TRAP1 enhances Warburg metabolism through modulation of PFK1 expression/activity and favors resistance to EGFR inhibitors in human colorectal carcinomas.

Metabolic rewiring is a mechanism of adaptation to unfavorable environmental conditions and tumor progression. TRAP1 is an HSP90 molecular chaperone upregulated in human colorectal carcinomas (CRCs) and responsible for downregulation of oxidative phosphorylation (OXPHOS) and adaptation to metabolic stress. The mechanism by which TRAP1 regulates glycolytic metabolism and the relevance of this regulation in resistance to EGFR inhibitors were investigated in patient-derived CRC spheres, human CRC cells, samples, and patients. A linear correlation was observed between TRAP1 levels and 18 F-fluoro-2-deoxy-glucose (18 F-FDG) uptake upon PET scan or GLUT1 expression in human CRCs. Consistently, TRAP1 enhances GLUT1 expression, glucose uptake, and lactate production and downregulates OXPHOS in CRC patient-derived spheroids and cell lines. Mechanistically, TRAP1 maximizes lactate production to balance low OXPHOS through the regulation of the glycolytic enzyme phosphofructokinase-1 (PFK1); this depends on the interaction between TRAP1 and PFK1, which favors PFK1 glycolytic activity and prevents its ubiquitination/degradation. By contrast, TRAP1/PFK1 interaction is lost in conditions of enhanced OXPHOS, which results in loss of TRAP1 regulation of PFK1 activity and lactate production. Notably, TRAP1 regulation of glycolysis is involved in resistance of RAS-wild-type CRCs to EGFR monoclonals. Indeed, either TRAP1 upregulation or high glycolytic metabolism impairs cetuximab activity in vitro, whereas TRAP1 targeting and/or inhibition of glycolytic pathway enhances cell response to cetuximab. Finally, a linear correlation between 18 F-FDG PET uptake and poor response to cetuximab in first-line therapy in human metastatic CRCs was observed. These results suggest that TRAP1 is a key determinant of CRC metabolic rewiring and favors resistance to EGFR inhibitors through regulation of glycolytic metabolism.

TRAP1 Regulates Wnt/ß-Catenin Pathway through LRP5/6 Receptors Expression Modulation.

Wnt/ß-Catenin signaling is involved in embryonic development, regeneration, and cellular differentiation and is responsible for cancer stemness maintenance. The HSP90 molecular chaperone TRAP1 is upregulated in 60-70% of human colorectal carcinomas (CRCs) and favors stem cells maintenance, modulating the Wnt/ß-Catenin pathway and preventing ß-Catenin phosphorylation/degradation. The role of TRAP1 in the regulation of Wnt/ß-Catenin signaling was further investigated in human CRC cell lines, patient-derived spheroids, and CRC specimens. TRAP1 relevance in the activation of Wnt/ß-Catenin signaling was highlighted by a TCF/LEF Cignal Reporter Assay in Wnt-off HEK293T and CRC HCT116 cell lines. Of note, this regulation occurs through the modulation of Wnt ligand receptors LRP5 and LRP6 that are both downregulated in TRAP1-silenced cell lines. However, while LRP5 mRNA is significantly downregulated upon TRAP1 silencing, LRP6 mRNA is unchanged, suggesting independent mechanisms of regulation by TRAP1. Indeed, LRP5 is regulated upon promoter methylation in CRC cell lines and human CRCs, whereas LRP6 is controlled at post-translational level by protein ubiquitination/degradation. Consistently, human CRCs with high TRAP1 expression are characterized by the co-upregulation of active ß-Catenin, LRP5 and LRP6. Altogether, these data suggest that Wnt/ß-Catenin signaling is modulated at multiple levels by TRAP1.

Comparative Gene Expression Profiling of Tobacco-Associated HPV-Positive versus Negative Oral Squamous Carcinoma Cell Lines.

