Franca Esposito

Adaptation to Oxidative Stress, Chemoresistance, and Cell Survival

The discovery of some additional properties and functions of reactive oxygen species (ROS), beyond their toxic effects, provides a novel scenario for the molecular basis and cell regulation of several pathophysiologic processes. ROS are generated by redox-sensitive, prosurvival signaling pathways and function as second messengers in the transduction of several extracellular signals. A complex intracellular redox buffering network has developed to adapt and protect cells against the dangerous effects of oxidative stress. However, pathways involved in ROS-adaptive response may also play a critical role in protecting cells against cytotoxic effects of anticancer agents, thus supporting the hypothesis of a correlation between adaptation/resistance to oxidative stress and resistance to anticancer drugs. This review summarizes the main systems involved in the adaptive responses: an overview on the pathophysiologic relevance of mitochondria on redox-sensitive transcription factors and genes and main antioxidant networks in tumor cells is provided. One of the major aims is to highlight the adaptive mechanisms and their interplay in the intricate connection between oncogenic signaling, oxidative stress, and chemoresistance. Clarification of these mechanisms has tremendous application potential, in terms of developing novel molecular-targeted anticancer therapies and innovative strategies for rational combination of these agents with chemotherapeutic or tumor-specific biologic drugs.

Tumor necrosis factor-associated protein 1 (TRAP-1) protects cells from oxidative stress and apoptosis.

TRAP-1 is a mitochondrial heat shock protein (HSP), recently identified in Saos-2 osteosarcoma cells adapted to mild oxidative stress induced by diethylmaleate (DEM). TRAP-1 mRNA expression is increased in DEM-adapted cells as well as in tumor cells resistant to 5-fluorouracil and to platin derivatives. Since a strong decrease of TRAP-1 protein levels, upon cisplatin treatment, is observed only in controls but not in the DEM-adapted counterpart, a possible role for this protein in the development of resistant phenotypes could be hypothesized. To characterize the protective role of TRAP-1 against oxidative stress and apoptosis, stable transfectants were generated and characterized for their response to different stress types. These stable clones expressing constitutively high TRAP-1 levels: (i) are more resistant to H2O2-induced DNA damage and to apoptosis by cisplatin; (ii) contain higher reduced glutathione (GSH) levels than control cells; and (iii) do not release the apoptosis-inducing factor into the nucleus upon cisplatin treatment. Furthermore, high TRAP-1 levels interfere with caspase 3 activation. These results confirm the anti-apoptotic role of TRAP-1, and suggest that increased expression of this mitochondrial HSP in DEM-adapted and chemoresistant cells could be part of a pro-survival signaling pathway aimed to evade toxic effects of oxidants and anticancer drugs.

TRAP1, a novel mitochondrial chaperone responsible for multi-drug resistance and protection from apoptotis in human colorectal carcinoma cells

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.

Mitochondrial chaperone Trap1 and the calcium binding protein Sorcin interact and protect cells against apoptosis induced by antiblastic agents.

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.

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.

High Mobility Group A1 (HMGA1) proteins interact with p53 and inhibit its apoptotic activity

HMGA gene overexpression and rearrangements are frequent in several tumours, but their oncogenic function is still unclear. Here we report of a physical and functional interaction between High Mobility Group A1 (HMGA1) protein and p53 oncosuppressor. We found that HMGA1 binds p53 in vitro and in vivo, and both proteins are present in the same complexes bound to the Bax gene promoter. HMGA1 interferes with the p53-mediated transcription of p53 effectors Bax and p21waf1 while cooperates with p53 in the transcriptional activation of the p53 inhibitor mdm2. This transcriptional modulation is associated with a reduced p53-dependent apoptosis in cells expressing exogenous HMGA1 and p53, or in cells expressing endogenously the proteins and in which p53 was activated by UV-irradiation. Furthermore, antisense inhibition of HMGA1b expression dramatically increases the UV-induced p53-mediated apoptosis. These data define a new physical and functional interaction between HMGA1 and p53 that modulates transcription of p53 target genes and inhibits apoptosis.

DNA binding activity of the glucocorticoid receptor is sensitive to redox changes in intact cells

The effect of changes of redox conditions on glucocorticoid receptor (GR) activity in intact cells has been studied using two approaches. One was to evaluate the GR-DNA binding in extracts of COS2 cells transiently overexpressing GR and in which reactive oxygen intermediates (ROI) accumulate as a consequence of glutathione (GSH) depletion. GR-DNA binding was significantly decreased in COS2 cells treated with diethylmaleate (DEM), which causes GSH depletion by forming GSH-DEM complexes. A similar effect was observed for Sp1, another Zn-finger transcription factor, whereas no difference was observed for the C/EBP transcription factor, which is known to be unaffected by redox changes in vitro. N-Acetylcysteine (NAC), which counteracts the effects of DEM by increasing GSH biosynthesis, prevents the decrease of GR-DNA binding in cells treated with DEM. The GR-DNA binding efficiency was similarly decreased using extracts from H2O2-treated COS2 cells and from COS2 cells treated with buthionine sulphoximine, which causes GSH depletion via a mechanism different from that of DEM. The other approach was to evaluate the efficiency of a GR-regulated promoter under different redox conditions. In HeLa cells, transfected with a plasmid containing the CAT gene under the control of the glucocorticoid responsive element (GRE) within the mouse mammary tumor virus promoter, and treated with dexamethasone to activate GR, exposure to DEM significantly impaired the activation of CAT gene expression induced by dexamethasone. Also in this case NAC treatment inhibited the effects of DEM.

Heat shock proteins, cell survival and drug resistance: The mitochondrial chaperone TRAP1, a potential novel target for ovarian cancer therapy

BACKGROUND Protein homeostasis is a highly complex network of molecular interactions governing the health and life span of the organism. Molecular chaperones, mainly heat shock proteins (HSP) and other stress-inducible proteins abundantly expressed in multiple compartments of the cell, are major modulators of protein homeostasis. TRAP1 is a mitochondrial HSP involved in protection against oxidant-induced DNA damage and apoptosis. It was recently described as a component of a mitochondrial pathway selectively up-regulated in tumor cells which antagonizes the proapoptotic activity of cyclophilin D, a mitochondrial permeability transition pore regulator, and is responsible for the maintenance of mitochondrial integrity, thus favoring cell survival. Interestingly, novel TRAP1 antagonists cause sudden collapse of mitochondrial function and selective tumor cell death, suggesting that this pathway may represent a novel molecular target to improve anticancer therapy. Preliminary data suggest that TRAP1 may be a valuable biomarker in ovarian cancers: in fact, TRAP1 levels are significantly higher in cisplatin-resistant ovarian tumors and ovarian carcinoma cell lines. CONCLUSIONS While major advances have been made in understanding the genetics and molecular biology of cancer, given the considerable heterogeneity of ovarian cancer, the introduction of novel targeted therapies and the consequent selection of treatments based on the molecular profile of each tumor may have a major impact on the management of this malignancy and might contribute to building a new era of personalized medicine.

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.