Michela Vitiello

PIT1 upregulation by HMGA proteins has a role in pituitary tumorigenesis.

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 of Gh, Prl, Ghrhr and Pit1 itself, playing a key role in pituitary gland development and physiology. Therefore, the aim of our study was to identify the role of Pit1 overexpression in pituitary tumour development induced by HMGA1B and HMGA2. First, we demonstrated that HMGA1B and HMGA2 directly interact with both PIT1 and its gene promoter in vivo, and that these proteins positively regulate Pit1 promoter activity, also co-operating with PIT1 itself. Subsequently, we showed, by colony-forming assays on two different pituitary adenoma cell lines, GH3 and αT3, that Pit1 overexpression increases pituitary cell proliferation. Finally, the expression analysis of HMGA1, HMGA2 and PIT1 in human pituitary adenomas of different histological types revealed a direct correlation between PIT1 and HMGA expression levels. Taken together, our data indicate a role of Pit1 upregulation by HMGA proteins in pituitary tumours.

PATZ1 interacts with p53 and regulates expression of p53-target genes enhancing apoptosis or cell survival based on the cellular context

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.

Embryonic defects and growth alteration in mice with homozygous disruption of the Patz1 gene

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. J. Cell. Physiol. 228: 646–653, 2013.

A polymorphism of HMGA1 protects against proliferative diabetic retinopathy by impairing HMGA1-induced VEGFA expression.

Diabetic retinopathy (DR) is a major complication of diabetes mellitus, and is the leading cause of blindness in working-age people. Usually, DR progresses from the asymptomatic non-proliferative DR that does not significantly alter vision, to proliferative DR (PDR), which can result in aberrant retinal neovessel formation and blindness. The High-Mobility-Group A1 (HMGA1) protein is a transcriptional master regulator of numerous genes, including metabolic and inflammatory genes, which, by modulating the expression of angiogenic factors, may induce retinal neovascularization, a hallmark of PDR. Herein, we examined the relationship between HMGA1 rs139876191 variant and DR. Results revealed that patients with type 2 diabetes, who were carriers of the HMGA1 rs139876191 variant had a significantly lower risk of developing PDR, compared to non-carrier diabetic patients. From a mechanistic point of view, our findings indicated that, by adversely affecting HMGA1 protein expression and function, the HMGA1 rs139876191 variant played a key role in this protective mechanism by downregulating the expression of vascular endothelial growth factor A (VEGFA), a major activator of neovascularization in DR. These data provide new insights into the pathogenesis and progression of DR, and may offer opportunities for discovering novel biomarkers and therapeutic targets for diagnosis, prevention and treatment of PDR.

HMGA2 cooperates with either p27kip1 deficiency or Cdk4R24C mutation in pituitary tumorigenesis

ABSTRACT 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.

PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells

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.

Transcriptional and post-transcriptional regulation of transmembrane protein 132A

Transmembrane protein 132A (TMEM132A) was first isolated from rat brain using PCR-selected cDNA subtraction, and it was found to be predominantly expressed in the brain. However, the transcriptional regulation of the TMEM132A gene has not been fully characterized. In this study, we characterized the promoter activity of the 880-bp region upstream of the mouse TMEM132A, identifying several putative sites recognized by transcription factors, which are highly conserved between the mouse and human TMEM132A genes. Using four different mouse cell lines (Neuro2a, NSC-34, NIH3T3, and Raw264.7), we first evaluated the intrinsic levels of TMEM132A mRNA and protein expression. Interestingly, TMEM132A mRNA was expressed in all four cell lines, whereas the protein was negligible in Raw264.7 cells even by transfection of TMEM132A gene. Then, we analyzed the TMEM132A promoter activity using serial deleted constructs, finding it was nearly same pattern in all four cell lines. A mutational analysis of the TMEM132A promoter identified a critical region for its activation just upstream of the transcriptional start site. Finally, we investigated the levels of TMEM132A mRNA and protein after exposure to five different neurotoxic stimuli, including thapsigargin, tunicamycin, serum starvation, homocysteine, and hydrogen peroxide. Treatment with thapsigargin, a calcium modulating agent, markedly attenuated the levels of TMEM132A mRNA and protein in NSC-34 cells. These results give new insight into the mechanisms involved in regulating TMEM132A expression, and suggest that several transcriptional and post-transcriptional pathways regulate TMEM132A expression under developmental and pathophysiological conditions.

PATZ1 is a new prognostic marker of glioblastoma associated with the stem-like phenotype and enriched in the proneural subtype

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.

PATZ1 expression correlates positively with BAX and negatively with BCL6 and survival in human diffuse large B cell lymphomas

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.

Loss of One or Two PATZ1 Alleles Has a Critical Role in the Progression of Thyroid Carcinomas Induced by the RET/PTC1 Oncogene

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.