Multifaceted Role of PRDM Proteins in Human Cancer.

The PR/SET domain family (PRDM) comprise a family of genes whose protein products share a conserved N-terminal PR [PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1)] homologous domain structurally and functionally similar to the catalytic SET [Su(var)3-9, enhancer-of-zeste and trithorax] domain of histone methyltransferases (HMTs). These genes are involved in epigenetic regulation of gene expression through their intrinsic HMTase activity or via interactions with other chromatin modifying enzymes. In this way they control a broad spectrum of biological processes, including proliferation and differentiation control, cell cycle progression, and maintenance of immune cell homeostasis. In cancer, tumor-specific dysfunctions of PRDM genes alter their expression by genetic and/or epigenetic modifications. A common characteristic of most PRDM genes is to encode for two main molecular variants with or without the PR domain. They are generated by either alternative splicing or alternative use of different promoters and play opposite roles, particularly in cancer where their imbalance can be often observed. In this scenario, PRDM proteins are involved in cancer onset, invasion, and metastasis and their altered expression is related to poor prognosis and clinical outcome. These functions strongly suggest their potential use in cancer management as diagnostic or prognostic tools and as new targets of therapeutic intervention.

Adiponectin as Link Factor between Adipose Tissue and Cancer.

Adipose tissue is a key regulator of energy balance playing an active role in lipid storage as well as in synthesizing several hormones directly involved in the pathogenesis of obesity. Obesity represents a peculiar risk factor for a growing list of cancers and is frequently associated to poor clinical outcome. The mechanism linking obesity and cancer is not completely understood, but, amongst the major players, there are both chronic low-grade inflammation and deregulation of adipokines secretion. In obesity, the adipose tissue is pervaded by an abnormal number of immune cells that create an inflammatory environment supporting tumor cell proliferation and invasion. Adiponectin (APN), the most abundant adipokine, shows anti-inflammatory, anti-proliferative and pro-apoptotic properties. Circulating levels of APN are drastically decreased in obesity, suggesting that APN may represent the link factor between obesity and cancer risk. The present review describes the recent advances on the involvement of APN and its receptors in the etiology of different types of cancer.

Adiponectin as novel regulator of cell proliferation in human glioblastoma.

Adiponectin (Acrp30) is an adipocyte-secreted hormone with pleiotropic metabolic effects, whose reduced levels were related to development and progression of several malignancies. We looked at the presence of Acrp30 receptors in human glioblastomas (GBM), hypothesizing a role for Acrp30 also in this untreatable cancer. Here we demonstrate that human GBM express Acrp30 receptors (AdipoR1 and AdipoR2), which are often co-expressed in GBM samples (70% of the analyzed tumors). To investigate the effects of Acrp30 on GBM growth, we used human GBM cell lines U87-MG and U251, expressing both AdipoR1 and AdipoR2 receptors. In these cells, Acrp30 treatment inhibits DNA synthesis and cell proliferation rate, inducing arrest in G1 phase of the cell cycle. These effects were correlated to a sustained activation of ERK1/2 and Akt kinases, upon Acrp30 treatment. Our results suggest that Acrp30 may represent a novel endogenous negative regulator of GBM cell proliferation, to be evaluated for the possible development of novel pharmacological approaches.

Identification of a functional estrogen-responsive enhancer element in the promoter 2 of PRDM2 gene in breast cancer cell lines.

The retinoblastoma protein-interacting zinc-finger (RIZ) gene, also known as PRDM2, encodes two protein products, RIZ1 and RIZ2, differing for the presence of a 202 aa domain, called PR domain, at the N-terminus of the RIZ1 molecule. While the histone H3 K9 methyltransferase activity of RIZ1 is associated with the negative control of cell proliferation, no information is currently available on either expression regulation of the RIZ2 form or on its biological activity. RIZ proteins act as ER co-activators and promote optimal estrogen response in female reproductive tissues. In estrogen-responsive cells, 17-ß estradiol modulates RIZ gene expression producing a shift in the balanced expression of the two forms. Here, we demonstrate that an estrogen-responsive element (ERE) within the RIZ promoter 2 is regulated in a ligand-specific manner by ERα, through both the AF1 and AF2 domains. The pattern of ERα binding, histone H4 acetylation, and histone H3 cyclical methylation of lysine 9 was comparable to other estrogen-regulated promoters. Association of topoisomerase IIß with the RIZ promoter 2 confirmed the transcriptional activation induced by estrogen. We hypothesize that RIZ2, acting as a negative regulator of RIZ1 function, mediates the proliferative effect of estrogen through regulation of survival and differentiation gene expression.

