January 16, 2016 Accepted: July 18, 2016 Published: August 01, 2016 ABSTRACT Non-Hodgkin lymphomas (NHLs) include a heterogeneous group of diseases, which differ in both cellular origin and clinical behavior. Among the aggressive malignancies of this group, the diffuse large B-cell lymphomas (DLBCLs) are the most frequently observed. They are themselves clinically and molecularly heterogeneous and have been further sub-divided in three sub-types according to different cell of origin, mechanisms of oncogenesis and clinical outcome. Among them, the germinal center B-cell-like (GCB) derives from the germinal center and expresses the BCL6 oncogene. We have previously shown that Patz1-knockout mice develop B-cell neoplasias, suggesting a tumor suppressor role for PATZ1 in human NHLs. Here, by immunohistochemical analysis of a tissue-microarray including 170 NHLs, we found that PATZ1 nuclear expression is down-regulated in follicular lymphomas and DLBCLs. Moreover, consistent with our previous results showing a PATZ1-dependent regulation of BCL6 and BAX transcription, we show that low PATZ1 nuclear expression significantly correlates with high BCL6 expression, mainly in DLBCLs, and with low BAX expression, also considering separately follicular lymphomas and DLBCLs. Finally, by analyzing overall and progression-free survival in DLBCL patients that underwent rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy, low levels of PATZ1 were significantly associated to a worst outcome and demonstrated an independent prognostic factor in multivariate analysis, including known prognostic factors of DLBCL, IPI score and cell of origin (GCB/non-GCB). Therefore, we propose PATZ1 as a new prognostic marker of DLBCLs, which may act as a tumor suppressor by enhancing apoptosis through inhibiting and enhancing transcription of BCL6 and BAX, respectively.

We have previously reported a critical role of HMGA proteins in pituitary tumorigenesis since either the Hmga1 or Hmga2 gene overexpression/activation induces the development of mixed growth hormone/prolactin cell pituitary adenomas by activating the E2F transcription factor 1, and then enhancing the G1/S transition of the cell cycle. Consistently, amplification and overexpression of the HMGA2 gene was found in human pituitary prolactinomas. Since impairment of the cell cycle control represents a feature of experimental and human pituitary adenomas, we have investigated the possible synergism between the alterations of other cell cycle regulators, such as p27 deficiency or Cdk4R24C mutation, with Hmga2 overexpression in pituitary tumorigenesis. Therefore, we crossed the Hmga2/T mice, overexpressing the truncated/active form of the Hmga2 gene, either with the knockout mice for p27kip1, or with the knockin mice for the Cdk4R24C mutation, both developing pituitary adenomas. Increased incidence and decreased latency in the development of pituitary lesions appeared in double mutant Hmga2/T;Cdk4R24C mice, and increased features of invasiveness and atypia were observed in pituitary tumors of both Hmga2/T;p27-ko and Hmga2/T;Cdk4R24C double mutant mice as compared with single mutant compounds. Interestingly, most of these mice develop pituitary adenomas with high Ki67 index, extrasellar expansion and brain tissue infiltration, representing good mouse models for human aggressive pituitary adenomas. Taken together, the results reported here indicate a cooperation between HMGA2 overexpression and either p27kip1 or CDK4 impairment in promoting pituitary tumor development and progression.

Thyroid cancer is one of the most frequent malignancies of the endocrine system, and its incidence is predicted to become the fourth leading cancer diagnosis by 2030. In spite of the progressive knowledge of the molecular mechanisms involved in thyroid transformation, its prognosis remains unpredictable and the identification of new biological markers are needed in addition to already known molecules, to correctly stratify patients at risk of recurrence and progression, eventually providing new targeted therapies. We recently showed that the transcriptional regulator PATZ1 is constantly down-regulated in human thyroid cancer and acts as a tumor suppressor in thyroid cancer cell lines by targeting p53-dependent genes involved in Epithelial-Mesenchymal Transition and cell migration. The aim of the present work was to elucidate the upstream signaling pathway regulating PATZ1 expression during thyroid transformation. We first identified miR-23b and miR-29b to specifically target PATZ1 expression, which was inversely correlated to their expression in rat thyroid cells stimulated to proliferate with Thyroid-stimulating hormone (TSH). Next, using an inducible cell system, we found that miR-29b was up-regulated by oncogenic Ras during transformation of FRTL-5 rat thyroid cells toward an undifferentiated phenotype resembling that of anaplastic carcinomas and characterized by the acquisition of a migratory and invasive behavior. Conversely, PATZ1 was down-regulated, with an inverse correlation compared to miR-29b expression, and was specifically targeted by miR-29b in untransformed FRTL-5 cells. Restoration of PATZ1 expression in FRTL-5 cells stably expressing oncogenic Ras inhibited cell proliferation and migration, indicating a key role of PATZ1 in Ras-driven thyroid transformation. These results confirm the tumor suppressor role of PATZ1 in thyroid cancer and suggest that its downregulation in thyroid cancer requires the activation of Ras GTPase signaling via miR-29b.

POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) is an emerging cancer-related gene that is downregulated in different human malignancies, including thyroid cancer, where its levels gradually decrease going from papillary thyroid carcinomas (PTC) to poorly differentiated and undifferentiated highly aggressive anaplastic carcinomas (ATC). The restoration of PATZ1 expression in thyroid cancer cells reverted their malignant phenotype by inducing mesenchymal-to-epithelial transition, thus validating a tumor suppressor role for PATZ1 and suggesting its involvement in thyroid cancer progression. Here, we investigated the consequences of the homozygous and heterozygous loss of PATZ1 in the context of a mouse modeling of PTC, represented by mice carrying the RET/PTC1 oncogene under the thyroid specific control of the thyroglobulin promoter RET/PTC1 (RET/PTC1TG). The phenotypic analysis of RET/PTC1TG mice intercrossed with Patz1-knockout mice revealed that deficiency of both Patz1 alleles enhanced thyroid cancer incidence in RET/PTC1TG mice, but not the heterozygous knockout of the Patz1 gene. However, both RET/PTC1TG;Patz1+/- and RET/PTC1TG;Patz1-/- mice developed a more aggressive thyroid cancer phenotype-characterized by higher Ki-67 expression, presence of ATCs, and increased incidence of solid variants of PTC-than that shown by RET/PTC1TG; Patz1+/+ compound mice. These results confirm that PATZ1 downregulation has a critical role in thyroid carcinogenesis, showing that it cooperates with RET/PTC1 in thyroid cancer progression.

We have previously reported a critical role of HMGA proteins in pituitary tumorigenesis since either the Hmga1 or Hmga2 gene overexpression/activation induces the development of mixed growth hormone/prolactin cell pituitary adenomas by activating the E2F transcription factor 1, and then enhancing the G1/S transition of the cell cycle. Consistently, amplification and overexpression of the HMGA2 gene was found in human pituitary prolactinomas. Since impairment of the cell cycle control represents a feature of experimental and human pituitary adenomas, we have investigated the possible synergism between the alterations of other cell cycle regulators, such as p27 deficiency or Cdk4(R24C) mutation, with Hmga2 overexpression in pituitary tumorigenesis. Therefore, we crossed the Hmga2/T mice, overexpressing the truncated/active form of the Hmga2 gene, either with the knockout mice for p27(kip1), or with the knockin mice for the Cdk4(R24C) mutation, both developing pituitary adenomas. Increased incidence and decreased latency in the development of pituitary lesions appeared in double mutant Hmga2/T;Cdk4(R24C) mice, and increased features of invasiveness and atypia were observed in pituitary tumors of both Hmga2/T;p27-ko and Hmga2/T;Cdk4(R24C) double mutant mice as compared with single mutant compounds. Interestingly, most of these mice develop pituitary adenomas with high Ki67 index, extrasellar expansion and brain tissue infiltration, representing good mouse models for human aggressive pituitary adenomas. Taken together, the results reported here indicate a cooperation between HMGA2 overexpression and either p27(kip1) or CDK4 impairment in promoting pituitary tumor development and progression.

