The molecular chaperone TRAP1 in cancer: From the basics of biology to pharmacological targeting.

TRAP1, the mitochondrial component of the Hsp90 family of molecular chaperones, displays important bioenergetic and proteostatic functions. In tumor cells, TRAP1 contributes to shape metabolism, dynamically tuning it with the changing environmental conditions, and to shield from noxious insults. Hence, TRAP1 activity has profound effects on the capability of neoplastic cells to evolve towards more malignant phenotypes. Here, we discuss our knowledge on the biochemical functions of TRAP1 in the context of a growing tumor mass, and we analyze the possibility of targeting its chaperone functions for developing novel anti-neoplastic approaches.

Hexokinase 2 in Cancer: A Prima Donna Playing Multiple Characters.

Hexokinases are a family of ubiquitous exose-phosphorylating enzymes that prime glucose for intracellular utilization. Hexokinase 2 (HK2) is the most active isozyme of the family, mainly expressed in insulin-sensitive tissues. HK2 induction in most neoplastic cells contributes to their metabolic rewiring towards aerobic glycolysis, and its genetic ablation inhibits malignant growth in mouse models. HK2 can dock to mitochondria, where it performs additional functions in autophagy regulation and cell death inhibition that are independent of its enzymatic activity. The recent definition of HK2 localization to contact points between mitochondria and endoplasmic reticulum called Mitochondria Associated Membranes (MAMs) has unveiled a novel HK2 role in regulating intracellular Ca2+ fluxes. Here, we propose that HK2 localization in MAMs of tumor cells is key in sustaining neoplastic progression, as it acts as an intersection node between metabolic and survival pathways. Disrupting these functions by targeting HK2 subcellular localization can constitute a promising anti-tumor strategy.

Modulation and Pharmacology of the Mitochondrial Permeability Transition: A Journey from F-ATP Synthase to ANT.

The permeability transition (PT) is an increased permeation of the inner mitochondrial membrane due to the opening of the PT pore (PTP), a Ca2+-activated high conductance channel involved in Ca2+ homeostasis and cell death. Alterations of the PTP have been associated with many pathological conditions and its targeting represents an incessant challenge in the field. Although the modulation of the PTP has been extensively explored, the lack of a clear picture of its molecular nature increases the degree of complexity for any target-based approach. Recent advances suggest the existence of at least two mitochondrial permeability pathways mediated by the F-ATP synthase and the ANT, although the exact molecular mechanism leading to channel formation remains elusive for both. A full comprehension of this to-pore conversion will help to assist in drug design and to develop pharmacological treatments for a fine-tuned PT regulation. Here, we will focus on regulatory mechanisms that impinge on the PTP and discuss the relevant literature of PTP targeting compounds with particular attention to F-ATP synthase and ANT.

STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia.

STAT3 and HIF1α are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. In mouse embryonic stem cells (ESCs) we manage to determine the specific subset of hypoxia-induced genes that need STAT3 to be properly transcribed and, among them, fundamental genes like Vegfa, Hk1, Hk2, Pfkp and Hilpda are worth mentioning. Unexpectedly, we also demonstrated that the absence of STAT3 does not affect the expression of Hif1α mRNA nor the stabilization of HIF1α protein, but the STAT3-driven regulation of the hypoxia-dependent subset of gene could rely on the physical interaction between STAT3 and HIF1α. To further elucidate the physiological roles of this STAT3 non-canonical nuclear activity, we used a CRISPR/Cas9 zebrafish stat3 knock-out line. Notably, hypoxia-related fluorescence of the hypoxia zebrafish reporter line (HRE:mCherry) cannot be induced when Stat3 is not active and, while Stat3 Y705 phosphorylation seems to have a pivotal role in this process, S727 does not affect the Stat3-dependent hypoxia response. Hypoxia is fundamental for vascularization, angiogenesis and immune cells mobilization; all processes that, surprisingly, cannot be induced by low oxygen levels when Stat3 is genetically ablated. All in all, here we report the specific STAT3/HIF1α-dependent subset of genes in vitro and, for the first time with an in vivo model, we determined some of the physiological roles of STAT3-hypoxia crosstalk.