Dual-specificity phosphatase (DUSP6) in human glioblastoma: epithelial-to-mesenchymal transition (EMT) involvement.

OBJECTIVE: Glioblastoma (GBM) is the most aggressive and common form of primary brain cancer. Survival is poor and improved treatment options are urgently needed. Dual specificity phosphatase-6 (DUSP6) is actively involved in oncogenesis showing unexpected tumor-promoting properties in human glioblastoma, contributing to the development and expression of the full malignant and invasive phenotype. The purpose of this study was to assess if DUSP6 activates epithelial-to-mesenchymal transition (EMT) in glioblastoma and its connection with the invasive capacity. RESULTS: We found high levels of transcripts mRNA by qPCR analysis in a panel of primary GBM compared to adult or fetal normal tissues. At translational levels, these data correlate with high protein expression and long half-life values by cycloheximide-chase assay in immunoblot experiments. Next, we demonstrate that DUSP6 gene is involved in epithelial-to-mesenchymal transition (EMT) in GBM by immunoblot characterization of the mesenchymal and epithelial markers. Vimentin, N-Cadherin, E-Cadherin and fibronectin were measured with and without DUSP6 over-expression, and in response to several stimuli such as chemotherapy treatment. In particular, the high levels of vimentin were blunted at increasing doses of cisplatin in condition of DUSP6 over-expression while N-Cadherin contextually increased. Finally, DUSP6 per se increased invasion capacity of GBM. Overall, our data unveil the DUSP6 involvement in invasive mesenchymal-like properties in GBM.

Expression of RIZ1 protein (Retinoblastoma-interacting zinc-finger protein 1) in prostate cancer epithelial cells changes with cancer grade progression and is modulated in vitro by DHT and E2.

The nuclear protein methyl-transferase Retinoblastoma-interacting zinc-finger protein 1 (RIZ1) is considered to be a downstream effector of estrogen action in target tissues. Silencing of RIZ1 expression is common in many tumors. We analyzed RIZ1 expression in normal and malignant prostate tissue and evaluated whether estradiol (E2) or dihydrotestosterone (DHT) treatment modulated RIZ1 in cultured prostate epithelial cells (PEC). Moreover, we studied the possible involvement of RIZ1 in estrogen action on the EPN prostate cell line, constitutively expressing both estrogen receptor (ER)-alpha and beta. RIZ1 protein, found in the nucleus of normal PECs by immunohistochemistry, was progressively lost in cancer tissues as the Gleason score increased and was only detected in the cytoplasmic compartment. RIZ1 transcript levels, as assayed by semi-quantitative RT-PCR in primary PEC cultures, were significantly reduced in cancer cells (P < 0.05). In EPN DHT treatment significantly increased RIZ1 transcript and protein levels (P < 0.05); E2 induced a reduction of S phase without significant changes of RIZ1 expression. In E2-treated EPN cell extracts RIZ co-immunoprecipitated with ERbeta and ERalpha. Our data demonstrate that RIZ1 is expressed in normal PECs and down-regulated in cancer cells, with a switch of its sub-cellular localization from the nucleus to the cytoplasm upon cancer grade progression. RIZ1 expression levels in the PECs were modulated by DHT or E2 treatment in vitro. Furthermore, the E2 effects on ER-expressing prostate cells involve RIZ1, which confirms a possible role for ER-mediated pathways in a non-classic E(2)-target tissue.

Early and Late Induction of KRAS and HRAS Proto-Oncogenes by Reactive Oxygen Species in Primary Astrocytes.

