TRAP1 controls cell cycle G2-M transition through the regulation of CDK1 and MAD2 expression/ubiquitination.

Regulation of tumour cell proliferation by molecular chaperones is still a complex issue. Here, the role of the HSP90 molecular chaperone TRAP1 in cell cycle regulation was investigated in a wide range of human breast, colorectal, and lung carcinoma cell lines, and tumour specimens. TRAP1 modulates the expression and/or the ubiquitination of key cell cycle regulators through a dual mechanism: (i) transcriptional regulation of CDK1, CYCLIN B1, and MAD2, as suggested by gene expression profiling of TRAP1-silenced breast carcinoma cells; and (ii) post-transcriptional quality control of CDK1 and MAD2, being the ubiquitination of these two proteins enhanced upon TRAP1 down-regulation. Mechanistically, TRAP1 quality control on CDK1 is crucial for its regulation of mitotic entry, since TRAP1 interacts with CDK1 and prevents CDK1 ubiquitination in cooperation with the proteasome regulatory particle TBP7, this representing the limiting factor in TRAP1 regulation of the G2-M transition. Indeed, TRAP1 silencing results in enhanced CDK1 ubiquitination, lack of nuclear translocation of CDK1/cyclin B1 complex, and increased MAD2 degradation, whereas CDK1 forced up-regulation partially rescues low cyclin B1 and MAD2 levels and G2-M transit in a TRAP1-poor background. Consistently, the CDK1 inhibitor RO-3306 is less active in a TRAP1-high background. Finally, a significant correlation was observed between TRAP1 and Ki67, CDK1 and/or MAD2 expression in breast, colorectal, and lung human tumour specimens. This study represents the first evidence that TRAP1 is relevant in the control of the complex machinery that governs cell cycle progression and mitotic entry and provides a strong rationale to regard TRAP1 as a biomarker to select tumours with deregulated cell cycle progression and thus likely poorly responsive to novel cell cycle inhibitors. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stress-Adaptive Response in Ovarian Cancer Drug Resistance: Role of TRAP1 in Oxidative Metabolism-Driven Inflammation.

Metabolic reprogramming is one of the most frequent stress-adaptive response of cancer cells to survive environmental changes and meet increasing nutrient requirements during their growth. These modifications involve cellular bioenergetics and cross talk with surrounding microenvironment, in a dynamic network that connect different molecular processes, such as energy production, inflammatory response, and drug resistance. Even though the Warburg effect has long been considered the main metabolic feature of cancer cells, recent reports identify mitochondrial oxidative metabolism as a driving force for tumor growth in an increasing number of cellular contexts. In recent years, oxidative phosphorylation has been linked to a remodeling of inflammatory response due to autocrine or paracrine secretion of interleukines that, in turn, induces a regulation of gene expression involving, among others, molecules responsible for the onset of drug resistance. This process is especially relevant in ovarian cancer, characterized by low survival, high frequency of disease relapse and chemoresistance. Recently, the molecular chaperone TRAP1 (tumor necrosis factor-associated protein 1) has been identified as a key junction molecule in these processes in ovarian cancer: in fact, TRAP1 mediates a metabolic switch toward oxidative phosphorylation that, in turn, triggers cytokines secretion, with consequent gene expression remodeling, finally leading to cisplatin resistance and epithelial-to-mesenchymal transition in ovarian cancer models. This review summarizes how metabolism, chemoresistance, inflammation, and epithelial-to-mesenchymal transition are strictly interconnected, and how TRAP1 stays at the crossroads of these processes, thus shedding new lights on molecular networks at the basis of ovarian cancer.

TRAP1 controls cell migration of cancer cells in metabolic stress conditions: Correlations with AKT/p70S6K pathways.

