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Supplementary Figure 4 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

Schematic representation of the transcriptional interactions among 6 cell cycle-related genes down-regulated in shTRAP1 cells according to the whole genome gene expression analysis and confirmed by subsequent Real Time validation analysis (query genes, visualized in green) and MAPK pathway (visualized in red). Transcriptional interactions from IPA software.

TRAP1 and BRAF protein levels in 41 human BRAF-wild type colorectal carcinomas expressed as time increase in tumor compared to correspondent non-infiltrated peritumoral mucosas.

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.

The Ca2+-binding protein sorcin regulates intracellular calcium homeostasis and plays a role in the induction of drug resistance in human cancers. Recently, an 18 kDa mitochondrial isoform of sorcin was reported to participate in antiapoptosis in human colorectal cancer (CRC), but information remains lacking about the functional role of the more abundant 22 kDa isoform of sorcin expressed in CRC. We found the 22 kDa isoform to be widely expressed in human CRC cells, whether or not they were drug resistant. Its upregulation in drug-sensitive cells induced resistance to 5-fluorouracil, oxaliplatin, and irinotecan, whereas its downregulation sensitized CRC cells to these chemotherapeutic agents. Sorcin enhances the accumulation of Ca2+ in the endoplasmic reticulum (ER), preventing ER stress, and, in support of this function, we found that the 22 kDa isoform of sorcin was upregulated under conditions of ER stress. In contrast, RNAi-mediated silencing of sorcin activated caspase-3, caspase-12, and GRP78/BiP, triggering apoptosis through the mitochondrial pathway. Our findings establish that CRC cells overexpress sorcin as an adaptive mechanism to prevent ER stress and escape apoptosis triggered by chemotherapeutic agents, prompting its further investigation as a novel molecular target to overcome MDR. Cancer Res; 71(24); 7659–69. ©2011 AACR.

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Supplementary Figure Legends 1- 4 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

Supplementary Figure 2 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

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Supplementary Figure 1 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

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Supplementary Table 1 from Mitochondrial Chaperone Trap1 and the Calcium Binding Protein Sorcin Interact and Protect Cells against Apoptosis Induced by Antiblastic Agents

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 staple line gastric leak (GL) is estimated to be the most serious complication of the sleeve gastrectomy. The use of indocyanine green (ICG) has been introduced in minimally invasive surgery to show the vascularization of the stomach in real time and its application to the gastroesophageal junction (GE) during Laparoscopic Sleeve Gastrectomy (LSG) seems very promising.We present the case of a 40-year-old female underwent laparoscopic sleeve gastrectomy. Intraoperative indocyanine green test showed a small dark area in the proximal third of the staple line reinforced with fibrin glue. Two weeks later the patient presented to the emergency room (ED) with abdominal pain, fever, vomiting, intolerance to oral intake and the evidence of a leak on the abdomen Computer Tomography (CT). The UIN for ClinicalTrial.gov Protocol Registration and Results System is: NCT05337644 for the Organization UFoggia.This case report shows that intraoperative ICG test can be helpful in determining which patients are at greater risk of the leak and, more importantly, the cause of the leak but further tests are needed to determine if the ICG predicts leak due to ischemia.