Background: HPV-positive oral squamous cell carcinomas (OSCCs) are specific biological and clinical entities, characterized by a more favorable prognosis compared to HPV-negative OSCCs and occurring generally in non-smoking and non-drinking younger individuals. However, poor information is available on the molecular and the clinical behavior of HPV-positive oral cancers occurring in smoking/drinking subjects. Thus, this study was designed to compare, at molecular level, two OSCC cell lines, both derived from drinking and smoking individuals and differing for presence/absence of HPV infection. Methods: HPV-negative UPCI-SCC-131 and HPV16-positive UPCI-SCC-154 cell lines were compared by whole genome gene expression profiling and subsequently studied for activation of Wnt/ßCatenin signaling pathway by the expression of several Wnt-target genes, ßCatenin intracellular localization, stem cell features and miRNA let-7e. Gene expression data were validated in head and neck squamous cell carcinoma (HNSCC) public datasets. Results: Gene expression analysis identified Wnt/ßCatenin pathway as the unique signaling pathway more active in HPV-negative compared to HPV-positive OSCC cells and this observation was confirmed upon evaluation of several Wnt-target genes (i.e., Cyclin D1, Cdh1, Cdkn2a, Cd44, Axin2, c-Myc and Tcf1). Interestingly, HPV-negative OSCC cells showed higher levels of total ßCatenin and its active form, increase of its nuclear accumulation and more prominent stem cell traits. Furthermore, miRNA let-7e was identified as potential upstream regulator responsible for the downregulation of Wnt/ßCatenin signaling cascade since its silencing in UPCI-SCC-154 cell resulted in upregulation of Wnt-target genes. Finally, the analysis of two independent gene expression public datasets of human HNSCC cell lines and tumors confirmed that Wnt/ßCatenin pathway is more active in HPV-negative compared to HPV-positive tumors derived from individuals with smoking habit. Conclusions: These data suggest that lack of HPV infection is associated with more prominent activation of Wnt/ßCatenin signaling pathway and gain of stem-like traits in tobacco-related OSCCs.

Heat shock proteins in cancer stem cell maintenance: A potential therapeutic target?

Cancer stem cells (CSCs) are a subpopulation of tumor cells with unlimited self-renewal capability, multilineage differentiation potential and long-term tumor repopulation capacity. CSCs reside in anatomically distinct regions within the tumor microenvironment, called niches, and this favors the maintenance of CSC properties and preserves their phenotypic plasticity. Indeed, CSCs are characterized by a flexible state based on their capacity to interconvert between a differentiated and a stem-like phenotype, and this depends on the activation of adaptive mechanisms in response to different environmental conditions. Heat Shock Proteins (HSPs) are molecular chaperones, upregulated upon cell exposure to several stress conditions and are responsible for normal maturation, localization and activity of intra and extracellular proteins. Noteworthy, HSPs play a central role in several cellular processes involved in tumor initiation and progression (i.e. cell viability, resistance to apoptosis, stress conditions and drug therapy, EMT, bioenergetics, invasiveness, metastasis formation) and, thus, are widely considered potential molecular targets. Furthermore, much evidence suggests a key regulatory function for HSPs in CSC maintenance and their upregulation has been proposed as a mechanism used by CSCs to adapt to unfavorable environmental conditions, such as nutrient deprivation, hypoxia, inflammation. This review discusses the relevance of HSPs in CSC biology, highlighting their role as novel potential molecular targets to develop anticancer strategies aimed at CSC targeting.

Heat shock proteins in thyroid malignancies: Potential therapeutic targets for poorly-differentiated and anaplastic tumours?

Thyroid cancer is the most common endocrine malignancy, with well-differentiated subtypes characterized by an excellent prognosis due to their optimal sensitivity to standard therapies whereas poorly differentiated and anaplastic tumours by chemo/radio-resistance and unfavourable outcome. Heat Shock Proteins (HSPs) are molecular chaperones overexpressed in thyroid malignancies and involved in crucial functions responsible for thyroid carcinogenesis, as protection from apoptosis, drug resistance and cell migration. Thus, HSPs inhibitors have been proposed as novel therapeutic agents in thyroid cancer to revert molecular mechanisms of tumour progression. In this review, we report an overview on the biological role of HSPs, and specifically HSP90s, in thyroid cancer and their potential involvement as biomarkers. We discuss the rationale to evaluate HSPs inhibitors as innovative anticancer agents in specific subtypes of thyroid cancer characterized by poor response to therapies with the objective to target single family chaperones to reduce, simultaneously, the expression/stability of multiple client proteins.