Cell motility is a highly dynamic phenomenon that is essential to physiological processes such as morphogenesis, wound healing and immune response, but also involved in pathological conditions such as metastatic dissemination of cancers. The involvement of the molecular chaperone TRAP1 in the regulation of cell motility, although still controversial, has been recently investigated along with some well-characterized roles in cancer cell survival and drug resistance in several tumour types. Among different functions, TRAP1-dependent regulation of protein synthesis seems to be involved in the migratory behaviour of cancer cells and, interestingly, the expression of p70S6K, a kinase responsible for translation initiation, playing a role in cell motility, is regulated by TRAP1. In this study, we demonstrate that TRAP1 silencing enhances cell motility in vitro but compromises the ability of cells to overcome stress conditions, and that this effect is mediated by the AKT/p70S6K pathway. In fact: i) inhibition of p70S6K activity specifically reduces migration in TRAP1 knock-down cells; ii) nutrient deprivation affects p70S6K activity thereby impairing cell migration only in TRAP1-deficient cells; iii) TRAP1 regulates the expression of both AKT and p70S6K at post-transcriptional level; and iii) TRAP1 silencing modulates the expression of genes involved in cell motility and epithelial-mesenchymal transition. Notably, a correlation between TRAP1 and AKT expression is found in vivo in human colorectal tumours. These results provide new insights into TRAP1 role in the regulation of cell migration in cancer cells, tumour progression and metastatic mechanisms.

Targeting TRAP1 as a downstream effector of BRAF cytoprotective pathway: a novel strategy for human BRAF-driven colorectal carcinoma.

The HSP90 chaperone TRAP1 is translational regulator of BRAF synthesis/ubiquitination, since BRAF down-regulation, ERK signaling inhibition and delay of cell cycle progression occur upon TRAP1 silencing/inhibition. Since TRAP1 is upregulated in human colorectal carcinomas (CRCs) and involved in protection from apoptosis and as human BRAF-driven CRCs are poorly responsive to anticancer therapies, the relationship between TRAP1 regulation of mitochondrial apoptotic pathway and BRAF antiapoptotic signaling has been further evaluated. This study reports that BRAF cytoprotective signaling involves TRAP1-dependent inhibition of the mitochondrial apoptotic pathway. It is worth noting that BRAF and TRAP1 interact and that the activation of BRAF signaling results in enhanced TRAP1 serine-phosphorylation, a condition associated with resistance to apoptosis. Consistently, a BRAF dominant-negative mutant prevents TRAP1 serine phosphorylation and restores drug sensitivity in BRAFV600E CRC drug-resistant cells with high TRAP1 levels. In addition, TRAP1 targeting by the mitochondria-directed HSP90 chaperones inhibitor gamitrinib induces apoptosis and inhibits colony formation in BRAF-driven CRC cells. Thus, TRAP1 is a downstream effector of BRAF cytoprotective pathway in mitochondria and TRAP1 targeting may represent a novel strategy to improve the activity of proapoptotic agents in BRAF-driven CRC cells.

TRAP1 is involved in BRAF regulation and downstream attenuation of ERK phosphorylation and cell-cycle progression: a novel target for BRAF-mutated colorectal tumors.

Human BRAF-driven tumors are aggressive malignancies with poor clinical outcome and lack of sensitivity to therapies. TRAP1 is a HSP90 molecular chaperone deregulated in human tumors and responsible for specific features of cancer cells, i.e., protection from apoptosis, drug resistance, metabolic regulation, and protein quality control/ubiquitination. The hypothesis that TRAP1 plays a regulatory function on the BRAF pathway, arising from the observation that BRAF levels are decreased upon TRAP1 interference, was tested in human breast and colorectal carcinoma in vitro and in vivo. This study shows that TRAP1 is involved in the regulation of BRAF synthesis/ubiquitination, without affecting its stability. Indeed, BRAF synthesis is facilitated in a TRAP1-rich background, whereas increased ubiquitination occurs upon disruption of the TRAP1 network that correlates with decreased protein levels. Remarkably, BRAF downstream pathway is modulated by TRAP1 regulatory activity: indeed, TRAP1 silencing induces (i) ERK phosphorylation attenuation, (ii) cell-cycle inhibition with cell accumulation in G0-G1 and G2-M transitions, and (iii) extensive reprogramming of gene expression. Interestingly, a genome-wide profiling of TRAP1-knockdown cells identified cell growth and cell-cycle regulation as the most significant biofunctions controlled by the TRAP1 network. It is worth noting that TRAP1 regulation on BRAF is conserved in human colorectal carcinomas, with the two proteins being frequently coexpressed. Finally, the dual HSP90/TRAP1 inhibitor HSP990 showed activity against the TRAP1 network and high cytostatic potential in BRAF-mutated colorectal carcinoma cells. Therefore, this novel TRAP1 function represents an attractive therapeutic window to target dependency of BRAF-driven tumors on TRAP1 translational/quality control machinery.