The management of cholelithiasis and choledocholithiasis combined is controversial. The more frequent approach is a two-stage procedure, with endoscopic sphincterotomy and stone removal from the bile duct followed by laparoscopic cholecystectomy. This study aims to demonstrate how, on the basis of the personal experience, the Rendez-vous technique, that combines the two techniques in a single-stage operation is better than the sequential treatment.Between June 2017 to December 2019, 40 consecutive patients with cholelithiasis and choledocholithiasis combined were enrolled for the study: 20 were treated with the sequential treatment and 20 with the Rendez-vous method. The preoperative diagnostic work-up was similar in the two group. The endpoints of the study included incidence of endoscopic and surgical complications, rate of hospitalization and cost analysis.The study showed no difference in demographic parameters between the two groups, but the success rate of clearance of CBD was significantly smaller for sequential arm, with the need of additional procedures. We found a statistical reduction of postoperative acute pancreatitis, hospital stay and charges in Rendez-vous group, at the expense of a prolonged total operating time.The data of the study confirm the superiority of the Rendez-vous technique because it resolves cholelithiasis associated with choledocholithiasis in a single surgical act, with greater acceptance of the patient who avoids a second invasive surgical act, and with a reduction in complications; moreover, it requires shorter hospitalization, resulting in reduced costs. We propose this option in the management of cases where preoperative ERCP-ES has failed.Common bile duct stones, Cholecysto-choledocholithiasis, Endoscopic retrograde cholangiopancreatography, Endoscopic sphincterotomy, Laparoscopic cholecystectomy, Laparo-endoscopic Rendez-vous.Diversi studi hanno dimostrato la superiorità della tecnica LERV rispetto alla strategia sequenziale nel management della calcolosi colecisto-coledocica in termini di riduzione delle complicanze postoperatorie, della degenza ospedaliera e dei costi complessivi. Tali risultati sono talmente promettenti che ci hanno spinto ad adottare la medesima tecnica nel nostro Istituto. Con uno studio retrospettivo abbiamo arruolato, tra Giugno 2017 e Dicembre 2019, 40 pazienti consecutivi affetti da calcolosi colecisto-coledocica; 20 del Gruppo A sono stati trattati con la strategia sequenziale, ERCP e SE seguite da colecistectomia laparoscopica, 20 del gruppo B sono stati trattati con la strategia One- Step, il Rendez-Vous Laparoendoscopico, con il quale in un solo atto chirurgico viene bonificata endoscopicamente la via biliare principale ed eseguito l’intervento di colecistectomia laparoscopica. I risultati così ottenuti hanno dimostrato tutti gli endpoints prefissati. Si è ottenuta una netta riduzione delle pancreatiti postoperatorie nel gruppo LERV, come dimostrazione del suo massimo vantaggio, ovvero un’annullazione selettiva della via biliare principale, lasciando integro ed illeso il Wirsung. Ciò associato alla possibilità di unire le due procedure in un unico solo atto chirurgico ha ridotto in maniera significativa la degenza ospedaliera, con conseguente riduzione anche dei costi. Rispetto alla classica tecnica sequenziale, quella LERV si avvale di un tasso di successo molto più alto nella clearance della via biliare, eliminando anche l’utilizzo di procedure aggiuntive che andrebbero a gravare sulla degenza complessiva e sui costi. Chiaramente il Rendez-vous ha delle chiare indicazioni, e va suggerito solo in pazienti selezionati; laddove sussistano i requisiti essenziali quali un endoscopista esperto e la possibilità logistica ed organizzativa di poter organizzare entrambe le procedure nella stessa sala operatoria. Queste limitazioni non ne hanno favorito la diffusione nonostante i risultati incoraggianti prodotti da numerosi Trials e meta-analisi. Le prove attuali sull’uso di questa tecnica presentate in questo articolo sono promettenti e dimostrano i principali vantaggi della procedura rispetto alla classica strategia sequenziale.

Wnt/&beta, 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&ndash, 70% of human colorectal carcinomas (CRCs) and favors stem cells maintenance, modulating the Wnt/&beta, Catenin pathway and preventing &beta, Catenin phosphorylation/degradation. The role of TRAP1 in the regulation of Wnt/&beta, Catenin signaling was further investigated in human CRC cell lines, patient-derived spheroids, and CRC specimens. TRAP1 relevance in the activation of Wnt/&beta, 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 &beta, Catenin, LRP5 and LRP6. Altogether, these data suggest that Wnt/&beta, Catenin signaling is modulated at multiple levels by TRAP1.

