miRNAs and Biomarkers in Testicular Germ Cell Tumors: An Update.

Testicular germ cell tumors (TGCTs) are the leading form of solid cancer and death affecting males between the ages of 20 and 40. Today, their surgical resection and chemotherapy are the treatments of first choice, even if sometimes this is not enough to save the lives of patients with TGCT. As seen for several tumors, the deregulation of microRNAs (miRNAs) is also a key feature in TGCTs. miRNAs are small molecules of RNA with biological activity that are released into biological fluids by testicular cancer cells. Their presence, therefore, can be detected and monitored by considering miRNAs as diagnostic and prognostic markers for TGCTs. The purpose of this review is to collect all the studies executed on miRNAs that have a potential role as biomarkers for testicular tumors.

HMGA1-Regulating microRNAs Let-7a and miR-26a are Downregulated in Human Seminomas.

BACKGROUND: Recent studies have underlined HMGA protein's key role in the onset of testicular germ cell tumors, where HMGA1 is differently expressed with respect to the state of differentiation, suggesting its fine regulation as master regulator in testicular tumorigenesis. Several studies have highlighted that the HMGA1 transcript is strictly regulated by a set of inhibitory microRNAs. Thus, the aim of this study is to test whether HMGA1 overexpression in human seminomas may be induced by the deregulation of miR-26a and Let-7a-two HMGA1-targeting microRNAs. METHODS: HMGA1 mRNA and Let-7a and miR-26a levels were measured in a seminoma dataset available in the Cancer Genome Atlas database and confirmed in a subset of seminomas by qRT-PCR and western blot. A TCam-2 seminoma cell line was then transfected with Let-7a and miR-26a and tested for proliferation and motility abilities. RESULTS: an inverse correlation was found between the expression of miR-26a and Let-7a and HMGA1 expression levels in seminomas samples, suggesting a critical role of these microRNAs in HMGA1 levels regulation. Accordingly, functional studies showed that miR-26a and Let-7a inhibited the proliferation, migration and invasion capabilities of the human seminoma derived cell line TCam-2. CONCLUSIONS: these data strongly support that the upregulation of HMGA1 levels occurring in seminoma is-at least in part-due to the downregulation of HMGA1-targeting microRNAs.

AMPA receptor expression in mouse testis and spermatogonial GC-1 cells: A study on its regulation by excitatory amino acids.

Excitatory amino acids (EAAs) are found present in the nervous and reproductive systems of animals. Numerous studies have demonstrated a regulatory role for Glutamate (Glu), d-aspartate ( d-Asp) and N-methyl- d-aspartate (NMDA) in the control of spermatogenesis. EAAs are able to stimulate the Glutamate receptors, including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Here in, we assess expression of the main AMPAR subunits, GluA1 and GluA2/3, in the mouse testis and in spermatogonial GC-1 cells. The results showed that both GluA1 and GluA2/3 were localized in mouse testis prevalently in spermatogonia. The subunit GluA2/3 was more highly expressed compared with GluA1 in both the testis and the GC-1 cells. Subsequently, GC-1 cells were incubated with medium containing l-Glu, d-Glu, d-Asp or NMDA to determine GluA1 and GluA2/3 expressions. At 30 minutes and 2 hours of incubation, EAA-treated GC-1 cells showed significantly higher expression levels of both GluA1 and GluA2/3. Furthermore, p-extracellular signal-regulated kinase (ERK), p-Akt, proliferating cell nuclear antigen (PCNA), and Aurora B expressions were assayed in l-Glu-, d-Glu-, and NMDA-treated GC-1 cells. At 30 minutes and 2 hours of incubation, treated GC-1 cells showed significantly higher expression levels of p-ERK and p-Akt. A consequent increase of PCNA and Aurora B expressions was induced by l-Glu and NMDA, but not by d-Glu. Our study demonstrates a direct effect of the EAAs on spermatogonial activity. In addition, the increased protein expression levels of GluA1 and GluA2/3 in EAA-treated GC-1 cells suggest that EAAs could activate ERK and Akt pathways through the AMPAR. Finally, the increased PCNA and Aurora B levels may imply an enhanced proliferative activity.