Mass spectrometric/bioinformatic identification of a protein subset that characterizes the cellular activity of anticancer peptides.

The preclinical study of the mechanism of action of anticancer small molecules is challenging due to the complexity of cancer biology and the fragmentary nature of available data. With the aim of identifying a protein subset characterizing the cellular activity of anticancer peptides, we used differential mass spectrometry to identify proteomic changes induced by two peptides, LR and [d-Gln(4)]LR, that inhibit cell growth and compared them with the changes induced by a known drug, pemetrexed, targeting the same enzyme, thymidylate synthase. The quantification of the proteome of an ovarian cancer cell model treated with LR yielded a differentially expressed protein data set with respect to untreated cells. This core set was expanded by bioinformatic data interpretation, the biologically relevant proteins were selected, and their differential expression was validated on three cis-platinum sensitive and resistant ovarian cancer cell lines. Via clustering of the protein network features, a broader view of the peptides' cellular activity was obtained. Differences from the mechanism of action of pemetrexed were inferred from different modulation of the selected proteins. The protein subset identification represents a method of general applicability to characterize the cellular activity of preclinical compounds and a tool for monitoring the cellular activity of novel drug candidates.

TRAP1 revisited: novel localizations and functions of a 'next-generation' biomarker (review).

In the last decade, the identification and characterization of novel molecular mechanisms and pathways involving the heat shock protein TRAP1/HSP75 in cancers and other diseases enhanced the scientific interest. Recent reports have shown that TRAP1 stays at the crossroad of multiple crucial processes in the onset of neoplastic transformation. In fact, TRAP1: i) contributes to the tumor's switch to aerobic glycolysis through the inhibition of succinate dehydrogenase, the complex II of the mitochondrial respiratory chain; ii) is part of a pro-survival signaling pathway aimed at evading the toxic effects of oxidants and anticancer drugs and protects mitochondria against damaging stimuli via a decrease of ROS generation; iii) controls protein homeostasis through a direct involvement in the regulation of protein synthesis and protein co-translational degradation. Therefore, TRAP1 seems to be a central regulatory protein with balancing functions at the intersection of different metabolic processes during the neoplastic transformation. For this reason, it can be considered at the same time an attractive target for the development of novel anticancer strategies and a promising study model to understand the biology of tumor cells at a systemic level. This review summarizes the most recent advances in TRAP1 biology and proposes a new comprehensive view of its functions.

TRAP1-dependent regulation of p70S6K is involved in the attenuation of protein synthesis and cell migration: relevance in human colorectal tumors.

