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Background Metabolic reprogramming is an important issue in tumor biology. A recently-identified actor in this regard is the molecular chaperone TRAP1, that is considered an oncogene in several cancers for its high expression but an oncosuppressor in others with predominant oxidative metabolism. TRAP1 is mainly localized in mitochondria, where it interacts with respiratory complexes, although alternative localizations have been described, particularly on the endoplasmic reticulum, where it interacts with the translational machinery with relevant roles in protein synthesis regulation. Results Herein we show that, inside mitochondria, TRAP1 binds the complex III core component UQCRC2 and regulates complex III activity. This decreases respiration rate during basal conditions but allows sustained oxidative phosphorylation when glucose is limiting, a condition in which the direct TRAP1-UQCRC2 binding is disrupted, but not TRAP1-complex III binding. Interestingly, several complex III components and assembly factors show an inverse correlation with survival and response to platinum-based therapy in high grade serous ovarian cancers, where TRAP1 inversely correlates with stage and grade and directly correlates with survival. Accordingly, drug-resistant ovarian cancer cells show high levels of complex III components and high sensitivity to complex III inhibitory drug antimycin A. Conclusions These results shed new light on the molecular mechanisms involved in TRAP1-dependent regulation of cancer cell metabolism and point out a potential novel target for metabolic therapy in ovarian cancer.

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/beta Catenin signaling pathway by the expression of several Wnt-target genes, PCatenin 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/beta Catenin 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 D I, Cdh I, Cdkn2a, Cd44, Axing, c-Myc and TcfI). Interestingly, HPV-negative OSCC cells showed higher levels of total beta Catenin 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/beta Catenin 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/beta Catenin 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/beta Catenin signaling pathway and gain of stem-like traits in tobacco-related OSCCs.

Metabolic rewiring fuels rapid cancer cell proliferation by promoting adjustments in energetic resources, and increasing glucose uptake and its conversion into lactate, even in the presence of oxygen. Furthermore, solid tumors often contain hypoxic areas and can rapidly adapt to low oxygen conditions by activating hypoxia inducible factor (HIF)-1α and several downstream pathways, thus sustaining cell survival and metabolic reprogramming. Since TNF receptor-associated protein 1 (TRAP1) is a HSP90 molecular chaperone upregulated in several human malignancies and is involved in cancer cell adaptation to unfavorable environments and metabolic reprogramming, in the present study, its role was investigated in the adaptive response to hypoxia in human colorectal cancer (CRC) cells and organoids. In the present study, glucose uptake, lactate production and the expression of key metabolic genes were evaluated in TRAP1-silenced CRC cell models under conditions of hypoxia/normoxia. Whole genome gene expression profiling was performed in TRAP1-silenced HCT116 cells exposed to hypoxia to establish the role of TRAP1 in adaptive responses to oxygen deprivation. The results revealed that TRAP1 was involved in regulating hypoxia-induced HIF-1α stabilization and glycolytic metabolism and that glucose transporter 1 expression, glucose uptake and lactate production were partially impaired in TRAP1-silenced CRC cells under hypoxic conditions. At the transcriptional level, the gene expression reprogramming of cancer cells driven by HIF-1α was partially inhibited in TRAP1-silenced CRC cells and organoids exposed to hypoxia. Moreover, Gene Set Enrichment Analysis of TRAP1-silenced HCT116 cells exposed to hypoxia demonstrated that TRAP1 was involved in the regulation of ribosome biogenesis and this occurred with the inhibition of the mTOR pathway. Therefore, as demonstrated herein, TRAP1 is a key factor in maintaining HIF-1α-induced genetic/metabolic program under hypoxic conditions and may represent a promising target for novel metabolic therapies.

