MAGE-I proteins and cancer-pathways: A bidirectional relationship.

Data emerged from the last 20 years of basic research on tumor antigens positioned the type I MAGE (Melanoma Antigen GEnes - I or MAGE-I) family as cancer driver factors. MAGE-I gene expression is mainly restricted to normal reproductive tissues. However, abnormal re-expression in cancer unbalances the cell status towards enhanced oncogenic activity or reduced tumor suppression. Anomalous MAGE-I gene re-expression in cancer is attributed to altered epigenetic-mediated chromatin silencing. Still, emerging data indicate that MAGE-I can be regulated at protein level. Results from different laboratories suggest that after its anomalous re-expression, specific MAGE-I proteins can be regulated by well-known signaling pathways or key cellular processes that finally potentiate the cancer cell phenotype. Thus, MAGE-I proteins both regulate and are regulated by cancer-related pathways. Here, we present an updated review highlighting the recent findings on the regulation of MAGE-I by oncogenic pathways and the potential consequences in the tumor cell behavior.

Differential regulation of the glucocorticoid receptor nucleocytoplasmic shuttling by TPR-domain proteins.

A dimer of the heat-shock protein of 90-kDa (Hsp90) represents the critical core of the chaperone complex associated to the glucocorticoid receptor (GR) oligomer. The C-terminal end of the Hsp90 dimer shapes a functional acceptor site for co-chaperones carrying tetratricopeptide repeat (TPR) domains, where they bind in a mutually exclusive and competitive manner. They impact on the biological properties of the GR•Hsp90 complex and are major players of the GR transport machinery. Recently, we showed that the overexpression of a chimeric TPR peptide influences the subcellular distribution of GR. In this study, the functional role of endogenous proteins carrying TPR or TPR-like sequences on GR subcellular distribution was characterized. It is demonstrated that, contrarily to the positive influence of FKBP52 on GR nuclear accumulation, FKBP51 and 14-3-3 impaired this property. While SGT1α showed no significant effect, the overexpression of the Ser/Thr phosphatase PP5 resulted in a nearly equal nuclear-cytoplasmic redistribution of GR rather than its typical cytoplasmic localization in the absence of steroid. This observation led to analyse the influence of the phosphorylation status of GR, which resulted not linked to its nucleo-cytoplasmic shuttling mechanism. Nonetheless, it was evidenced that both PP5 and FKBP52 are related to the anchorage of the GR to nucleoskeleton structures. The influence of these TPR domain proteins on the steroid-dependent transcriptional activity of GR was also characterized. It is postulated that the pleiotropic actions of the GR in different cell types may be the consequence of the relative abundance of different TPR-domain interacting co-chaperones.

Expression of the tumor-expressed protein MageB2 enhances rRNA transcription.

An essential requirement for cells to sustain a high proliferating rate is to be paired with enhanced protein synthesis through the production of ribosomes. For this reason, part of the growth-factor signaling pathways, are devoted to activate ribosome biogenesis. Enhanced production of ribosomes is a hallmark in cancer cells, which is boosted by different mechanisms. Here we report that the nucleolar tumor-protein MageB2, whose expression is associated with cell proliferation, also participates in ribosome biogenesis. Studies carried out in both siRNA-mediated MageB2 silenced cells and CRISPR/CAS9-mediated MageB2 knockout (KO) cells showed that its expression is linked to rRNA transcription increase independently of the cell proliferation status. Mechanistically, MageB2 interacts with phospho-UBF, a protein which causes the recruitment of RNA Pol I pre-initiation complex required for rRNA transcription. In addition, cells expressing MageB2 displays enhanced phospho-UBF occupancy at the rDNA gene promoter. Proteomic studies performed in MageB2 KO cells revealed impairment in ribosomal protein (RPs) content. Functionally, enhancement in rRNA production in MageB2 expressing cells, was directly associated with an increased dynamic in protein synthesis. Altogether our results unveil a novel function for a tumor-expressed protein from the MAGE-I family. Findings reported here suggest that nucleolar MageB2 might play a role in enhancing ribosome biogenesis as part of its repertoire to support cancer cell proliferation.