Background. Epigenetic remodeling is responsible for tumor progression and drug resistance in human colorectal carcinoma (CRC). A subgroup of human CRCs exhibits the CIMP status, with extensive hypermethylation events in promoter regions of several genes, even though the prognostic significance of CIMP is controversial. This study addressed the hypothesis that DNA methylation profiling may identify metastatic CRC (mCRC) subtypes with different clinical behavior. Methods. Global methylation profile was comparatively analyzed between 24 first-line primary-resistant and 12 drug-sensitive mCRCs (in-house cohort), two subgroups of tumors with significantly different outcome. Methylation and gene expression data from 33 mCRCs of the TCGA COAD dataset (TCGA COAD cohort) were used to identify, among differentially methylated genes, a prognostic signature of functionally methylated genes. Clusters of mCRCs with different methylation patterns were further characterized for DNA mutational load, gene copy number and gene expression profiles. Human CRC HT29 and HCT116 cell lines were adapted to growth in presence of oxaliplatin and irinotecan and used as in vitro model to validate gene expression data.Results. Twelve functionally methylated genes yielded a hierarchical clustering of patients in two well-defined clusters with hypermethylated tumors characterized by a significantly worse relapse-free and overall survival compared to hypomethylated cancers and this was reproduced in both the in-house and the TCGA COAD cohorts. Interestingly, the hypermethylated poor prognosis cluster was enriched of CIMP-high and MSI-like cases. Furthermore, methylation events were enriched in genes located on q-arm of chromosomes 13 and 20, two chromosomal regions with gain/loss alterations strongly associated with adenoma-to-carcinoma progression. Finally, the expression of the 12-genes signature and MSI-enriching genes was confirmed in two independent oxaliplatin- and irinotecan-resistant CRC cell lines. Conclusions. These data represent the proof of concept that the hypermethylation of specific sets of genes may provide prognostic information being able to identify a subgroup of mCRCs with poor prognosis.

Minimally invasive surgery for colorectal cancer has been demonstrated to have the same oncological results as open surgery, with better clinical outcomes. Robotic surgery is an evolution of minimally invasive technique. This study aims to evaluate surgical and oncological short-term outcomes of robotic right colon resection in comparison with the laparoscopic approach.Between January 2014 and May 2017, fifteen laparoscopic right hemicolectomies were compared to seven robotic ones. The primary data points included operation time, length of hospital stay, extraction site incision length, complications, and conversions. When malignancy was the indication for surgery, additional data points have been added.The study showed no difference in parameters between the two groups, but estimated blood loss was significantly smaller for Robotic arm. We found a prolonged total operative room time in the robotic arm, while the surgical time is similar in two groups. The data collected about specimen length and number of lymph nodes suggest that robotic procedure is oncologically similar to laparoscopic one.Robotic approach allows performance of adequate dissection of the right colon with radical lymphadenectomy as in laparoscopic surgery, confirming the safety and oncological efficacy of this technique, with acceptable results and short-term outcomes.Da Vinci surgery, XI, Laparoscopic colorectal surgery, Right hemicolectomy, Robot.Le tecniche chirurgiche mininvasive, per il trattamento delle patologie del colon retto hanno dimostrato di essere caratterizzate dagli stessi risultati delle tecniche tradizionali in merito agli scopi oncologici, ma sono contraddistinte da migliori risultati clinici. La chirurgia robotica, grazie all’impiego del robot DaVinci Xi, rappresenta un’evoluzione del classico approccio mininvasivo laparoscopico. In questo studio si ha come scopo quello di individuare risultati chirurgici e oncologici a breve termine, dell’emicolectomia destra robotica e compararli a quelli ottenuti nella corrispettiva procedura laparoscopica. Sono stati analizzati un numero complessivo di 22 casi, di cui 15 sottoposti a procedura laparoscopica e 7 a quella robotica. I principali dati presi in considerazione in questo studio, sono stati, la durata dell’intervento chirurgico, i giorni di degenza postoperatoria, la lunghezza del sito d’estrazione del pezzo operatorio, la presenza di complicanze e il tasso di conversione. Solo nei casi in cui il trattamento era eseguito per patologia maligna sono stati introdotti ulteriori dati. I risultati di questo studio dimostrano come non ci siano particolari differenze tra i due gruppi analizzati, sebbene si sia osservato una significativa minore perdita stimata di sangue nei pazienti sottoposti a procedura robotica. È stato inoltre osservato un tempo operatorio più lungo nelle procedure robotiche, mentre la durata della procedura chirurgica è risultata essere simile nei due gruppi. I dati raccolti sulla lunghezza del campione e sul numero di linfonodi suggeriscono che la procedura robotica è oncologicamente simile a quella laparoscopica. L’approccio robotico consente l’esecuzione di un’adeguata dissezione del colon destro con linfoadenectomia radicale come nella chirurgia laparoscopica, confermando la sicurezza e l’efficacia oncologica di questa tecnica.