// Elia Guadagno 1 , Michela Vitiello 2 , Paola Francesca 2 , Gaetano Cali 2 , Federica Caponnetto 3 , Daniela Cesselli 3 , Simona Camorani 2 , Giorgio Borrelli 1 , Marialuisa Califano 1 , Paolo Cappabianca 4 , Claudio Arra 5 , Elvira Crescenzi 2 , Laura Cerchia 2 , Maria Laura Del Basso De Caro 1 and Monica Fedele 2 1 Department of Advanced Biomedical Sciences, Pathology Section, University of Naples Federico II, 80131 Naples, Italy 2 Institute of Experimental Endocrinology and Oncology (IEOS) “Gaetano Salvatore”, National Council of Research, 80131 Naples, Italy 3 Department of Medical and Biological Sciences, University of Udine, 33100 Udine, Italy 4 Division of Neurosurgery, Department of Neurosciences, Reproductive and Odontostomatological Sciences, University of Naples Federico II, 80131 Naples, Italy 5 Department of Experimental Oncology, National Cancer Institute “Fondazione Giovanni Pascale”, IRCCS, 80131 Naples, Italy Correspondence to: Monica Fedele, email: mfedele@unina.it , monica.fedele@cnr.it Maria Laura Del Basso De Caro, email: marialaura.delbasso@unina.it Keywords: glioma, glioma stem cells, PATZ1, tumor heterogeneity, biomarker Received: January 18, 2017 Accepted: June 19, 2017 Published: July 25, 2017 ABSTRACT Glioblastoma (GBM), the most malignant of the brain tumors, has been classified on the basis of molecular signature into four subtypes: classical, mesenchymal, proneural and neural, among which the mesenchymal and proneural subtypes have the shortest and longest survival, respectively. Here we show that the transcription factor PATZ1 gene is upregulated in gliomas compared to normal brain and, among GBMs, is particularly enriched in the proneural subtype and co-localize with stemness markers. Accordingly, in GBM-derived glioma-initiating stem cells (GSCs) PATZ1 is overexpressed compared to differentiated tumor cells and its expression significantly correlates with the characteristic stem cell capacity to grow as neurospheres in vitro . Interestingly, survival analysis demonstrated that PATZ1 lower levels informed poor prognosis in GBM and, specifically, in the proneural subgroup, suggesting it may serve a role as diagnostic and prognostic biomarker for intra-subtype heterogeneity of proneural GBM. We also show that PATZ1 suppresses the expression of the mesenchyme-inducer CXCR4, and that PATZ1 and CXCR4 are inversely correlated in GSC and proneural GBM. Overall these findings support a central role of PATZ1 in regulating malignancy of GBM.

The regulatory transcriptional factor PATZ1 is constantly downregulated in human thyroid cancer where it acts as a tumour suppressor by targeting p53-dependent genes involved in Epithelial-Mesenchymal Transition and cell migration. The aim of the present work was to elucidate the upstream signalling mechanisms regulating PATZ1 expression in thyroid cancer cells. The bioinformatics search for microRNAs able to potentially target PATZ1 led to the identification of several miRNAs. Among them we focused on the miR-29b since it was found upregulated in rat thyroid differentiated cells transformed by the Ha-Ras oncogene towards a high proliferating and high migratory phenotype resembling that of anaplastic carcinomas. Functional assays confirmed PATZ1 as a target of miR-29b, and, consistently, an inverse correlation between miR-29b and PATZ1 protein levels was found upon induction of Ha-Ras oncogene expression in these cells. Interestingly, restoration of PATZ1 expression in rat thyroid cells stably expressing the Ha-Ras oncogene decreased cell proliferation and migration, indicating a key role of PATZ1 in Ras-driven thyroid transformation. Together, these results suggest a novel mechanism regulating PATZ1 expression based on the upregulation of miR-29b expression induced by Ras oncogene.