MageC2 protein is upregulated by oncogenic activation of MAPK pathway and causes impairment of the p53 transactivation function.

Normal-to-tumor cell transition is accompanied by changes in gene expression and signal transduction that turns the balance toward cancer-cell phenotype, eluding by different mechanisms, the response of tumor-suppressor genes. Here, we observed that MageC2, a MAGE-I protein able to regulate the p53 tumor-suppressor, is accumulated upon MEK/ERK MAPK activation. Overexpression of H-RasV12 oncogene causes an increase in MageC2 protein that is prevented by pharmacologic inhibition of MEK. Similarly, decrease in MageC2 protein levels is shown in A375 melanoma cells (which harbor B-RafV600E oncogenic mutation) treated with MEK inhibitors. MageC2 protein levels decrease when p14ARF is expressed, causing an Mdm2-independent upregulation of p53 transactivation. However, MageC2 is refractory to p14ARF-driven downregulation when H-RasV12 is co-expressed. Using MageC2 knockout A375 cells generated by CRISPR/CAS9 technology, we demonstrated the relevance of MageC2 protein in reducing p53 transcriptional activity in cells containing hyperactive MEK/ERK signaling. Furthermore, gene expression analysis performed in cancer-genomic databases, supports the correlation of reduced p53 transcriptional activity and high MageC2 expression, in melanoma cells containing Ras or B-Raf driver mutations. Data presented here suggest that MageC2 can be a functional target of the oncogenic MEK/ERK pathway to regulate p53.

Dengue Non-structural Protein 5 Polymerase Complexes With Promyelocytic Leukemia Protein (PML) Isoforms III and IV to Disrupt PML-Nuclear Bodies in Infected Cells.

Dengue virus (DENV) threatens almost 70% of the world's population, with no therapeutic currently available. The severe, potentially lethal forms of DENV disease (dengue hemorrhagic fever/dengue shock syndrome) are associated with the production of high level of cytokines, elicited as part of the host antiviral response, although the molecular mechanisms have not been fully elucidated. We previously showed that infection by DENV serotype 2 (DENV2) disrupts promyelocytic leukemia (PML) gene product nuclear bodies (PML-NBs) after viral protein translation in infected cells. Apart from playing a key role as the nucleating agent in forming PML-NBs, PML has antiviral activity against various viruses, including DENV. The present study builds on this work, showing for the first time that all four DENV serotypes elicit PML-NB breakdown. Importantly, we show for the first time that of the nuclear localizing proteins of DENV, DENV non-structural protein (NS) 5 polymerase alone is sufficient to elicit PML-NB disassembly, in part through complexing with PML isoforms III and IV, but not other PML isoforms or other PML-NB components. The results raise the possibility that PML-NB disruption by nuclear localized NS5 contributes to DENV's suppression of the host antiviral response.

GTSE1: a novel TEAD4-E2F1 target gene involved in cell protrusions formation in triple-negative breast cancer cell models.

GTSE1 over-expression has been reported as a potential marker for metastasis in various types of malignancies, including breast cancer. Despite this, the transcriptional regulation of this protein and the causes of its misregulation in tumors remain largely unknown. The aims of this work were to elucidate how GTSE1 is regulated at the transcriptional level and to clarify the mechanism underlying GTSE1-dependent cell functions in triple-negative breast cancer (TNBC). Here, we identified GTSE1 as a novel target gene of the TEAD4 transcription factor, highlighting a role for the YAP and TAZ coactivators in the transcriptional regulation of GTSE1. Moreover, we found that TEAD4 controls the formation of cell protrusions required for cell migration through GTSE1, unveiling a relevant effector role for this protein in the TEAD-dependent cellular functions and confirming TEAD4 role in promoting invasion and metastasis in breast cancer. Finally, we highlighted a role for the pRb-E2F1 pathway in the control of GTSE1 transcription and observed that treatment with drugs targeting the pRb-E2F1 or YAP/TAZ-TEAD pathways dramatically downregulated the expression levels of GTSE1 and of other genes involved in the formation of metastasis, suggesting their potential use in the treatment of TNBC.