BRAF Inhibitors in Thyroid Cancer: Clinical Impact, Mechanisms of Resistance and Future Perspectives.

The Kirsten rat sarcoma viral oncogene homolog (RAS)/v-raf-1 murine leukemia viral oncogene homolog 1 (RAF)/mitogen-activated protein kinase 1 (MAPK) signaling cascade is the most important oncogenic pathway in human cancers. Tumors leading mutations in the gene encoding for v-raf murine sarcoma viral oncogene homolog B (BRAF) serine-threonine kinase are reliant on the MAPK signaling pathway for their growth and survival. Indeed, the constitutive activation of MAPK pathway results in continuous stimulation of cell proliferation, enhancement of the apoptotic threshold and induction of a migratory and metastatic phenotype. In a clinical perspective, this scenario opens to the possibility of targeting BRAF pathway for therapy. Thyroid carcinomas (TCs) bearing BRAF mutations represent approximately 29-83% of human thyroid malignancies and, differently from melanomas, are less sensitive to BRAF inhibitors and develop primary or acquired resistance due to mutational events or activation of alternative signaling pathways able to reactivate ERK signaling. In this review, we provide an overview on the current knowledge concerning the mechanisms leading to resistance to BRAF inhibitors in human thyroid carcinomas and discuss the potential therapeutic strategies, including combinations of BRAF inhibitors with other targeted agents, which might be employed to overcome drug resistance and potentiate the activity of single agent BRAF inhibitors.

HSP90 Molecular Chaperones, Metabolic Rewiring, and Epigenetics: Impact on Tumor Progression and Perspective for Anticancer Therapy.

Heat shock protein 90 (HSP90) molecular chaperones are a family of ubiquitous proteins participating in several cellular functions through the regulation of folding and/or assembly of large multiprotein complexes and client proteins. Thus, HSP90s chaperones are, directly or indirectly, master regulators of a variety of cellular processes, such as adaptation to stress, cell proliferation, motility, angiogenesis, and signal transduction. In recent years, it has been proposed that HSP90s play a crucial role in carcinogenesis as regulators of genotype-to-phenotype interplay. Indeed, HSP90 chaperones control metabolic rewiring, a hallmark of cancer cells, and influence the transcription of several of the key-genes responsible for tumorigenesis and cancer progression, through either direct binding to chromatin or through the quality control of transcription factors and epigenetic effectors. In this review, we will revise evidence suggesting how this interplay between epigenetics and metabolism may affect oncogenesis. We will examine the effect of metabolic rewiring on the accumulation of specific metabolites, and the changes in the availability of epigenetic co-factors and how this process can be controlled by HSP90 molecular chaperones. Understanding deeply the relationship between epigenetic and metabolism could disclose novel therapeutic scenarios that may lead to improvements in cancer treatment.

TRAP1: a viable therapeutic target for future cancer treatments?

INTRODUCTION: HSP90 molecular chaperones (i.e., HSP90α, HSP90ß, GRP94 and TRAP1) are potential therapeutic targets to design novel anticancer agents. However, despite numerous designed HSP90 inhibitors, most of them have failed due to unfavorable toxicity profiles and lack of specificity toward different HSP90 paralogs. Indeed, a major limitation in this field is the high structural homology between different HSP90 chaperones, which significantly limits our capacity to design paralog-specific inhibitors. Area covered: This review examines the relevance of TRAP1 in tumor development and progression, with an emphasis on its oncogenic/oncosuppressive role in specific human malignancies and its multifaceted and context-dependent functions in cancer cells. Herein, we discuss the rationale for considering TRAP1 as a potential molecular target and the strategies used to date, to achieve its compartmentalized inhibition directly in mitochondria. Expert opinion: TRAP1 targeting may represent a promising strategy for cancer therapy, based on the increasing and compelling evidence supporting TRAP1 involvement in human carcinogenesis. However, considering the complexity of TRAP1 biology, future strategies of drug discovery need to improve selectivity and specificity toward TRAP1 respect to other HSP90 paralogs. The characterization of specific human malignancies suitable for TRAP1 targeting is also mandatory.