The tumor microenvironment (TME) includes immune (T, B, NK, dendritic), stromal, mesenchymal, endothelial, adipocytic cells, extracellular matrix, and cytokines/chemokines/soluble factors regulating various intracellular signaling pathways (ISP) in tumor cells. TME influences the survival/progression of prostate cancer (PC), enabling tumor cell immune-evasion also through the activation of the PD-1/PD-L1 axis. We have performed a systematic literature review according to the PRISMA guidelines, to investigate how the PD-1/PD-L1 pathway is influenced by TME and ISPs. Tumor immune-escape mechanisms include suppression/exhaustion of tumor infiltrating cytotoxic T lymphocytes, inhibition of tumor suppressive NK cells, increase in immune-suppressive immune cells (regulatory T, M2 macrophagic, myeloid-derived suppressor, dendritic, stromal, and adipocytic cells). IFN-γ (the most investigated factor), TGF-β, TNF-α, IL-6, IL-17, IL-15, IL-27, complement factor C5a, and other soluble molecules secreted by TME components (and sometimes increased in patients’ serum), as well as and hypoxia, influenced the regulation of PD-L1. Experimental studies using human and mouse PC cell lines (derived from either androgen-sensitive or androgen-resistant tumors) revealed that the intracellular ERK/MEK, Akt-mTOR, NF-kB, WNT and JAK/STAT pathways were involved in PD-L1 upregulation in PC. Blocking the PD-1/PD-L1 signaling by using immunotherapy drugs can prevent tumor immune-escape, increasing the anti-tumor activity of immune cells.

In prostate cancer (PC), the PD-1/PD-L1 axis regulates various signaling pathways and it is influenced by extracellular factors. Pre-clinical experimental studies investigating the effects of various treatments (alone or combined) may discover how to overcome the immunotherapy-resistance in PC-patients. We performed a systematic literature review (PRISMA guidelines) to delineate the landscape of pre-clinical studies (including cell lines and mouse models) that tested treatments with effects on PD-L1 signaling in PC. NF-kB, MEK, JAK, or STAT inhibitors on human/mouse, primary/metastatic PC-cell lines variably down-modulated PD-L1-expression, reducing chemoresistance and tumor cell migration. If PC-cells were co-cultured with NK, CD8+ T-cells or CAR-T cells, the immune cell cytotoxicity increased when PD-L1 was downregulated (opposite effects for PD-L1 upregulation). In mouse models, radiotherapy, CDK4/6-inhibitors, and RB deletion induced PD-L1-upregulation, causing PC-immune-evasion. Epigenetic drugs may reduce PD-L1 expression. In some PC experimental models, blocking only the PD-1/PD-L1 pathway had limited efficacy in reducing the tumor growth. Anti-tumor effects could be increased by combining the PD-1/PD-L1 blockade with other approaches (inhibitors of tyrosine kinase, PI3K/mTOR or JAK/STAT3 pathways, p300/CBP; anti-RANKL and/or anti-CTLA-4 antibodies; cytokines; nitroxoline; DNA/cell vaccines; radiotherapy/Radium-223).

Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP1) is a heat shock protein 90 (HSP90) molecular chaperone overexpressed in 60&ndash, 70% human colorectal carcinomas (CRCs) and the co-upregulation of TRAP1 and associated 6-related proteins identifies metastatic CRCs with poor prognosis. Since the molecular mechanisms responsible for TRAP1 regulation are still unknown, the significance of TRAP1 gene copy number (CN) and the role of post-transductional protein modifications were addressed. TRAP1 gene aneuploidy accounted for 34.5% of cases in a cohort of 58 human CRCs and TRAP1 CN correlated with its mRNA and protein expression, suggesting that transcriptional mechanisms are responsible for TRAP1 upregulation. Furthermore, the analysis of the National Cancer Institute&rsquo, s Clinical Proteomic Tumor Analysis Consortium/The Cancer Genome Atlas (CPTAC/TCGA) CRC database showed that TRAP1 polysomy significantly correlates with lymph node involvement. However, a subgroup of tumors showed TRAP1 protein levels independent from its CN. Of note, a direct correlation was observed between TRAP1 protein levels and the expression of S-nitrosoglutathione reductase (GSNOR), a denitrosylase involved in the regulation of protein S-nitrosylation. Furthermore, CRC cell lines exposed to hypoxia or dichloroacetate treatment showed the downregulation of TRAP1 upon GSNOR silencing and this resulted in increased TRAP1 mono/polyubiquitination. These data suggest that transcriptional and post-transductional mechanisms account for TRAP1 expression in human CRCs and GSNOR protects TRAP1 from S-nitrosylation and consequent proteasome degradation mostly in conditions of stress.