An Overview on Predictive Biomarkers of Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) are frequent solid malignant tumors and cause of death in men between 20-40 years of age. Genetic and environmental factors play an important role in the origin and development of TGCTs. Although the majority of TGCTs are responsive to chemotherapy, about 20% of patient presents incomplete response or tumors relapse. In addition, the current treatments cause acute toxicity and several chronic collateral effects, including sterility. The present mini-review collectively summarize the most recent findings on the new discovered molecular biomarkers such as tyrosine kinases, HMGAs, Aurora B kinase, and GPR30 receptor predictive of TGCTs and as emerging new possible molecular targets for therapeutic strategies. J. Cell. Physiol. 232: 276-280, 2017. © 2016 Wiley Periodicals, Inc.

D-Aspartate Induces Proliferative Pathways in Spermatogonial GC-1 Cells.

D-aspartate (D-Asp) is an endogenous amino acid present in vertebrate tissues, with particularly high levels in the testis. In vivo studies indicate that D-Asp indirectly stimulates spermatogenesis through the hypothalamic-pituitary-gonadal axis. Moreover, in vitro studies have demonstrated that D-Asp up-regulates testosterone production in Leydig cells by enhancing expression of the steroidogenic acute regulatory protein. In this study, a cell line derived from immortalized type-B mouse spermatogonia retaining markers of mitotic germ cells (GC-1) was employed to explore more direct involvement of D-Asp in spermatogenesis. Activity and protein expression of markers of cell proliferation were determined at intervals during incubation in D-Asp-containing medium. D-Asp induced phosphorylation of ERK and Akt proteins, stimulated expression of PCNA and Aurora B, and enhanced mRNA synthesis and protein expression of P450 aromatase and protein expression of Estrogen Receptor ß (ERß). These results are the first demonstration of a direct effect of D-Asp on spermatogonial mitotic activity. Considering that spermatogonia express the NR1 subunit of the N-Methyl-D-Aspartic Acid receptor (NMDAR), we suggest that their response to D-Asp depends on NMDAR-mediated activation of the ERK and Akt pathways and is further enhanced by activation of the P450 aromatase/ERß pathway.

The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival.

NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 in cancer cells lead to dysregulation of the centrosome cycle and aneuploidy. Thus, a tight regulation of NEK2 function is needed during cell cycle progression. In this study, we found that NEK2 localizes in the nucleus of cancer cells derived from several tissues. In particular, NEK2 co-localizes in splicing speckles with SRSF1 and SRSF2. Moreover, NEK2 interacts with several splicing factors and phosphorylates some of them, including the oncogenic SRSF1 protein. Overexpression of NEK2 induces phosphorylation of endogenous SR proteins and affects the splicing activity of SRSF1 toward reporter minigenes and endogenous targets, independently of SRPK1. Conversely, knockdown of NEK2, like that of SRSF1, induces expression of pro-apoptotic variants from SRSF1-target genes and sensitizes cells to apoptosis. Our results identify NEK2 as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells.

Interaction between HMGA1 and retinoblastoma protein is required for adipocyte differentiation.

[This retracts the article on p. 25993 in vol. 284, PMID: 19633359.].

High levels of GPR30 protein in human testicular carcinoma in situ and seminomas correlate with low levels of estrogen receptor-beta and indicate a switch in estrogen responsiveness.

The G protein-coupled estrogen receptor (GPR30) is suggested to be involved in non-nuclear estrogen signalling and is expressed in a variety of hormone dependent cancer entities. It is well established that oestrogens are involved in pathological germ cell proliferation including testicular germ cell tumours. This study was performed to further elucidate the role of this receptor and the possible correlation with the estrogen receptor ß in human testicular carcinoma in situ (CIS), seminomas and in GC1 and TCam-2 germ cell lines; in addition, a Tissue Micro-Array was built using the most representative areas from 25 cases of human testicular seminomas and 20 cases of CIS. The expression of ERß and GPR30 were observed by using Western blot analysis in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that down regulation of ERß associates with GPR30 over-expression both in human testicular CIS and seminomas. In addition, we show that 17ß-oestradiol induces the ERK1/2 activation and increases c-Fos expression through GPR30 associated with ERß down-regulation in TCam-2 cell line. The present results suggest that exposure to oestrogens or oestrogen-mimics, in some as of yet undefined manner, diminishes the ERß-mediated growth restraint in CIS and in human testicular seminoma, probably due to ERß down-regulation associated to GPR30 increased expression indicating that GPR30 could be a potential therapeutic target to design specific inhibitors.