Functional interaction between co-expressed MAGE-A proteins.

MAGE-A (Melanoma Antigen Genes-A) are tumor-associated proteins with expression in a broad spectrum of human tumors and normal germ cells. MAGE-A gene expression and function are being increasingly investigated to better understand the mechanisms by which MAGE proteins collaborate in tumorigenesis and whether their detection could be useful for disease prognosis purposes. Alterations in epigenetic mechanisms involved in MAGE gene silencing cause their frequent co-expression in tumor cells. Here, we have analyzed the effect of MAGE-A gene co-expression and our results suggest that MageA6 can potentiate the androgen receptor (AR) co-activation function of MageA11. Database search confirmed that MageA11 and MageA6 are co-expressed in human prostate cancer samples. We demonstrate that MageA6 and MageA11 form a protein complex resulting in the stabilization of MageA11 and consequently the enhancement of AR activity. The mechanism involves association of the Mage A6-MHD domain to MageA11, prevention of MageA11 ubiquitinylation on lysines 240 and 245 and decreased proteasome-dependent degradation. We experimentally demonstrate here for the first time that two MAGE-A proteins can act together in a non-redundant way to potentiate a specific oncogenic function. Overall, our results highlight the complexity of the MAGE gene networking in regulating cancer cell behavior.

Human MageB2 Protein Expression Enhances E2F Transcriptional Activity, Cell Proliferation, and Resistance to Ribotoxic Stress.

MageB2 belongs to the melanoma antigen gene (MAGE-I) family of tumor-specific antigens. Expression of this gene has been detected in human tumors of different origins. However, little is known about the protein function and how its expression affects tumor cell phenotypes. In this work, we found that human MageB2 protein promotes tumor cell proliferation in a p53-independent fashion, as observed both in cultured cells and growing tumors in mice. Gene expression analysis showed that MageB2 enhances the activity of E2F transcription factors. Mechanistically, the activation of E2Fs is related to the ability of MageB2 to interact with the E2F inhibitor HDAC1. Cellular distribution of MageB2 protein includes the nucleoli. Nevertheless, ribotoxic drugs rapidly promote its nucleolar exit. We show that MageB2 counteracts E2F inhibition by ribosomal proteins independently of Mdm2 expression. Importantly, MageB2 plays a critical role in impairing cell cycle arrest in response to Actinomycin D. The data presented here support a relevant function for human MageB2 in cancer cells both under cycling and stressed conditions, presenting a distinct functional feature with respect to other characterized MAGE-I proteins.

GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.

Tumor-specific MAGE proteins as regulators of p53 function.

Since its discovery in 1991, the knowledge about the tumor specific melanoma antigen gene (MAGE-I) family has been continuously increasing. Initially, MAGE-I proteins were considered as selective targets for immunotherapy. More recently, emerging data obtained from different cellular mechanisms controlled by MAGE-I proteins suggest a key role in the regulation of important pathways linked to cell proliferation. This is in part due to the ability of some MAGE-I proteins to control the p53 tumor suppressor. In this review, we focus on the mechanisms proposed to explain how MAGE-I proteins affect p53 functions.

Interaction of p53 with tumor suppressive and oncogenic signaling pathways to control cellular reactive oxygen species production.

p53 is a crucial transcription factor with tumor suppressive properties that elicits its function through specific target genes. It constitutes a pivotal system that integrates information received by many signaling pathways and subsequently orchestrates cell fate decisions, namely, growth-arrest, senescence, or apoptosis. Reactive oxygen species (ROS) production in cells can play a key role in signal transduction, being able to trigger different processes as cell death or cell proliferation. Sustained oxidative stress can induce genomic instability and collaborates with cancer development, whereas acute enhancement of high ROS levels leads to toxic oxidative cell damage and cell death. Here, it has been considered p53 broad potential contribution through its ability to regulate selected key cancer signaling pathways, where ROS participate as inductors or effectors of the final biological outcome. Further, we have discussed how p53 could play a role in preventing potentially harmful oxidative state and cell proliferation by pro-oncogenic pathways such as PI3K/AKT/mTOR and WNT/ß-catenin or under hypoxia state. In addition, we have considered potential mechanisms by which p53 could collaborate with signal transduction pathways such as transforming growth factor-ß (TGF-ß) and stress-activated protein kinases (SAPK) that produce ROS, to stop or eliminate uncontrolled proliferating cells.