HIF1α-dependent induction of the mitochondrial chaperone TRAP1 regulates bioenergetic adaptations to hypoxia.

The mitochondrial paralog of the Hsp90 chaperone family TRAP1 is often induced in tumors, but the mechanisms controlling its expression, as well as its physiological functions remain poorly understood. Here, we find that TRAP1 is highly expressed in the early stages of Zebrafish development, and its ablation delays embryogenesis while increasing mitochondrial respiration of fish larvae. TRAP1 expression is enhanced by hypoxic conditions both in developing embryos and in cancer models of Zebrafish and mammals. The TRAP1 promoter contains evolutionary conserved hypoxic responsive elements, and HIF1α stabilization increases TRAP1 levels. TRAP1 inhibition by selective compounds or by genetic knock-out maintains a high level of respiration in Zebrafish embryos after exposure to hypoxia. Our data identify TRAP1 as a primary regulator of mitochondrial bioenergetics in highly proliferating cells following reduction in oxygen tension and HIF1α stabilization.

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.

Rational Design of Allosteric and Selective Inhibitors of the Molecular Chaperone TRAP1.

TRAP1 is the mitochondrial paralog of the heat shock protein 90 (HSP90) chaperone family. Its activity as an energy metabolism regulator has important implications in cancer, neurodegeneration, and ischemia. Selective inhibitors of TRAP1 could inform on its mechanisms of action and set the stage for targeted drug development, but their identification was hampered by the similarity among active sites in HSP90 homologs. We use a dynamics-based approach to identify a TRAP1 allosteric pocket distal to its active site that can host drug-like molecules, and we select small molecules with optimal stereochemical features to target the pocket. These leads inhibit TRAP1, but not HSP90, ATPase activity and revert TRAP1-dependent downregulation of succinate dehydrogenase activity in cancer cells and in zebrafish larvae. TRAP1 inhibitors are not toxic per se, but they abolish tumorigenic growth of neoplastic cells. Our results indicate that exploiting conformational dynamics can expand the chemical space of chaperone antagonists to TRAP1-specific inhibitors with wide therapeutic opportunities.

The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria.

Mitochondrial DNA depletion syndromes (MDS) are a group of rare autosomal recessive disorders with early onset and no cure available. MDS are caused by mutations in nuclear genes involved in mitochondrial DNA (mtDNA) maintenance, and characterized by both a strong reduction in mtDNA content and severe mitochondrial defects in affected tissues. Mutations in MPV17, a nuclear gene encoding a mitochondrial inner membrane protein, have been associated with hepatocerebral forms of MDS. The zebrafish mpv17 null mutant lacks the guanine-based reflective skin cells named iridophores and represents a promising model to clarify the role of Mpv17. In this study, we characterized the mitochondrial phenotype of mpv17-/- larvae and found early and severe ultrastructural alterations in liver mitochondria, as well as significant impairment of the respiratory chain, leading to activation of the mitochondrial quality control. Our results provide evidence for zebrafish Mpv17 being essential for maintaining mitochondrial structure and functionality, while its effects on mtDNA copy number seem to be subordinate. Considering that a role in nucleotide availability had already been postulated for MPV17, that embryos blocked in pyrimidine synthesis do phenocopy mpv17-/- knockouts (KOs) and that mpv17-/- KOs have impaired Dihydroorotate dehydrogenase activity, we provided mpv17 mutants with the pyrimidine precursor orotic acid (OA). Treatment with OA, an easily available food supplement, significantly increased both iridophore number and mtDNA content in mpv17-/- mutants, thus linking the loss of Mpv17 to pyrimidine de novo synthesis and opening a new simple therapeutic approach for MPV17-related MDS.