Testicular/paratesticular mesothelial tumours: Uncommon histopathologic entities in a very complex anatomical site.

Mesothelial tumours of the testicular/paratesticular region are uncommon, poorly characterised and difficult-to-diagnose lesions. They encompass entirely benign proliferations (adenomatoid tumour) and malignant, very aggressive tumours (mesothelioma) whose morphological features can be overlapping, highly variable and confounding. Moreover, testicular/paratesticular mesothelial tumours comprise relatively new entities with indolent behaviour (well-differentiated papillary mesothelial tumour) as well as tumours which cannot be correctly included in any of the aforementioned categories and whose classification is still controversial. The molecular profile of such tumours represents an open issue. In fact, despite the recent discoveries about the genomic landscape of mesothelial proliferations at other sites (pleura, peritoneum), testicular/paratesticular mesothelial tumours, and namely mesotheliomas, are too rare to be extensively studied on large case series and they could arguably hide relevant differences in their molecular background when compared to the more common pleural/peritoneal counterparts.The aim of this review is to provide a guide for the pathological assessment of testicular/paratesticular mesothelial tumours. Herein, we describe the most recent updates on this topic according to the latest (year 2022) World Health Organisation Classification of Urinary and Male Genital Tumours (5th edition) and current literature. The diagnostic criteria, the main differentials and the role of ancillary techniques in the diagnosis of mesothelial testicular/paratesticular tumours are discussed.

Molecular biomarkers as potential targets for therapeutic strategies in human testicular germ cell tumors: an overview.

Testicular germ cell tumors (TGCTs), the most common malignancy in males between 15 and 34 years of age and the most frequent cause of death from solid tumors in this age group. TGCTs can be subdivided into seminoma and non-seminoma germ cell tumors (NSGCTs), including embryonal cell carcinoma, choriocarcinoma, yolk sac tumor, and teratoma. Seminomas and NSGCTs do not only present distinctive clinical features, but they also show significant differences as far as therapy and prognosis are concerned. Seminomas are highly sensitive to both radiation and chemotherapy, with a good prognosis, non-seminomas are sensitive to platinum-based combination chemotherapy and are less susceptible to radiation, with the exception of teratomas. The different therapeutic outcome might be explained by inherent properties of the cells from which testicular neoplasia originate. The unique treatment sensitivity of TGCTs is unexplained so far, but it is likely to be related to intrinsic molecular characteristics of the PGCs/gonocytes, from which these tumors originate. Many discovered bio-markers including OCT3/4, SOX2, SOX17, HMGA1, HMGA2, PATZ1, GPR30, Aurora B, estrogen receptor ß, and others have given further advantages to discriminate between histological subgroups. In addition, therapeutic approaches for the treatment of TGCTs have been proposed: humanized antibodies against receptors/surface molecules on cancer cells, inhibitors of serine-threonine, and tyrosine kinases, and others. The mini-review will be an overview on the molecular alterations identified in TGCTs and on novel targeted antineoplastic strategies that might help to treat chemotherapy resistant TGCTs.

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.

Critical role of CCDC6 in the neoplastic growth of testicular germ cell tumors.

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.

Drug-induced inhibition of HMGA and EZH2 activity as a possible therapy for anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal neoplasms in humans, and just limited progresses have been made to extend patient survival and decrease ATC-associated mortality. Thus, the identification of novel therapeutic strategies for treating ATC is needed. Recently, our group has identified two proteins with oncogenic activity, namely HMGA1 and EZH2, with pivotal roles in ATC cancer progression. Therefore, we tested the ability of trabectedin, a HMGA1-targeting drug, and GSK126, an inhibitor of EZH2 enzymatic activity, to impair cell viability of four ATC-derived cell lines. In the present study, we first confirmed the overexpression of HMGA1 and EZH2 in all ATC-derived cell lines and tissues compared to the normal primary thyroid cells and tissues. Then, treatment of the ATC cell lines with trabectedin and GSK126 resulted in a drastic induction of apoptotic cell death, which increased when the ATC cell lines were treated with a combination of both drugs. Conversely, normal primary human thyroid cells did not show any significant reduction in their viability when exposed to the same drugs. Noteworthy, both drugs induced the deregulation of EZH2- and HMGA1-controlled genes. Altogether, these findings propose the combination of trabectedin and GSK126 as possible novel strategy for ATC therapy.