// Gennaro Chiappetta 1, * , Teresa Valentino 2, * , Michela Vitiello 2 , Rosa Pasquinelli 1 , Mario Monaco 1 , Giuseppe Palma 3 , Romina Sepe 2, 4 , Antonio Luciano 3 , Pierlorenzo Pallante 2 , Dario Palmieri 5 , Concetta Aiello 1 , Domenica Rea 3 , Simona Nunzia Losito 1 , Claudio Arra 3 , Alfredo Fusco 2, 4 , Monica Fedele 2 1 Department of Experimental Oncology, Functional Genomic Unit, National Cancer Institute “Fondazione Giovanni Pascale”, IRCCS, 80131 Naples, Italy 2 Institute of Experimental Endocrinology and Oncology (IEOS), National Research Counsil (CNR), 80131 Naples, Italy 3 Animal Facility, National Cancer Institute “Fondazione Giovanni Pascale”, IRCCS, 80131 Naples, Italy 4 Department of Molecular Medicine and Medical Biotechnologies, University of Naples “Federico II”, 80131 Naples, Italy 5 Departments of Molecular Virology, Immunology and Human Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA * These authors have contributed equally to this work Correspondence to: Monica Fedele, e-mail: mfedele@unina.it Gennaro Chiappetta, e-mail: g.chiappetta@istitutotumori.na.it Keywords: thyroid cancer, PATZ1, Epithelial-Mesenchymal Transition, cell migration Received: July 22, 2014 Accepted: November 18, 2014 Published: December 16, 2014 ABSTRACT PATZ1, a POZ-Zinc finger protein, is emerging as an important regulator of development and cancer, but its cancer-related function as oncogene or tumor-suppressor is still debated. Here, we investigated its possible role in thyroid carcinogenesis. We demonstrated PATZ1 is down-regulated in thyroid carcinomas compared to normal thyroid tissues, with an inverse correlation to the degree of cell differentiation. In fact, PATZ1 expression was significantly further down-regulated in poorly differentiated and anaplastic thyroid cancers compared to the papillary histotype, and it resulted increasingly delocalized from the nucleus to the cytoplasm proceeding from differentiated to undifferentiated thyroid carcinomas. Restoration of PATZ1 expression in three thyroid cancer-derived cell lines, all characterized by fully dedifferentiated cells, significantly inhibited their malignant behaviors, including in vitro proliferation, anchorage-independent growth, migration and invasion, as well as in vivo tumor growth. Consistent with recent studies showing a role for PATZ1 in the p53 pathway, we showed that ectopic expression of PATZ1 in thyroid cancer cells activates p53-dependent pathways opposing epithelial-mesenchymal transition and cell migration to prevent invasiveness. These results provide insights into a potential tumor-suppressor role of PATZ1 in thyroid cancer progression, and thus may have potential clinical relevance for the prognosis and therapy of thyroid cancer.

We have previously demonstrated that HMGA1B and HMGA2 overexpression in mice induces the development of GH and prolactin (PRL) pituitary adenomas mainly by increasing E2F1 transcriptional activity. Interestingly, these adenomas showed very high expression levels of PIT1, a transcriptional factor that regulates the gene expression ofGh,Prl,GhrhrandPit1itself, playing a key role in pituitary gland development and physiology. Therefore, the aim of our study was to identify the role ofPit1overexpression in pituitary tumour development induced by HMGA1B and HMGA2. First, we demonstrated that HMGA1B and HMGA2 directly interact with both PIT1 and its gene promoterin vivo, and that these proteins positively regulatePit1promoter activity, also co-operating with PIT1 itself. Subsequently, we showed, by colony-forming assays on two different pituitary adenoma cell lines, GH3 and αT3, thatPit1overexpression increases pituitary cell proliferation. Finally, the expression analysis ofHMGA1,HMGA2andPIT1in human pituitary adenomas of different histological types revealed a direct correlation betweenPIT1and HMGA expression levels. Taken together, our data indicate a role ofPit1upregulation by HMGA proteins in pituitary tumours.

PATZ1 is an emerging cancer-related gene coding for a POZ/AT-hook/kruppel Zinc finger transcription factor, which is lost or misexpressed in human neoplasias. Here, we investigated its role in development exploring wild-type and Patz1-knockout mice during embryogenesis. We report that the Patz1 gene is ubiquitously expressed at early stages of development and becomes more restricted at later stages, with high levels of expression in actively proliferating neuroblasts belonging to the ventricular zones of the central nervous system (CNS). The analysis of embryos in which Patz1 was disrupted revealed the presence of severe defects in the CNS and in the cardiac outflow tract, which eventually lead to a pre-mature in utero death during late gestation or soon after birth. Moreover, the Patz1-null mice showed a general growth retardation, which was consistent with the slower growth rate and the increased susceptibility to senescence of Patz1(-/-) mouse embryonic fibroblasts (MEFs) compared to wild-type controls. Therefore, these results indicate a critical role of PATZ1 in the control of cell growth and embryonic development.