Here, we show that TRAP1 modulates glycolytic metabolism by regulating PFK1 activity/stability. In a high TRAP1 background, TRAP1 inhibits cellular respiration and interacts with PFK1 on the ER and this enables PFK1 glycolytic activity preventing its ubiquitination/degradation. In a low TRAP1 background, cellular respiration is upregulated and PFK1 activity reduced due to increased ubiquitination/degradation and this results in loss of TRAP1 control on glycolytic cascade. The increased levels of citrate, observed in conditions of enhanced cellular respiration, are responsible for the inhibition of PFK1 activity, and this results in enhancement of PFK1 ubiquitination/degradation., 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 18F‐fluoro‐2‐deoxy‐glucose (18F‐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 18F‐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.

Cholangiocarcinoma is a primary malignancy of the biliary tract characterized by late and unspecific symptoms, unfavorable prognosis, and few treatment options. The advent of next-generation sequencing has revealed potential targetable or actionable molecular alterations in biliary tumors. Among several identified genetic alterations, the IDH1 mutation is arousing interest due to its role in epigenetic and metabolic remodeling. Indeed, some IDH1 point mutations induce widespread epigenetic alterations by means of a gain-of-function of the enzyme, which becomes able to produce the oncometabolite 2-hydroxyglutarate, with inhibitory activity on α-ketoglutarate-dependent enzymes, such as DNA and histone demethylases. Thus, its accumulation produces changes in the expression of several key genes involved in cell differentiation and survival. At present, small-molecule inhibitors of IDH1 mutated enzyme are under investigation in preclinical and clinical phases as promising innovative treatments for IDH1-mutated intrahepatic cholangiocarcinomas. This review examines the molecular rationale and the results of preclinical and early-phase studies on novel pharmacological agents targeting mutant IDH1 in cholangiocarcinoma patients. Contextually, it will offer a starting point for discussion on combined therapies with metabolic and epigenetic drugs, to provide molecular support to target the interplay between metabolism and epigenetics, two hallmarks of cancer onset and progression.

Simple Summary The global methylation profile of two human metastatic colorectal carcinoma subgroups with significantly different outcomes (primary-resistant versus drug-sensitive tumors) was analyzed and compared with the gene expression and methylation data from The Cancer Genome Atlas COlon ADenocarcinoma (TCGA COAD) metastatic colorectal carcinoma dataset with the aim to identify a prognostic signature of functionally methylated genes. A novel epigenetic eight-gene signature, with hypermethylation of the promoter regions, was identified and validated for its capacity to predict poor outcome, which had a CpG-island methylator phenotype (CIMP)-high status and microsatellite instability (MSI)-like phenotype. Abstract Epigenetics is involved in tumor progression and drug resistance in human colorectal carcinoma (CRC). This study addressed the hypothesis that the DNA methylation profiling may predict the clinical behavior of metastatic CRCs (mCRCs). The global methylation profile of two human mCRC subgroups with significantly different outcome was analyzed and compared with gene expression and methylation data from The Cancer Genome Atlas COlon ADenocarcinoma (TCGA COAD) and the NCBI GENE expression Omnibus repository (GEO) GSE48684 mCRCs datasets to identify a prognostic signature of functionally methylated genes. A novel epigenetic signature of eight hypermethylated genes was characterized that was able to identify mCRCs with poor prognosis, which had a CpG-island methylator phenotype (CIMP)-high and microsatellite instability (MSI)-like phenotype. Interestingly, methylation events were enriched in genes located on the q-arm of chromosomes 13 and 20, two chromosomal regions with gain/loss alterations associated with adenoma-to-carcinoma progression. Finally, the expression of the eight-genes signature and MSI-enriching genes was confirmed in oxaliplatin- and irinotecan-resistant CRC cell lines. These data reveal that the hypermethylation of specific genes may provide prognostic information that is able to identify a subgroup of mCRCs with poor prognosis.