The lncRNA RMST is drastically downregulated in anaplastic thyroid carcinomas where exerts a tumor suppressor activity impairing epithelial-mesenchymal transition and stemness.

Thyroid cancer is the most prevalent endocrine malignancy and comprises a wide range of lesions subdivided into differentiated (DTC) and undifferentiated thyroid cancer (UTC), mainly represented by the anaplastic thyroid carcinoma (ATC). This is one of the most lethal malignancies in humankind leading invariably to patient death in few months. Then, a better comprehension of the mechanisms underlying the development of ATC is required to set up new therapeutic approaches. Long non-coding RNAs (lncRNAs) are transcripts over 200 nucleotides in length that do not code for proteins. They show a strong regulatory function at both transcriptional and post-transcriptional level and are emerging as key players in regulating developmental processes. Their aberrant expression has been linked to several biological processes, including cancer, making them potential diagnostic and prognostic markers. We have recently analyzed the lncRNA expression profile in ATC through a microarray technique and have identified rhabdomyosarcoma 2-associated transcript (RMST) as one of the most downregulated lncRNA in ATC. RMST has been reported to be deregulated in a series of human cancers, to play an anti-oncogenic role in triple-negative breast cancer, and to modulate neurogenesis by interacting with SOX2. Therefore, these findings prompted us to investigate the role of RMST in ATC development. In this study we show that RMST levels are strongly decreased in ATC, but only slightly in DTC, indicating that the loss of this lncRNA could be related to the loss of the differentiation and high aggressiveness. We also report a concomitant increase of SOX2 levels in the same subset of ATC, that inversely correlated with RMST levels, further supporting the RMST/SOX2 relationship. Finally, functional studies demonstrate that the restoration of RMST in ATC cells reduces cell growth, migration and the stemness properties of ATC stem cells. In conclusion, these findings support a critical role of RMST downregulation in ATC development.

Analysis of microsatellite instability (MSI) in pediatric gonadal and extra-gonadal germ cell tumors.

Gonadal and extragonadal pediatric germ cell tumors (GCTs) are rare neoplasms with different clinical behavior. Although surgery and cisplatin-based chemotherapy are resolutive in most cases, some patients do not respond to chemotherapy and have a worse outcome. Microsatellite instability (MSI) was correlated to resistance to chemotherapy and sensitivity to immunotherapy in different neoplasms. A series of 21 pediatric GCTs were tested by immuno-histochemistry and PCR to evaluate MSI status. Next generation sequencing was applied to further evaluate cases with discordant results between immunohistochemistry and PCR. Twenty-one cases of pediatric GCT were included in the series. The mean age ranged between 1 and 10 years. Nine cases were gonadal GCTs and the remaining 12 were extra-gonadal GCTs. By immunohistochemistry, one case showed a deficit of Mismatch repair (MMR) proteins. This case was a 1-year-old children affected by gonadal yolk sac tumor. However, all cases resulted microsatellite stable (MSS) by PCR and NGS. MSI was not detected in our series of pediatric GCTs, as well as the data present in literature about adult patients with GCTs. Molecular techniques could have a role to confirm the MSI status in case of dMMR by immunohistochemistry.

The high-mobility group A1-estrogen receptor ß nuclear interaction is impaired in human testicular seminomas.

It is well established that estrogens participate in the control of normal spermatogenesis and endogenous or environmental estrogens are involved in pathological germ cell proliferation including testicular germ cell tumors. The effects of estrogen are now known to be mediated by estrogen receptor-α (ERα) and ERß subtypes, but only ERß has been found in human germ cells of normal testis. However, its expression was markedly diminished in human testicular seminomas. The expression and the possible interaction of ERß and HMGA1 were studied in normal germ cells and in human testicular seminomas. GC1 and TCam-2 germ cell lines, were used; in addition, a tissue micro-array (TMA) was built using the most representative areas from 35 cases of human testicular seminomas. The expression and the interaction of ERß and HMGA1 were observed by using immunoprecipitation and Western blot analyses in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that ERß interacts with HMGA1 in normal germ cells, while down regulation of ERß associates with transcriptional co-regulator HMGA1 over-expression and cytoplasmic localization both in human testicular seminomas and in TCam-2 cell line. In addition, we show that 17ß-oestradiol induces an HMGA1 increased cytoplasmic expression associated to an ERß down-regulation in TCam-2 cell line. Taken together, our results suggest that exposure to estrogens or estrogen-mimics, in some as of yet undefined manner, diminishes the ERß-mediated growth restraint in human testicular seminoma, probably due to the HMGA1 cytoplasmic delocalization associated with ERß down-regulation.