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.

TRAP1 protein signature predicts outcome in human metastatic colorectal carcinoma.

TRAP1 is a HSP90 molecular chaperone upregulated in colorectal carcinomas and involved in control of intracellular signaling, cell cycle, apoptosis and drug resistance, stemness and bioenergetics through co-traslational regulation of a network of client proteins. Thus, the clinical significance of TRAP1 protein network was analyzed in human colorectal cancers. TRAP1 and/or its client proteins were quantified, by immunoblot analysis, in 60 surgical specimens of colorectal carcinomas at different stages and, by immunohistochemistry, in 9 colorectal adenomatous polyps, 11 in situ carcinomas and 55 metastatic colorectal tumors. TRAP1 is upregulated at the transition between low- and high-grade adenomas, in in situ carcinomas and in about 60% of human colorectal carcinomas, being downregulated only in a small cohort of tumors. The analysis of TCGA database showed that a subgroup of colorectal tumors is characterized by gain/loss of TRAP1 copy number, this correlating with its mRNA and protein expression. Interestingly, TRAP1 is co-expressed with the majority of its client proteins and hierarchical cluster analysis showed that the upregulation of TRAP1 and associated 6-protein signature (i.e., IF2α, eF1A, TBP7, MAD2, CDK1 and ßCatenin) identifies a cohort of metastatic colorectal carcinomas with a significantly shorter overall survival (HR 5.4; 95% C.I. 1.1-26.6; p=0.037). Consistently, the prognostic relevance of TRAP1 was confirmed in a cohort of 55 metastatic colorectal tumors. Finally, TRAP1 positive expression and its prognostic value are more evident in left colon cancers. These data suggest that TRAP1 protein network may provide a prognostic signature in human metastatic colorectal carcinomas.

TRAP1 downregulation in human ovarian cancer enhances invasion and epithelial-mesenchymal transition.

Ovarian cancer (OC) is the second leading cause of gynecological cancer death worldwide. Although the list of biomarkers is still growing, molecular mechanisms involved in OC development and progression remain elusive. We recently demonstrated that lower expression of the molecular chaperone TRAP1 in OC patients correlates with higher tumor grade and stage, and platinum resistance. Herein we show that TRAP1 is often deleted in high-grade serous OC patients (N=579), and that TRAP1 expression is correlated with the copy number, suggesting this could be one of the driving mechanisms for the loss of TRAP1 expression in OC. At molecular level, downregulation of TRAP1 associates with higher expression of p70S6K, a kinase frequently active in OC with emerging roles in cell migration and tumor metastasis. Indeed, TRAP1 silencing in different OC cells induces upregulation of p70S6K expression and activity, enhancement of cell motility and epithelial-mesenchymal transition (EMT). Consistently, in a large cohort of OC patients, TRAP1 expression is reduced in tumor metastases and directly correlates with the epithelial marker E-Cadherin, whereas it inversely correlates with the transcription factor Slug and the matrix metallopeptidases 2 and 9. Strikingly, pharmacological inhibition of p70S6K reverts the high motility phenotype of TRAP1 knock-down cells. However, although p70S6K inhibition or silencing reduces the expression of the transcription factors Snail and Slug, thus inducing upregulation of E-Cadherin expression, it is unable to revert EMT induced by TRAP1 silencing; furthermore, p70S6K did not show any significant correlation with EMT genes in patients, nor with overall survival or tumor stage, suggesting an independent and predominant role for TRAP1 in OC progression. Altogether, these results may provide novel approaches in OC with reduced TRAP1 expression, which could be resistant to therapeutic strategies based on the inhibition of the p70S6K pathway, with potential future intervention in OC invasion and metastasis.

TRAP1 regulates stemness through Wnt/ß-catenin pathway in human colorectal carcinoma.