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.

Metabolic reprogramming is an important issue in tumor biology. An unexpected inter- and intra-tumor metabolic heterogeneity has been strictly correlated to tumor outcome. Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP1) is a molecular chaperone involved in the regulation of energetic metabolism in cancer cells. This protein is highly expressed in several cancers, such as glioblastoma, colon, breast, prostate and lung cancers and is often associated with drug resistance. However, TRAP1 is also downregulated in specific tumors, such as ovarian, bladder and renal cancers, where its lower expression is correlated with the worst prognoses and chemoresistance. TRAP1 is the only mitochondrial member of the Heat Shock Protein 90 (HSP90) family that directly interacts with respiratory complexes, contributing to their stability and activity but it is still unclear if such interactions lead to reduced or increased respiratory capacity. The role of TRAP1 is to enhance or suppress oxidative phosphorylation; the effects of such regulation on tumor development and progression are controversial. These observations encourage the study of the mechanisms responsible for the dualist role of TRAP1 as an oncogene or oncosuppressor in specific tumor types. In this review, TRAP1 puzzling functions were recapitulated with a special focus on the correlation between metabolic reprogramming and tumor outcome. We wanted to investigate whether metabolism-targeting drugs can efficiently interfere with tumor progression and whether they might be combined with chemotherapeutics or molecular-targeted agents to counteract drug resistance and reduce therapeutic failure.

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. Cancer Res; 74(22); 6693–704. ©2014 AACR.

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a heat shock protein 90 (HSP90) molecular chaperone upregulated in several human malignancies and involved in protection from apoptosis and drug resistance, cell cycle progression, cell metabolism and quality control of specific client proteins. TRAP1 role in thyroid carcinoma (TC), still unaddressed at present, was investigated by analyzing its expression in a cohort of 86 human TCs and evaluating its involvement in cancer cell survival and proliferationin vitro. Indeed, TRAP1 levels progressively increased from normal peritumoral thyroid gland, to papillary TCs (PTCs), follicular variants of PTCs (FV-PTCs) and poorly differentiated TCs (PDTCs). By contrast, anaplastic thyroid tumors exhibited a dual pattern, the majority being characterized by high TRAP1 levels, while a small subgroup completely negative. Consistently with a potential involvement of TRAP1 in thyroid carcinogenesis, TRAP1 silencing resulted in increased sensitivity to paclitaxel-induced apoptosis, inhibition of cell cycle progression and attenuation of ERK signaling. Noteworthy, the inhibition of TRAP1 ATPase activity by pharmacological agents resulted in attenuation of cell proliferation, inhibition of ERK signaling and reversion of drug resistance. These data suggest that TRAP1 inhibition may be regarded as potential strategy to target specific features of human TCs, i.e., cell proliferation and resistance to apoptosis.

Tumour cells have long been considered defective in mitochondrial respiration and mostly dependent on glycolytic metabolism. However, this assumption is currently challenged by several lines of evidence in a growing number of tumours. Ovarian cancer (OC) is one of the most lethal cancers worldwide, but it continues to be a poorly understood disease and its metabolic features are far to be elucidated. In this context, we investigated the role of tumour necrosis factor receptor-associated protein 1 (TRAP1), which is found upregulated in several cancer types and is a key modulator of tumour cell metabolism. Surprisingly, we found that TRAP1 expression inversely correlated with grade, stage and lower survival in a large cohort of OC patients. Accordingly, TRAP1 silencing induced resistance to cisplatin, resistant cells showed increased oxidative metabolism compared with their sensitive counterpart, and the bioenergetics cellular index of higher grade tumours indicated increased mitochondrial respiration. Strikingly, cisplatin resistance was reversible upon pharmacological inhibition of mitochondrial oxidative phosphorylation by metformin/oligomycin. At molecular level, increased oxidative metabolism in low TRAP1-expressing OC cells and tissues enhanced production of inflammatory mediators such as interleukin (IL)-6 and IL-8. Mechanistically, we identified members of the multidrug resistance complex (MDR) as key mediators of such metabolism-driven, inflammation-induced process. Indeed, treatment of OC cell lines with TNFα and IL6 induced a selective increase in the expression of TAP1 and multidrug resistance protein 1, whereas TAP1 silencing sensitized cells to cisplatin-induced apoptosis. Our results unveil a novel role for TRAP1 and oxidative metabolism in cancer progression and suggest the targeting of mitochondrial bioenergetics to increase cisplatin efficacy in human OC.Cell Death and Differentiation advance online publication, 20 May 2016; doi:10.1038/cdd.2016.39.