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.

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.

HSP90 molecular chaperones (i.e., HSP90α, HSP90β, 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.

Cancer has been considered, for a long time, a genetic disease where mutations in key regulatory genes drive tumor initiation, growth, metastasis, and drug resistance. Instead, the advent of high-throughput technologies has revolutionized cancer research, allowing to investigate molecular alterations at multiple levels, including genome, epigenome, transcriptome, proteome, and metabolome and showing the multifaceted aspects of this disease. The multi-omics approaches revealed an intricate molecular landscape where different cellular functions are interconnected and cooperatively contribute to shaping the malignant phenotype. Recent evidence has brought to light how metabolism and epigenetics are highly intertwined, and their aberrant crosstalk can contribute to tumorigenesis. The oncogene-driven metabolic plasticity of tumor cells supports the energetic and anabolic demands of proliferative tumor programs and secondary can alter the epigenetic landscape via modulating the production and/or the activity of epigenetic metabolites. Conversely, epigenetic mechanisms can regulate the expression of metabolic genes, thereby altering the metabolome, eliciting adaptive responses to rapidly changing environmental conditions, and sustaining malignant cell survival and progression in hostile niches. Thus, cancer cells take advantage of the epigenetics-metabolism crosstalk to acquire aggressive traits, promote cell proliferation, metastasis, and pluripotency, and shape tumor microenvironment. Understanding this bidirectional relationship is crucial to identify potential novel molecular targets for the implementation of robust anti-cancer therapeutic strategies.

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.

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.

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/beta-catenin pathway. Mechanistically, TRAP1 maintenance of stemness is mediated by the regulation of Wnt/beta-catenin signaling, through the modulation of the expression of frizzled receptor ligands and the control of beta-catenin ubiquitination/phosphorylation. Remarkably, TRAP1 is associated with higher expression of beta-catenin and several Wnt/beta-catenin target genes in human CRCs, thus supporting the relevance of TRAP1 regulation of beta-catenin in human pathology. This study is the first demonstration that TRAP1 regulates stemness and Wnt/beta-catenin pathway in CRC and provides novel landmarks in cancer biology and therapeutics.

Despite initial chemotherapy response, ovarian cancer is the deadliest gynecologic cancer, due to frequent relapse and onset of drug resistance. To date, there is no affordable diagnostic/prognostic biomarker for early detection of the disease. However, it has been recently shown that high grade serous ovarian cancers show peculiar oxidative metabolism, which is in turn responsible for inflammatory response and drug resistance. The molecular chaperone TRAP1 plays pivotal roles in such metabolic adaptations, due to the involvement in the regulation of mitochondrial respiration. Here, we show that platinum-resistant ovarian cancer cells also show reduced cholesterol biosynthesis, and mostly rely on the uptake of exogenous cholesterol for their needs. Expression of FDPS and OSC, enzymes involved in cholesterol synthesis, are decreased both in drug-resistant cells and upon TRAP1 silencing, whereas the expression of LDL receptor, the main mediator of extracellular cholesterol uptake, is increased. Strikingly, treatment with statins to inhibit cholesterol synthesis reduces cisplatin-induced apoptosis, whereas silencing of LIPG, an enzyme involved in lipid metabolism, or withdrawal of lipids from the culture medium, increases sensitivity to the drug. These results suggest caveats for the use of statins in ovarian cancer patients and highlights the importance of lipid metabolism in ovarian cancer treatment. This research was funded by POR CAMPANIA FESR 2014/2020 [project “SATIN” (Sviluppo di Approcci Terapeutici INnovativi per patologie neoplastiche resistenti ai trattamenti)]

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 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.

Ovarian cancer (OC) is a deadly disease, representing the fifth leading cause of cancer death worldwide. In such a context, the major barrier to improve OC survival is resistance to chemotherapy, including platinum. Indeed, the majority of OC patients respond to initial therapy with tumor cytoreductive surgery and platinum-based chemotherapy, but approximately 70% of advanced stage patients will develop tumor recurrence and eventually succumb to recurrent disease, typically characterized by multiple drug resistance.

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.