Undifferentiated cells and embryos express high levels of endogenous non-telomerase reverse transcriptase (RT) of retroposon/retroviral origin. We previously found that RT inhibitors modulate cell growth and differentiation in several cell lines. We have now sought to establish whether high levels of RT activity are directly linked to cell transformation. To address this possibility, we have employed two different approaches to inhibit RT activity in melanoma and prostate carcinoma cell lines: pharmacological inhibition by two characterized RT inhibitors, nevirapine and efavirenz, and downregulation of expression of RT-encoding LINE-1 elements by RNA interference (RNAi). Both treatments reduced proliferation, induced morphological differentiation and reprogrammed gene expression. These features are reversible upon discontinuation of the anti-RT treatment, suggesting that RT contributes to an epigenetic level of control. Most importantly, inhibition of RT activity in vivo antagonized tumor growth in animal experiments. Moreover, pretreatment with RT inhibitors attenuated the tumorigenic phenotype of prostate carcinoma cells inoculated in nude mice. Based on these data, the endogenous RT can be regarded as an epigenetic regulator of cell differentiation and proliferation and may represent a novel target in cancer therapy.

S100A13, a member of the S100 gene family of Ca 2؉ -binding proteins has been previously characterized as a component of a brain-derived heparin-binding multiprotein aggregate/complex containing fibroblast growth factor 1 (FGF1). We report that while expression of S100A13 in NIH 3T3 cells results in the constitutive release of S100A13 into the extracellular compartment at 37°C, co-expression of S100A13 with FGF1 represses the constitutive release of S100A13 and enables NIH 3T3 cells to release S100A13 in response to temperature stress. S100A13 release in response to stress occurs with kinetics similar to that observed for the stress-induced release of FGF1, but S100A13 expression is able to reverse the sensitivity of FGF1 release to inhibitors of transcription and translation. The release of FGF1 and S100A13 in response to heat shock results in the solubility of FGF1 at 100% (w/v) ammonium sulfate saturation, and the expression of a S100A13 deletion mutant lacking its novel basic residue-rich domain acts as a dominant negative effector of FGF1 release in vitro. Surprisingly, the expression of S100A13 also results in the stress-induced release of a Cys-free FGF1 mutant, which is normally not released from NIH 3T3 cells in response to heat shock. These data suggest that S100A13 may be a component of the pathway for the release of the signal peptide-less polypeptide, FGF1, and may involve a role for S100A13 in the formation of a noncovalent FGF1 homodimer. FGF11 and FGF2 are the prototype members of a large family of heparin-binding growth factor genes that regulate numerous biological processes such as neurogenesis, mesoderm formation, and angiogenesis (1, 2). FGF1 and FGF2 lack a classical signal peptide sequence that provides access to the conventional endoplasmic reticulum (ER)-Golgi secretion pathway, a characteristic that led to the hypothesis that the release of these polypeptides may proceed through novel release/export pathways (2). Our laboratory previously demonstrated that FGF1, but not FGF2, is released as a latent homodimer by a transcription-and translation-dependent mechanism in response to a variety of cellular stresses including heat shock (3), hypoxia (4), and serum starvation (5). Conversely, the disruption of communication between the ER and Golgi apparatus by brefeldin A does not prevent the release of FGF1 from NIH 3T3 cells, confirming that FGF1 release may occur through a nonconventional pathway (6).FGF1 is released in vitro as a reducing agent-and denaturant-sensitive complex, which contains the p40 extravesicular domain of the Ca 2ϩ -binding protein, p65 synaptotagmin (Syt)1 (7). The release of FGF1 in response to stress is dependent on Syt1 expression, since the expression of either a deletion mutant lacking 95 amino acids from the extravesicular portion of Syt1 or an antisense-Syt1 gene is able to repress FGF1 release in NIH 3T3 cells (7,8). In addition, FGF1 purified from ovine brain as a high molecular weight aggregate exists as a component of a noncovalent heparin-binding complex wit...

SummaryWe report that the mitochondrial chaperone TRAP1, which is induced in most tumor types, is required for neoplastic growth and confers transforming potential to noncancerous cells. TRAP1 binds to and inhibits succinate dehydrogenase (SDH), the complex II of the respiratory chain. The respiratory downregulation elicited by TRAP1 interaction with SDH promotes tumorigenesis by priming the succinate-dependent stabilization of the proneoplastic transcription factor HIF1α independently of hypoxic conditions. These findings provide a mechanistic clue to explain the switch to aerobic glycolysis of tumors and identify TRAP1 as a promising antineoplastic target.