Down-regulation of oestrogen receptor-ß associates with transcriptional co-regulator PATZ1 delocalization in human testicular seminomas.

Oestrogen exposure has been linked to a risk for the development of testicular germ cell cancers. The effects of oestrogen are now known to be mediated by oestrogen receptor-α (ERα) and ERß subtypes, but only ERß has been found in human germ cells of normal testis. However, its expression was markedly diminished in seminomas, embryonal cell carcinomas and mixed germ cell tumours, but remains high in teratomas. PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. We have previously described that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mislocalization could be associated with the development of testicular germ cell tumours. Here we show that ERß interacts with PATZ1 in normal germ cells, while down-regulation of ERß associates with transcriptional co-regulator PATZ1 delocalization in human testicular seminomas. In addition, we show that the translocation of PATZ1 from the cytoplasm into the nucleus is regulated by cAMP, which also induces increased expression and nuclear localization of ERß, while this effect is counteracted by using the anti-oestrogen ICI 182-780.

Leptin modulates the survival of autoreactive CD4+ T cells through the nutrient/energy-sensing mammalian target of rapamycin signaling pathway.

Chronic inflammation can associate with autoreactive immune responses, including CD4(+) T cell responses to self-Ags. In this paper, we show that the adipocyte-derived proinflammatory hormone leptin can affect the survival and proliferation of autoreactive CD4(+) T cells in experimental autoimmune encephalomyelitis, an animal model of human multiple sclerosis. We found that myelin olygodendrocyte glycoprotein peptide 35-55 (MOG(35-55))-specific CD4(+) T cells from C57BL/6J wild-type mice could not transfer experimental autoimmune encephalomyelitis into leptin-deficient ob/ob mice. Such a finding was associated with a reduced proliferation of the transferred MOG(35-55)-reactive CD4(+) T cells, which had a reduced degradation of the cyclin-dependent kinase inhibitor p27(kip1) and ERK1/2 phosphorylation. The transferred cells displayed reduced Th1/Th17 responses and reduced delayed-type hypersensitivity. Moreover, MOG(35-55)-reactive CD4(+) T cells in ob/ob mice underwent apoptosis that associated with a downmodulation of Bcl-2. Similar results were observed in transgenic AND-TCR- mice carrying a TCR specific for the pigeon cytochrome c 88-104 peptide. These molecular events reveal a reduced activity of the nutrient/energy-sensing AKT/mammalian target of rapamycin pathway, which can be restored in vivo by exogenous leptin replacement. These results may help to explain a link between chronic inflammation and autoimmune T cell reactivity.

The role of HMGA1 protein in gastroenteropancreatic neuroendocrine tumors.

Neuroendocrine tumors (NETs) are neoplasms derived from neuroendocrine cells. One of their main features is to often remain asymptomatic and clinically undetectable. High Mobility Group A (HMGA) proteins belong to a family of non-histone chromatinic proteins able to modulate gene expression through the interaction with DNA and transcription factors. They are overexpressed in most of the human malignancies, playing a critical role in carcinogenesis. However, their expression levels and their role in neuroendocrine carcinogenesis has not been exhaustively evaluated until now. Therefore, in this study, we have addressed the validity of using the expression of HMGA1 as a diagnostic marker and have investigated its role in NET carcinogenesis. The expression of HMGA1 has been evaluated by qRT-PCR and immunohistochemistry, using NET tissue microarrays, in a cohort of gastroenteropancreatic (GEP)-NET samples. The expression levels of HMGA1 have been then correlated with the main clinical features of NET samples. Finally, the contribution of HMGA1 overexpression to NET development has been addressed as far as the modulation of proliferation and migration abilities of NET cells is concerned. Here, we report that HMGA1 is overexpressed in GEP-NET samples, at both mRNA and protein levels, and that the silencing of HMGA1 protein expression interferes with the ability of NET cells to proliferate and migrate through the downregulation of Cyclin E, Cyclin B1 and EZH2. These results propose the HMGA proteins as new diagnostic and prognostic markers.