Danilo Swann Matassa

Whole-exome resequencing reveals recessive mutations in TRAP1 in individuals with CAKUT and VACTERL association

Congenital abnormalities of the kidney and urinary tract (CAKUT) account for approximately half of children with chronic kidney disease and they are the most frequent cause of end-stage renal disease in children in the US. However, its genetic etiology remains mostly elusive. VACTERL association is a rare disorder that involves congenital abnormalities in multiple organs including the kidney and urinary tract in up to 60% of the cases. By homozygosity mapping and whole exome resequencing combined with high-throughput mutation analysis by array-based multiplex PCR and next-generation sequencing, we identified recessive mutations in the gene TNF receptor-associated protein 1 (TRAP1) in two families with isolated CAKUT and three families with VACTERL association. TRAP1 is a heat shock protein 90-related mitochondrial chaperone possibly involved in antiapoptotic and endoplasmic reticulum-stress signaling. Trap1 is expressed in renal epithelia of developing mouse kidney E13.5 and in the kidney of adult rats, most prominently in proximal tubules and in thick medullary ascending limbs of Henle’s loop. Thus, we identified mutations in TRAP1 as highly likely causing CAKUT or CAKUT in VACTERL association.

TRAP1 and the proteasome regulatory particle TBP7/Rpt3 interact in the endoplasmic reticulum and control cellular ubiquitination of specific mitochondrial proteins

Tumor necrosis factor receptor-associated protein-1 (TRAP1) is a mitochondrial (MITO) antiapoptotic heat-shock protein. The information available on the TRAP1 pathway describes just a few well-characterized functions of this protein in mitochondria. However, our groups use of mass-spectrometric analysis identified TBP7, an AAA-ATPase of the 19S proteasomal subunit, as a putative TRAP1-interacting protein. Surprisingly, TRAP1 and TBP7 colocalize in the endoplasmic reticulum (ER), as demonstrated by biochemical and confocal/electron microscopic analyses, and interact directly, as confirmed by fluorescence resonance energy transfer analysis. This is the first demonstration of TRAP1s presence in this cellular compartment. TRAP1 silencing by short-hairpin RNAs, in cells exposed to thapsigargin-induced ER stress, correlates with upregulation of BiP/Grp78, thus suggesting a role of TRAP1 in the refolding of damaged proteins and in ER stress protection. Consistently, TRAP1 and/or TBP7 interference enhanced stress-induced cell death and increased intracellular protein ubiquitination. These experiments led us to hypothesize an involvement of TRAP1 in protein quality control for mistargeted/misfolded mitochondria-destined proteins, through interaction with the regulatory proteasome protein TBP7. Remarkably, expression of specific MITO proteins decreased upon TRAP1 interference as a consequence of increased ubiquitination. The proposed TRAP1 network has an impact in vivo, as it is conserved in human colorectal cancers, is controlled by ER-localized TRAP1 interacting with TBP7 and provides a novel model of the ER–mitochondria crosstalk.

Sorcin Induces a Drug-Resistant Phenotype in Human Colorectal Cancer by Modulating Ca2+ Homeostasis

The Ca(2+)-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 Ca(2+) 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.

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.

Oxidative metabolism drives inflammation-induced platinum resistance in human ovarian cancer

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.

Translational control in the stress adaptive response of cancer cells: a novel role for the heat shock protein TRAP1

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 eIF2α pathway either under basal conditions or under stress, favoring the activation of GCN2 and PERK kinases, with consequent phosphorylation of eIF2α 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 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 tumors switch to aerobic glycolysis through the inhibition of succinate dehydrogenase, the complex II of the mitochondrial respiratory chain; ii) is part of a pro-survival signaling pathway aimed at evading the toxic effects of oxidants and anticancer drugs and protects mitochondria against damaging stimuli via a decrease of ROS generation; iii) controls protein homeostasis through a direct involvement in the regulation of protein synthesis and protein co-translational degradation. Therefore, TRAP1 seems to be a central regulatory protein with balancing functions at the intersection of different metabolic processes during the neoplastic transformation. For this reason, it can be considered at the same time an attractive target for the development of novel anticancer strategies and a promising study model to understand the biology of tumor cells at a systemic level. This review summarizes the most recent advances in TRAP1 biology and proposes a new comprehensive view of its functions.

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

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

Crucial role of androgen receptor in vascular H2S biosynthesis induced by testosterone

Hydrogen sulphide (H2S) is a gaseous mediator strongly involved in cardiovascular homeostasis, where it provokes vasodilatation. Having previously shown that H2S contributes to testosterone‐induced vasorelaxation, here we aim to uncover the mechanisms underlying this effect.