An unknown primary tumor (UPT) is defined by the presence of a metastatic cancer without a known primary site of origin despite a standardized diagnostic workup. Clinically, UPTs show rapid progression and early dissemination, with signs and symptoms related to the metastatic site. The molecular bases of their biology remain largely unknown, with no evidence as to whether they represent a distinct biological entity. Immunohistochemistry remain the best diagnostic tool in term of cost-effectiveness, but the time-consuming "algorithmic process" it relies on has led to the application of new molecular techniques for the identification of the primary site of UPTs. For example, several microarray or miRNA classifications of UPTs have been used, with an accuracy in the prediction of the primary site as high as 90%. It should be noted that validating a prediction of tissue origin is challenging in these patients, since most of them will never have a primary site identified. Moreover, prospective studies to determine whether selection of treatment options based on such profiling methods actually improves patient outcome are still missing. In the last few years functional imaging (i.e. FDG-PET/CT) has gained a main role in the detection of the site of origin of UPTs and is currently recommended by the European Association of Nuclear Medicine. However, despite recent refinements in the diagnostic workup, the site of origin of UPT often remains elusive. As a consequence, treatment of patients with UPT is still empirical and inadequate.

Despite marked tumor shrinkage after 5-FU treatment, the frequency of colon cancer relapse indicates that a fraction of tumor cells survives treatment causing tumor recurrence. The majority of cancer cells divert metabolites into anabolic pathways through Warburg behavior giving an advantage in terms of tumor growth. Here, we report that treatment of colon cancer cell with 5-FU selects for cells with mesenchymal stem-like properties that undergo a metabolic reprogramming resulting in addiction to OXPHOS to meet energy demands. 5-FU treatment-resistant cells show a de novo expression of pyruvate kinase M1 (PKM1) and repression of PKM2, correlating with repression of the pentose phosphate pathway, decrease in NADPH level and in antioxidant defenses, promoting PKM2 oxidation and acquisition of stem-like phenotype. Response to 5-FU in a xenotransplantation model of human colon cancer confirms activation of mitochondrial function. Combined treatment with 5-FU and a pharmacological inhibitor of OXPHOS abolished the spherogenic potential of colon cancer cells and diminished the expression of stem-like markers. These findings suggest that inhibition of OXPHOS in combination with 5-FU is a rational combination strategy to achieve durable treatment response in colon cancer.

ABSTRACT: Aim: To evaluate whether pyrosequencing (PS) improves the KRAS mutational status predictive value. Patients & methods: A retrospective analysis of KRAS mutations by PS and direct sequencing (DS) in 192 metastatic colorectal carcinomas (mCRCs), subgrouped in 51 KRAS mutated at PS and 141 KRAS wild-type at DS. Results: DS failed to detect low-frequency KRAS mutations in four out of 51 mCRCs, whereas PS detected 12 additional low-frequency KRAS mutations in 141 mCRCs KRAS wild-type at DS. After reanalyzing by PS 97 KRAS wild-type tumors treated with anti-EGF receptor (EGFR) antibodies, nine additional mutations were revealed in nonresponders, whereas none of responders exhibited a KRAS-mutated genotype. Of note, KRAS-mutated tumors upon PS showed a worst progression-free survival after EGFR therapy. Finally, PS allowed the detection of additional NRAS, BRAF and exon 20 PIK3CA mutations mostly in KRAS wild-type mCRCs resistant to EGFR therapy. Conclusion: PS detection of low-frequency mutations may improve the KRAS predictive value for EGFR therapy selection.