Astrocytes, one of the predominant types of glial cells, function as both supportive and metabolic cells for the brain. Among mammalian tissues, the highest levels of p21Ras protein are detected in the brain. Here, we investigated the expression of KRAS and HRAS proto-oncogenes in primary astrocytes following acute oxidative stimulation. Reactive oxygen species (ROS) changed the expression of proto-oncogenes at both transcriptional and translational levels. De novo protein synthesis analysis measured approximate values of proteins half-life, ranging from 1-4 h, of the different H- and K- isoforms by western blot analysis. Quantitative gene expression analysis of KRAS and HRAS revealed an unexpected short-term induction of KRAS mRNA in primary astrocytes in response to acute stimulation. Indeed, cultured astrocytes responded to proteasomal inhibition by preventing the reduction of c-K-Ras. A fraction of K-Ras protein accumulated in the presence of ROS and cycloheximide, while a substantial proportion was continuously synthesized. These data indicate that ROS regulate in a complementary fashion p21Ras isoforms in primary astrocytes: K-Ras is rapidly and transiently induced by post-translational and post-transcriptional mechanisms, while H-Ras is stably induced by mRNA accumulation. We suggest that K-Ras and H-Ras are ROS sensors that adapt cells to metabolic needs and oxidative stress.

Critical Function of PRDM2 in the Neoplastic Growth of Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) derive from primordial germ cells. Their maturation is blocked at different stages, reflecting histological tumor subtypes. A common genetic alteration in TGCT is a deletion of the chromosome 1 short arm, where the PRDM2 gene, belonging to the Positive Regulatory domain gene (PRDM) family, is located. Expression of PRDM2 gene is shifted in different human tumors, where the expression of the two principal protein forms coded by PRDM2 gene, RIZ1 and RIZ2, is frequently unbalanced. Therefore, PRDM2 is actually considered a candidate tumor suppressor gene in different types of cancer. Although recent studies have demonstrated that PRDM gene family members have a pivotal role during the early stages of testicular development, no information are actually available on the involvement of these genes in TGCTs. In this article we show by qRT-PCR analysis that PRDM2 expression level is modulated by proliferation and differentiation agents such as estradiol, whose exposure during fetal life is probably an important risk factor for TGCTs development in adulthood. Furthermore in normal and cancer germ cell lines, PRDM2 binds estradiol receptor α (ERα) and influences proliferation, survival and apoptosis, as previously reported using MCF-7 breast cancer cell line, suggesting a potential tumor-suppressor role in TGCT formation.

Prostate cancer stem cells: the role of androgen and estrogen receptors.

Prostate cancer is one of the most commonly diagnosed cancers in men, and androgen deprivation therapy still represents the primary treatment for prostate cancer patients. This approach, however, frequently fails and patients develop castration-resistant prostate cancer, which is almost untreatable.Cancer cells are characterized by a hierarchical organization, and stem/progenitor cells are endowed with tumor-initiating activity. Accumulating evidence indicates that prostate cancer stem cells lack the androgen receptor and are, indeed, resistant to androgen deprivation therapy. In contrast, these cells express classical (α and/or ß) and novel (GPR30) estrogen receptors, which may represent new putative targets in prostate cancer treatment.In the present review, we discuss the still-debated mechanisms, both genomic and non-genomic, by which androgen and estradiol receptors (classical and novel) mediate the hormonal control of prostate cell stemness, transformation, and the continued growth of prostate cancer. Recent preclinical and clinical findings obtained using new androgen receptor antagonists, anti-estrogens, or compounds such as enhancers of androgen receptor degradation and peptides inhibiting non-genomic androgen functions are also presented. These new drugs will likely lead to significant advances in prostate cancer therapy.

The Zn-finger domain of RIZ protein promotes MCF-7 cell proliferation.