Human GTSE-1 regulates p21(CIP1/WAF1) stability conferring resistance to paclitaxel treatment.

p21(CIP1/WAF1) belongs to the CIP/KIP family of Cdk inhibitors, and its expression is tightly controlled during the cell cycle, mainly by transcriptional and post-translational mechanisms. Fine regulation of p21(CIP1/WAF1) levels is critical for cell cycle control and for cellular response to stress. In the present work, we describe a novel mechanism to modulate p21(CIP1/WAF1) levels mediated by the human GTSE-1 (G(2) and S phase-expressed-1) protein. Our results provide evidence that hGTSE-1 protects p21(CIP1/WAF1) from proteasome-dependent degradation as part of a functional complex containing the Hsp90-binding TPR protein WISp39. We further show that the hGTSE-1 N-terminal portion is sufficient for p21(CIP1/WAF1) binding and stabilization. Finally, we demonstrate that hGTSE-1 mediated-p21(CIP1/WAF1) stabilization is clearly involved in the ability of cells to counteract cytotoxicity induced by the microtubule poison paclitaxel.

The hsp90-FKBP52 complex links the mineralocorticoid receptor to motor proteins and persists bound to the receptor in early nuclear events.

In this study, we demonstrate that the subcellular localization of the mineralocorticoid receptor (MR) is regulated by tetratricopeptide domain (TPR) proteins. The high-molecular-weight immunophilin (IMM) FKBP52 links the MR-hsp90 complex to dynein/dynactin motors favoring the cytoplasmic transport of MR to the nucleus. Replacement of this hsp90-binding IMM by FKBP51 or the TPR peptide favored the cytoplasmic localization of MR. The complete movement machinery, including dynein and tubulin, could be recovered from paclitaxel/GTP-stabilized cytosol and was fully reassembled on stripped MR immune pellets. The whole MR-hsp90-based heterocomplex was transiently recovered in the soluble fraction of the nucleus after 10 min of incubation with aldosterone. Moreover, cross-linked MR-hsp90 heterocomplexes accumulated in the nucleus in a hormone-dependent manner, demonstrating that the heterocomplex can pass undissociated through the nuclear pore. On the other hand, a peptide that comprises the DNA-binding domain of MR impaired the nuclear export of MR, suggesting the involvement of this domain in the process. This study represents the first report describing the entire molecular system that commands MR nucleocytoplasmic trafficking and proposes that the MR-hsp90-TPR protein heterocomplex is dissociated in the nucleus rather than in the cytoplasm.

MAGE-A tumor antigens target p53 transactivation function through histone deacetylase recruitment and confer resistance to chemotherapeutic agents.

The MAGE gene family is characterized by a conserved domain (MAGE Homology Domain). A subset of highly homologous MAGE genes (group A; MAGE-A) belong to the chromosome X-clustered cancer/testis antigens. MAGE-A genes are normally expressed in the human germ line and overexpressed in various tumor types; however, their biological function is largely unknown. Here we present evidence indicating that MageA2 protein, belonging to the MAGE-A subfamily, confers wild-type-p53-sensitive resistance to etoposide (ET) by inducing a novel p53 inhibitory loop involving recruitment of histone deacetylase 3 (HDAC3) to MageA2/p53 complex, thus strongly down-regulating p53 transactivation function. In fact, enhanced MageA2 protein levels, in addition to ET resistance, correlate with impaired acetylation of both p53 and histones surrounding p53-binding sites. Association between MAGE-A expression levels and resistance to ET treatment is clearly shown in short-term cell lines obtained from melanoma biopsies harboring wild-type-p53, whereas cells naturally, or siRNA-mediated expressing low MAGE-A levels, correlate with enhanced p53-dependent sensitivity to ET. In addition, combined trichostatin A/ET treatment in melanoma cells expressing high MAGE-A levels reestablishes p53 response and reverts the chemoresistance.