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.

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.

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.

TNF receptor-associated protein 1 (TRAP1), the main mitochondrial member of the heat shock protein (HSP) 90 family, is induced in most tumor types and is involved in the regulation of proteostasis in the mitochondria of tumor cells through the control of folding and stability of selective proteins, such as Cyclophilin D and Sorcin. Notably, we have recently demonstrated that TRAP1 also interacts with the regulatory protein particle TBP7 in the endoplasmic reticulum (ER), where it is involved in a further extra-mitochondrial quality control of nuclear-encoded mitochondrial proteins through the regulation of their ubiquitination/degradation. Here we show that TRAP1 is involved in the translational control of cancer cells through an attenuation of global protein synthesis, as evidenced by an inverse correlation between TRAP1 expression and ubiquitination/degradation of nascent stress-protective client proteins. This study demonstrates for the first time that TRAP1 is associated with ribosomes and with several translation factors in colon carcinoma cells and, remarkably, is found co-upregulated with some components of the translational apparatus (eIF4A, eIF4E, eEF1A and eEF1G) in human colorectal cancers, with potential new opportunities for therapeutic intervention in humans. Moreover, TRAP1 regulates the rate of protein synthesis through the eIF2?? pathway either under basal conditions or under stress, favoring the activation of GCN2 and PERK kinases, with consequent phosphorylation of eIF2?? and attenuation of cap-dependent translation. This enhances the synthesis of selective stress-responsive proteins, such as the transcription factor ATF4 and its downstream effectors BiP/Grp78, and the cystine antiporter system xCT, thereby providing protection against ER stress, oxidative damage and nutrient deprivation. Accordingly, TRAP1 silencing sensitizes cells to apoptosis induced by novel antitumoral drugs that inhibit cap-dependent translation, such as ribavirin or 4EGI-1, and reduces the ability of cells to migrate through the pores of transwell filters. These new findings target the TRAP1 network in the development of novel anti-cancer strategies.

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 Ca2+-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. Cancer Res; 70(16); 6577–86. ©2010 AACR.

TRAP1 is a component of a pro-survival mitochondrial pathway up-regulated in tumor cells. The evaluation of TRAP1 expression in 26 human colorectal carcinomas showed up-regulation in 17/26 tumors. Accordingly, TRAP1 levels were increased in HT-29 colorectal carcinoma cells resistant to 5-fluorouracil, oxaliplatin and irinotecan. Thus, we investigated the role of TRAP1 in multi-drug resistance in human colorectal cancer. Interestingly, TRAP1 overexpression leads to 5-fluorouracil-, oxaliplatin- and irinotecan-resistant phenotypes in different neoplastic cells. Conversely, the inhibition of TRAP1 activity by TRAP1 ATPase antagonist, shepherdin, increased the sensitivity to oxaliplatin and irinotecan in colorectal carcinoma cells resistant to the single agents. These results suggest that the increased expression of TRAP1 could be part of a pro-survival pathway responsible for multi-drug resistance. © 2009 Elsevier Ireland Ltd. All rights reserved.

// Valentina Condelli 1, * , Francesca Maddalena 1, * , Lorenza Sisinni 1 , Giacomo Lettini 1 , Danilo Swann Matassa 2 , Annamaria Piscazzi 3 , Giuseppe Palladino 3 , Maria Rosaria Amoroso 2 , Franca Esposito 2 , Matteo Landriscina 3 1 Laboratory of Pre-Clinical and Translational Research, IRCCS, Referral Cancer Center of Basilicata, Rionero in Vulture, PZ, Italy 2 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy 3 Medical Oncology Unit, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy * These authors have contributed equally to this work Correspondence to: Matteo Landriscina, e-mail: matteo.landriscina@unifg.it Franca Esposito, e-mail: franca.esposito@unina.it Keywords: BRAF, TRAP1, apoptosis, colon cancer, drug resistance Received: April 13, 2015 Accepted: June 01, 2015 Published: June 13, 2015 ABSTRACT 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.