Colorectal carcinoma (CRC) is a common cause of cancer-related death worldwide. Indeed, treatment failures are triggered by cancer stem cells (CSCs) that give rise to tumor repopulation upon initial remission. Thus, the role of the heat shock protein TRAP1 in stemness was investigated in CRC cell lines and human specimens, based on its involvement in colorectal carcinogenesis, through regulation of apoptosis, protein homeostasis and bioenergetics. Strikingly, co-expression between TRAP1 and stem cell markers was observed in stem cells located at the bottom of intestinal crypts and in CSCs sorted from CRC cell lines. Noteworthy, TRAP1 knockdown reduced the expression of stem cell markers and impaired colony formation, being the CSC phenotype and the anchorage-independent growth conserved in TRAP1-rich cancer cells. Consistently, the gene expression profiling of HCT116 cells showed that TRAP1 silencing results in the loss of the stem-like signature with acquisition of a more-differentiated phenotype and the downregulation of genes encoding for activating ligands and target proteins of Wnt/ß-catenin pathway. Mechanistically, TRAP1 maintenance of stemness is mediated by the regulation of Wnt/ß-catenin signaling, through the modulation of the expression of frizzled receptor ligands and the control of ß-catenin ubiquitination/phosphorylation. Remarkably, TRAP1 is associated with higher expression of ß-catenin and several Wnt/ß-catenin target genes in human CRCs, thus supporting the relevance of TRAP1 regulation of ß-catenin in human pathology. This study is the first demonstration that TRAP1 regulates stemness and Wnt/ß-catenin pathway in CRC and provides novel landmarks in cancer biology and therapeutics.

Evaluation of Glucose Uptake in Normal and Cancer Cell Lines by Positron Emission Tomography.

To date, there is no definitive demonstration of the utility of positron emission tomography (PET) in studying glucose metabolism in cultured cell lines. Thus, this study was designed to compare PET to more standardized methods for the quantitative assessment of glucose uptake in nontransformed and transformed living cells and to validate PET for metabolic studies in vitro. Human colon and breast carcinoma cell lines and mouse embryo fibroblasts were evaluated for [(18)F]fluorodeoxyglucose ([(18)F]FDG) uptake by PET and autoradiography and 2-deoxyglucose (2-DG) incorporation by colorimetric assay and analyzed for the radiotoxic effects of [(18)F]FDG and the expression levels of glucose transporters. Indeed, [(18)F]FDG incorporation on PET was comparable to [(18)F]FDG uptake by autoradiography and 2-DG incorporation by colorimetric assay, although radiotracer-based methods exhibited more pronounced differences between individual cell lines. As expected, these data correlated with glucose transporters 1 to 4 and hexokinase II expression in tumor cell lines and mouse fibroblasts. Notably, [(18)F]FDG incorporation resulted in low apoptotic rates, with fibroblasts being slightly more sensitive to radiotracer-induced cell death. The quantitative analysis of [(18)F]FDG uptake in living cells by PET represents a valuable and reproducible method to study tumor cell metabolism in vitro, being representative of the differences in the molecular profile of normal and tumor cell lines.

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.

[The occupational medicine specialist advice in support of the competent physician in the elaboration of the judgement of suitability to the job specification: evaluation method and analysis of case studies]. / La consulenza specialistica di Medicina del Lavoro a supporto del Medico Competente nella elaborazione del giudizio di idoneità alla mansione specifica: metodo di valutazione e analisi di una casistica.

INTRODUCTION: Still today, one of the most problematic, qualified and awkward assignments for the occupational physician, also for its ethical and socio-economic involvements, is to provide for each worker a fitness judgment to a specific work task. AIM: The aim of this study is to describe and to support results and effectiveness of a "second level" expert advice in occupational medicine, among workers with a "problematic" judgment of fitness to work. MATERIALS AND METHODS: We considered 80 requests for a "second level" advice. They were all requested, during 6 years, to the same Occupational Medicine Unit (UOOML) of a single hospital in north of Italy by a single large metalwork company, following the art. 39 of the 81/2008 legislative decree. RESULTS: The study underlined the effectiveness of "second level" advices in evaluating the adequacy of worker's health conditions related to the specific occupational task. Moreover, this study contributed to develop all operative protocol to carry out a systematic and effective process when "second level" advices are addressed to an Occupational Medicine Unit (UOOML). In particular, our operative proposal suggests, as a key point in the assessment process, a careful visit to the occupational environment, to directly study each single task and to deepen the needs of each single occupational emplacement.