The calpain system is involved in the constitutive regulation of beta-catenin signaling functions.

Beta-catenin is a multifunctional protein serving both as a structural element in cell adhesion and as a signaling component in the Wnt pathway, regulating embryogenesis and tumorigenesis. The signaling fraction of beta-catenin is tightly controlled by the adenomatous polyposis coli-axin-glycogen synthase kinase 3beta complex, which targets it for proteasomal degradation. It has been recently shown that Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Here we have highlighted the critical relevance of constitutive calpain pathway in the control of beta-catenin levels and functions, showing that small interference RNA knock down of endogenous calpain per se (i.e. in the absence of external stimuli) induces an increase in the free transcriptional competent pool of endogenous beta-catenin. We further characterized the role of the known calpain inhibitors, Gas2 and Calpastatin, demonstrating that they can also control levels, function, and localization of beta-catenin through endogenous calpain regulation. Finally we present Gas2 dominant negative (Gas2DN) as a new tool for regulating calpain activity, providing evidence that it counteracts the described effects of both Gas2 and Calpastatin on beta-catenin and that it works via calpain independently of the classical glycogen synthase kinase 3beta and proteasome pathway. Moreover, we provide in vitro biochemical evidence showing that Gas2DN can increase the activity of calpain and that in vivo it can induce degradation of stabilized/mutated beta-catenin. In fact, in a context where the classical proteasome pathway is impaired, as in colon cancer cells, Gas2DN biological effects accounted for a significant reduction in proliferation and anchorage-independent growth of colon cancer.

hGTSE-1 expression stimulates cytoplasmic localization of p53.

hGTSE-1 (human G(2) and S phase-expressed-1) is a cell cycle-regulated protein mainly localized in the cytoplasm and apparently associated with the microtubules. hGTSE-1 is able to down-regulate levels and activity of the p53 tumor suppressor protein: it binds the C-terminal region of p53 and represses its ability to induce apoptosis after DNA damage. Here we report that, after DNA damage, hGTSE-1 becomes stabilized in a p53-independent way and accumulated in the nucleus. Further characterization of hGTSE-1 localization revealed increased nuclear staining in unstressed cells after treatment with the nuclear export inhibitor leptomycin B, or when a nuclear export signal (NES) located in its C-terminal region was mutated. Finally, we provide evidence that hGTSE-1 ectopic expression, in addition to p53 protein levels down-regulation, is able to enhance cytoplasmic localization of p53. Interestingly, NES-mutated hGTSE-1 accumulates in the nucleus, binds p53 but looses its ability to enhance cytoplasmic redistribution of p53 and to regulate p53 protein levels. Similarly, when wild type hGTSE-1 functions on p53 were analyzed in cells lacking Mdm2, it failed in regulating both p53 localization and protein levels, thus indicating that hGTSE-1 requires an intact NES and functional Mdm2 for the regulation of p53. Our results provide new insights into the mechanism of hGTSE-1 function, whereby its characterized nucleo-cytoplasmic shuttling ability is required to regulate p53.

The cell cycle-regulated protein human GTSE-1 controls DNA damage-induced apoptosis by affecting p53 function.

GTSE-1 (G2 and S phase-expressed-1) protein is specifically expressed during S and G2 phases of the cell cycle. It is mainly localized to the microtubules and when overexpressed delays the G2 to M transition. Here we report that human GTSE-1 (hGTSE-1) protein can negatively regulate p53 transactivation function, protein levels, and p53-dependent apoptosis. We identified a physical interaction between the C-terminal regulatory domain of p53 and the C-terminal region of hGTSE-1 that is necessary and sufficient to down-regulate p53 activity. Furthermore, we provide evidence that hGTSE-1 is able to control p53 function in a cell cycle-dependent fashion. hGTSE-1 knock-down by small interfering RNA resulted in a S/G2-specific increase of p53 levels as well as cell sensitization to DNA damage-induced apoptosis during these phases of the cell cycle. Altogether, this work suggests a physiological role of hGTSE-1 in apoptosis control after DNA damage during S and G2 phases through regulation of p53 function.