TNF receptor-associated protein 1 (TRAP1) is an HSP90 chaperone involved in stress protection and apoptosis in mitochondrial and extramitochondrial compartments. Remarkably, aberrant deregulation of TRAP1 function has been observed in several cancer types with potential new opportunities for therapeutic intervention in humans. Although previous studies by our group identified novel roles of TRAP1 in quality control of mitochondria-destined proteins through the attenuation of protein synthesis, molecular mechanisms are still largely unknown. To shed further light on the signaling pathways regulated by TRAP1 in the attenuation of protein synthesis, this study demonstrates that the entire pathway of cap-mediated translation is activated in cells following TRAP1 interference: consistently, expression and consequent phosphorylation of p70S6K and RSK1, two translation activating kinases, are increased upon TRAP1 silencing. Furthermore, we show that these regulatory functions affect the response to translational stress and cell migration in wound healing assays, processes involving both kinases. Notably, the regulatory mechanisms controlled by TRAP1 are conserved in colorectal cancer tissues, since an inverse correlation between TRAP1 and p70S6K expression is found in tumor tissues, thereby supporting the relevant role of TRAP1 translational regulation in vivo. Taken as a whole, these new findings candidate TRAP1 network for new anti-cancer strategies aimed at targeting the translational/quality control machinery of tumor cells.

Resistance to paclitxel in breast carcinoma cells requires a quality control of mitochondrial antiapoptotic proteins by TRAP1.

TRAP1 is a mitochondrial antiapoptotic protein up-regulated in several human malignancies. However, recent evidences suggest that TRAP1 is also localized in the endoplasmic reticulum (ER) where it is involved in ER stress protection and protein quality control of tumor cells. Based on the mechanistic link between ER stress, protection from apoptosis and drug resistance, we questioned whether these novel roles of TRAP1 are relevant for its antiapoptotic function. Here, we show for the first time that: i) TRAP1 expression is increased in about 50% of human breast carcinomas (BC), and ii) the ER stress protecting activity of TRAP1 is conserved in human tumors since TRAP1 is co-upregulated with the ER stress marker, BiP/Grp78. Notably, ER-associated TRAP1 modulates mitochondrial apoptosis by exerting a quality control on 18 kDa Sorcin, a TRAP1 mitochondrial client protein involved in TRAP1 cytoprotective pathway. Furthermore, this TRAP1 function is relevant in favoring resistance to paclitaxel, a microtubule stabilizing/ER stress inducer agent widely used in BC therapy. Indeed, the transfection of a TRAP1 deletion mutant, whose localization is restricted to the ER, in shTRAP1 cells enhances the expression of mitochondrial Sorcin and protects from apoptosis induced by ER stress agents and paclitaxel. Furthermore, BC cells adapted to paclitaxel or ER stress inducers share common resistance mechanisms: both cell models exhibit cross-resistance to single agents and the inhibition of TRAP1 by siRNAs or gamitrinib, a mitochondria-directed HSP90 family inhibitor, in paclitaxel-resistant cells rescues the sensitivity to paclitaxel. These results support the hypothesis that ER-associated TRAP1 is responsible for an extramitochondrial control of apoptosis and, therefore, an interference of ER stress adaptation through TRAP1 inhibition outside of mitochondria may be considered a further compartment-specific molecular approach to rescue drug-resistance.

New insights into TRAP1 pathway.

Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP1) is a mitochondrial heat shock protein involved in the protection from DNA damages and apoptosis induced by oxidants and several other stress conditions. Despite the well-characterized role in the regulation of mitochondrial integrity, through the interaction with cyclophilin D, a mitochondrial permeability transition pore regulator, several recent studies contributed to draw a more complex "picture" of the TRAP1 pathway: most of these updated functions arise from the identification of novel specific TRAP1 "client" proteins and from the recent discovery of multiple subcellular localizations/functions for this chaperone. This review briefly highlights some general features of TRAP1, and among others its role in cytoprotection, summarizing many different functions, which contribute to its protective role upon several stress inducers. Of note, particular emphasis is given to the recent findings on the regulation of Endoplasmic Reticulum stress and protein quality control by TRAP1, as well as to its role in regulating calcium homeostasis throughout its client protein Sorcin. Starting from the above observations a preliminary "TRAP1 signature" is provided and a new intriguing and interesting field to explore is discussed. Several questions are still open given the complexity of such mechanisms. However, by translating these recent insights at the molecular and cellular levels into personalized individual anticancer treatments, designing novel strategies based on the simultaneous inhibition of multiple tumor-specific pathways, and contemplating subcellular-targeted approaches aimed at reverting drug resistance and improving antitumor activity the struggle to combat cancer become more successful and closer.