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.

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.

Validation of vacuum-based refrigerated system for biobanking tissue preservation: analysis of cellular morphology, protein stability, and RNA quality.

Biobanks of fresh, unfixed human normal and malignant tissues represent a valuable source for gene expression analysis in translational cancer research and molecular pathology. However, the success of molecular and cellular analysis in both clinical and translational research is strongly dependent on the collection, handling, storage, and quality control of fresh human tissue samples. The aim of this study was to evaluate an innovative vacuum-based refrigerated system, as a logistically feasible technology to increase the collection of tissue specimens, preserving the integrity of cellular and molecular components. We tested randomly-selected tissues stored under vacuum at 4°C by using endpoints important for research and diagnosis, including tissue morphology, epitope stability, and RNA integrity. Gene expression was evaluated by qualitative and quantitative RT analysis of selected housekeeping and tissue-specific genes. Tissue morphology and overall protein stability were generally well preserved, being compromised only in gallbladder tissue. By contrast, phosphoprotein and RNA analysis demonstrated a time-dependent degree of degradation, with progressive loss of stability from 24 to 72 hours. However, this reduction in RNA quality did not represent a limitation for successful expression analysis of selected genes. Indeed, a comparative qualitative and quantitative RT-PCR analysis showed that RNA extracted from tissues stored under vacuum is suitable for gene expression profiling, but requires highly sensitive technologies, such as quantitative RT-PCR. These data suggest that the refrigerated vacuum-based system represents a suitable and feasible technology for routine transport of fresh specimens from surgery to biobanks, thus increasing the opportunity to collect biospecimens.

TRAP1 role in endoplasmic reticulum stress protection favors resistance to anthracyclins in breast carcinoma cells.

Adaptation to endoplasmic reticulum (ER) stress through the upregulation of the ER chaperone BiP/Grp78 favors resistance of cancer cells to anthracyclins. We recently demonstrated that the mitochondrial HSP90 chaperone TNF receptor-associated protein 1 (TRAP1) is also localized in the ER, where it is responsible for protection from ER stress and quality control on specific mitochondrial proteins contributing to its anti-apoptotic function and the regulation of the mitochondrial apoptotic pathway. Based on the evidence that Bip/Grp78 and TRAP1 are co-upregulated in about 50% of human breast carcinomas (BCs), and considering that the expression of TRAP1 is critical in favoring resistant phenotypes to different antitumor agents, we hypothesized that ER-associated TRAP1 is also favoring resistance to anthracyclins. Indeed, anthracyclins induce ER stress in BC cells and cross-resistance between ER stress agents and anthracyclins was observed in bortezomib- and anthracyclin-resistant cells. Several lines of evidence suggest a mechanistic link between the ER-stress protecting function of TRAP1 and resistance to anthracyclins: i) ER stress- and anthracyclin-resistant cell lines are characterized by the upregulation of TRAP1; ii) TRAP1 silencing in both drug-resistant cell models restored the sensitivity to bortezomib and anthracyclins; iii) the transfection of a TRAP1 deletion mutant, whose localization is restricted to the ER, in TRAP1 KD cells protected from apoptosis induced by anthracyclins; iv) the disruption of the ER-associated TRAP1/TBP7 pathway by a TBP7 dominant negative deletion mutant re-established drug sensitivity in drug-resistant cells. This process is likely mediated by the ability of TRAP1 to modulate the PERK pathway as TRAP1 KD cells failed to induce the phosphorylation of PERK in response to anthracyclins. Moreover, the downregulation of TRAP1 in combination with ER stress agents produced high cytotoxic effects in BC cells. These results suggest that ER-associated TRAP1 plays a role in protecting tumor cells against DNA damaging agents by modulating the PERK pathway.

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.