In order to understand the oncogenic properties of retinoblastoma-interacting zinc-finger (RIZ) gene products, we produced an MCF-7-derived cell line expressing a fusion protein containing the zinc-finger (aa 359-497) domain of RIZ protein (MCF-7/znf). The Zn-finger domain contains three of the eight putative Zn-finger motifs and is located in proximity of the E1A-like domain containing the Rb protein-binding motif. The MCF-7/znf cells showed a higher growth rate than the parental or the control cell lines, both in hormone-deprived conditions or upon estrogen stimulation. Furthermore, they were less sensitive to the growth inhibitory effect of anti-estrogens and showed a higher level of expression of cyclin D1 and A. The expressed Zn-finger domain recombinant product was localized in the nucleus and in the nucleoli and its expression modified the pattern of actin staining in the cytoplasm. In conclusion the presented results indicated that the Zn-finger domain could be endowed with the putative oncogenic activity of RIZ2 gene product.

Neuronal differentiation dictates estrogen-dependent survival and ERK1/2 kinetic by means of caveolin-1.

Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E2 stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival.

PRDM Proteins: Molecular Mechanisms in Signal Transduction and Transcriptional Regulation.

PRDM (PRDI-BF1 and RIZ homology domain containing) protein family members are characterized by the presence of a PR domain and a variable number of Zn-finger repeats. Experimental evidence has shown that the PRDM proteins play an important role in gene expression regulation, modifying the chromatin structure either directly, through the intrinsic methyltransferase activity, or indirectly through the recruitment of chromatin remodeling complexes. PRDM proteins have a dual action: they mediate the effect induced by different cell signals like steroid hormones and control the expression of growth factors. PRDM proteins therefore have a pivotal role in the transduction of signals that control cell proliferation and differentiation and consequently neoplastic transformation. In this review, we describe pathways in which PRDM proteins are involved and the molecular mechanism of their transcriptional regulation.

Highlighting chromosome loops in DNA-picked chromatin (DPC).

Growing evidence supports the concept that dynamic intra- and inter-chromosomal links between specific loci contribute to the creation of cell-type specific gene expression profiles. Therefore, analysis of the establishment of peculiar functional correlations between sites, also distant on linear DNA, that govern the transcriptional process appears to be of fundamental relevance. We propose here an experimental approach showing that 17ß-estradiol-induced transcription associates to formation of loops between the promoter and termination regions of hormone-responsive genes. This strategy reveals as a tool to be also suitably used, in conjunction with automated techniques, for an extensive analysis of sites shared by multiple genes for induced expression.

Modulation of RIZ gene expression is associated to estradiol control of MCF-7 breast cancer cell proliferation.

The retinoblastoma protein-interacting zinc-finger (RIZ) gene, a member of the nuclear protein methyltransferase superfamily, is characterized by the presence of the N-terminal PR domain. The RIZ gene encodes for two proteins, RIZ1 and RIZ2. While RIZ1 contains the PR (PRDI-BF1 and RIZ homologous) domain, RIZ2 lacks it. RIZ gene expression is altered in a variety of human cancers and RIZ1 is now considered to be a candidate tumor suppressor. Estradiol treatment of MCF-7 cells produced a selective decrease of RIZ1 transcript and an increase of total RIZ mRNA. Experiments of chromatin immunoprecipitation indicated that RIZ2 protein expression was controlled by estrogen receptor and RIZ1 had a direct repressor function on c-myc gene expression. To investigate the role of RIZ gene products as regulators of the proliferation/differentiation transition, we analyzed the effects of forced suppression of RIZ1 induced in MCF-7 cells by siRNA of the PR domain-containing form. Silencing of RIZ1 expression stimulated cell proliferation, similar to the effect of estradiol on these cells, associated with a transient increase of c-myc expression.

Proteomic analysis of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of retinoblastoma-interacting-zinc-finger protein.

To identify a growth-promoting activity related to retinoblastoma-interacting-zinc-finger (RIZ) protein, differential protein expression of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of RIZ protein was analyzed by a robust bottom-up mass-spectrometry proteomic approach. Spots corresponding to qualitative and quantitative differences in protein expression have been selected and identified. Some of these proteins have been previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Knowledge of specific differentially expressed proteins by MCF-7-derived cell lines expressing RIZ different domains will provide the basis for identifying a growth-promoting activity related to RIZ gene products.