The rheumatic adverse effects accompanying treatment with aromatase inhibitors (AIs) in hormone-dependent breast cancer represent an area of clinical relevance and emerging concern. This report describes these rheumatic complaints detailing their clinical pattern.During 1-year period, 18 consecutive postmenopausal women (mean age, 58.33 years; range, 52-66 years) in treatment with AIs for hormone-dependent breast cancer (mean duration of therapy, 12.0 months; range, 9.1-17.7 months) were referred for evaluation in the outpatient clinic of the rheumatology unit in relation to rheumatic complaints. According to a routine protocol planned with oncologists, patient evaluations consisted of a complete clinical examination with careful assessment of rheumatic complaints and related physical symptoms, followed by laboratory testing and a bone scintiscan. In no cases were rheumatic complaints present before AI therapy.On the basis of clinical data and investigations and by applying accepted diagnostic criteria, a diagnosis of an undifferentiated spondyloarthropathy was reached in 10 (55.5%) of the 18 patients studied, and an oligoarthritis was shown in 2 more patients (11.1%), whereas a simple arthralgia was found in the remaining 6 patients (33.3%). In the patients meeting criteria as belonging to a spondyloarthritic subset, a family history positive for psoriasis and celiac disease was shown in 2 and 1 instance, respectively, whereas HLA-CW6 and HLA-B27 were detected in 3 and 1 case. A high serum level of anti-cyclic citrullinated peptide antibodies was shown in 1 patient with oligoarthritis. Most of the patients (16/18) were treated with nonsteroidal anti-inflammatory drugs or with corticosteroids. Methotrexate (10 mg weekly) was added in 3 of these patients, nonresponders. Aromatase inhibitor discontinuation was needed in the remaining 2 cases with spontaneous resolution of symptoms over time.Data from the present study emphasize a previously unsuspected high prevalence of defined arthritides underlying these rheumatic complaints. Therefore, investigative efforts should be addressed to better clarify the clinical and pathogenetic significance of these important consequences of AI therapy. An accurate monitoring of rheumatic complaints has to be suggested to patients taking AI therapy, with a rapid referral to a rheumatologist in the case of consistent suspicion of an inflammatory arthritis.

PATZ1 is a transcriptional factor functioning either as an activator or a repressor of gene transcription depending upon the cellular context. It appears to have a dual oncogenic/anti-oncogenic activity. Indeed, it is overexpressed in colon carcinomas, and its silencing inhibits colon cancer cell proliferation or increases sensitivity to apoptotic stimuli of glioma cells, suggesting an oncogenic role. Conversely, the development of B-cell lymphomas, sarcomas, hepatocellular carcinomas and lung adenomas in Patz1-knockout (ko) mice supports its tumour suppressor function. PATZ1 role in mouse lymphomagenesis is mainly because of the involvement of PATZ1 in BCL6-negative autoregulation. However, this does not exclude that PATZ1 may be involved in tumorigenesis by other mechanisms. Here, we report that PATZ1 interacts with the tumour suppressor p53 and binds p53-dependent gene promoters, including those of BAX, CDKN1A and MDM2. Knockdown of PATZ1 in HEK293 cells reduces promoter activity of these genes and inhibits their expression, suggesting a role of PATZ in enhancing p53 transcriptional activity. Consistently, Patz1-ko mouse embryonic fibroblasts (MEFs) show decreased expression of Bax, Cdkn1a and Mdm2 compared with wild-type (wt) MEFs. Moreover, Patz1-ko MEFs show a decreased percentage of apoptotic cells, either spontaneous or induced by treatment with 5-fluorouracil (5FU), compared with wt controls, suggesting a pro-apoptotic role for PATZ1 in these cells. However, PATZ1 binds p53-target genes also independently from p53, exerting, in the absence of p53, an opposite function on their expression. Indeed, knockdown of PATZ1 in p53-null osteosarcoma cells upregulates BAX expression and decreases survival of 5FU-treated cells, then suggesting an anti-apoptotic role of PATZ1 in p53-null cancer cells. Therefore, these data support a PATZ1 tumour-suppressive function based on its ability to enhance p53-dependent transcription and apoptosis. Conversely, its opposite and anti-apoptotic role in p53-null cancer cells provides the perspective of PATZ1 silencing as a possible adjuvant in the treatment of p53-null cancer. © 2013 Macmillan Publishers Limited All rights reserved.