The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei.

The data presented here show for the first time that the protein known as "ribonuclease (RNase) inhibitor" (RI or RNH1) is present not only in the cell cytosol, but also in mitochondria, the central organelles in cell redox homeostasis. This finding directly correlates with the reported ability of RI to protect the cell from oxidative stress, with its sensitivity to oxidation and reactivity as a reactive oxygen species scavenger. While this study was carried out we also surprisingly discovered the presence of RI in the cell nucleus. We deem that these data open new views in the investigation on the cellular role(s) of the RI.

Mitochondrial chaperone Trap1 and the calcium binding protein Sorcin interact and protect cells against apoptosis induced by antiblastic agents.

TRAP1, a mitochondrial chaperone (Hsp75) with antioxidant and antiapoptotic functions, is involved in multidrug resistance in human colorectal carcinoma cells. Through a proteomic analysis of TRAP1 coimmunoprecipitation complexes, the Ca(2+)-binding protein Sorcin was identified as a new TRAP1 interactor. This result prompted us to investigate the presence and role of Sorcin in mitochondria from human colon carcinoma cells. Using fluorescence microscopy and Western blot analysis of purified mitochondria and submitochondrial fractions, we showed the mitochondrial localization of an isoform of Sorcin with an electrophoretic motility lower than 20 kDa that specifically interacts with TRAP1. Furthermore, the effects of overexpressing or downregulating Sorcin and/or TRAP1 allowed us to demonstrate a reciprocal regulation between these two proteins and to show that their interaction is required for Sorcin mitochondrial localization and TRAP1 stability. Indeed, the depletion of TRAP1 by short hairpin RNA in colorectal carcinoma cells lowered Sorcin levels in mitochondria, whereas the depletion of Sorcin by small interfering RNA increased TRAP1 degradation. We also report several lines of evidence suggesting that intramitochondrial Sorcin plays a role in TRAP1 cytoprotection. Finally, preliminary evidence that TRAP1 and Sorcin are both implicated in multidrug resistance and are coupregulated in human colorectal carcinomas is provided. These novel findings highlight a new role for Sorcin, suggesting that some of its previously reported cytoprotective functions may be explained by involvement in mitochondrial metabolism through the TRAP1 pathway.

Heat shock proteins, cell survival and drug resistance: the mitochondrial chaperone TRAP1, a potential novel target for ovarian cancer therapy.

BACKGROUND: Protein homeostasis is a highly complex network of molecular interactions governing the health and life span of the organism. Molecular chaperones, mainly heat shock proteins (HSP) and other stress-inducible proteins abundantly expressed in multiple compartments of the cell, are major modulators of protein homeostasis. TRAP1 is a mitochondrial HSP involved in protection against oxidant-induced DNA damage and apoptosis. It was recently described as a component of a mitochondrial pathway selectively up-regulated in tumor cells which antagonizes the proapoptotic activity of cyclophilin D, a mitochondrial permeability transition pore regulator, and is responsible for the maintenance of mitochondrial integrity, thus favoring cell survival. Interestingly, novel TRAP1 antagonists cause sudden collapse of mitochondrial function and selective tumor cell death, suggesting that this pathway may represent a novel molecular target to improve anticancer therapy. Preliminary data suggest that TRAP1 may be a valuable biomarker in ovarian cancers: in fact, TRAP1 levels are significantly higher in cisplatin-resistant ovarian tumors and ovarian carcinoma cell lines. CONCLUSIONS: While major advances have been made in understanding the genetics and molecular biology of cancer, given the considerable heterogeneity of ovarian cancer, the introduction of novel targeted therapies and the consequent selection of treatments based on the molecular profile of each tumor may have a major impact on the management of this malignancy and might contribute to building a new era of personalized medicine.