Non-classical protein release independent of the ER-Golgi pathway has been reported for an increasing number of proteins lacking an N-terminal signal sequence. The export of FGF1 and IL-1α, two pro-angiogenic polypeptides, provides two such examples. In both cases, export is based on the Cu 2+ -dependent formation of multiprotein complexes containing the S100A13 protein and might involve translocation of the protein across the membrane as a 'molten globule'. FGF1 and IL-1α are involved in pathological processes such as restenosis and tumor formation. Inhibition of their export by Cu 2+ chelators is thus an effective strategy for treatment of several diseases.

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.

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 Ca 2+ -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.

A growing number of proteins devoid of signal peptides have been demonstrated to be released through the non-classical pathways independent of endoplasmic reticulum and Golgi. Among them are two potent proangiogenic cytokines FGF1 and IL1α. Stress-induced transmembrane translocation of these proteins requires the assembly of copper-dependent multiprotein release complexes. It involves the interaction of exported proteins with the acidic phospholipids of the inner leaflet of the cell membrane and membrane destabilization. Not only stress, but also thrombin treatment and inhibition of Notch signaling stimulate the export of FGF1. Non-classical release of FGF1 and IL1α presents a promising target for treatment of cardiovascular, oncologic, and inflammatory disorders. Keywords non-classical secretion; FGF1; IL1α VARIETY OF NON-CLASSICALLY RELEASED PROTEINSThe familiar textbook scheme of protein secretion starts with the cotranslational protein translocation into the endoplasmic reticulum (ER). This translocation requires a molecular exit visa, a hydrophobic signal peptide located usually at the N-terminus of a secretable protein [Blobel, 1995]. After the protein is translocated through the ER membrane, its folding, transport, sorting, and covalent modifications occur in the ER and Golgi. Finally, the protein is released from the cell as a result of the fusion of an exocytotic vesicle with the cell membrane.

Fibroblast growth factor (FGF) 1 is known to be released in response to stress conditions as a component of a multiprotein aggregate containing the p40 extravescicular domain of p65 synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a Cu 2؉ -binding protein and Cu 2؉is known to induce its dimerization, we evaluated the capacity of recombinant FGF1, p40 Syt1, and S100A13 to interact in a cell-free system and the role of Cu 2؉ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu 2؉ with similar affinity and to interact in the presence of Cu 2؉ to form a multiprotein aggregate which is resistant to low concentrations of SDS and sensitive to reducing conditions and ultracentrifugation. The formation of this aggregate in the presence of Cu 2؉ is dependent on the presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1. Interestingly, S100A13 is also able to interact in the presence of Cu 2؉with Cys-free FGF1 and this observation may account for the ability of S100A13 to export Cys-free FGF1 in response to stress. Lastly, tetrathiomolybdate, a Cu 2؉ chelator, significantly represses in a dose-dependent manner the heat shock-induced release of FGF1 and S100A13. These data suggest that S100A13 may be involved in the assembly of the multiprotein aggregate required for the release of FGF1 and that Cu 2؉ oxidation may be an essential post-translational intracellular modifier of this process. FGF11 and FGF2, the prototype members of a family of heparin-binding growth factors involved in the regulation of neurogenesis, mesoderm formation, and angiogenesis (1, 2), function as extracellular mitogens for diverse populations of target cells, yet they both lack the presence of a classical signal peptide sequence that enables their secretion through the endoplasmic reticulum-Golgi apparatus (1, 2). FGF1, but not FGF2 (3), is released from NIH 3T3 cells in response to heat shock (4), hypoxia (5), and serum starvation (6) in a transcription-and translation-dependent manner, as a biologically inactive homodimer with decreased affinity for heparin (4). Recent studies have suggested that FGF1 may be released as a multiprotein aggregate containing FGF1 and the p40 extravesicular domain of p65 synaptotagmin (Syt)1 (7, 8). These studies are consistent with the observation that FGF1 is present in neuronal tissue as a component of a non-covalent aggregate containing p40 Syt1 and S100A13 (9). It is also known that (i) both p40 Syt1 and S100A13 are constitutively released at 37°C from NIH 3T3 (8, 10), (ii) the expression of FGF1 is able to repress the constitutive release of S100A13 but not p40 Syt1 at 37°C (8, 10), and (iii) FGF1 and S100A13 NIH 3T3 cell cotransfectants are able to release FGF1 and S100A13 in response to temperature stress in a form which alters the solubility of FGF1 at 100% (w/v) ammonium sulfate (10). Moreover, S100A13 appears to play a role in the regulation of FGF1 release since (i) a deletion mutant of S100A13, lacking the carboxyl...