TRAP1 Expression Research Articles

The METTL3/TRAP1 axis as a key regulator of 5-fluorouracil chemosensitivity in colorectal cancer.

Colorectal cancer (CRC) remains a significant clinical challenge, with 5-Fluorouracil (5-FU) being the frontline chemotherapy. However, chemoresistance remains a major obstacle to effective treatment. METTL3, a key methyltransferase involved in RNA methylation processes, has been implicated in CRC carcinogenesis. However, its role in modulating CRC sensitivity to 5-FU remains elusive. In this study, we aimed to investigate the role and mechanisms of METTL3 in regulating 5-FU chemosensitivity in CRC cells. Initially, we observed that 5-FU treatment inhibited cell viability and induced apoptosis, accompanied by a reduction in METTL3 expression in HCT-116 and HCT-8 cells. Subsequent assays including drug sensitivity, EdU, colony formation, TUNEL staining, and flow cytometry revealed that METTL3 depletion enhanced 5-FU sensitivity and increased apoptosis induction both in vitro and in vivo. Conversely, METTL3 overexpression conferred resistance to 5-FU in both cell lines. Moreover, knockdown of METTL3 in 5-FU-resistant CRC cell lines HCT-116/FU and HCT-15/FU significantly decreased 5-FU tolerance and induced apoptosis upon 5-FU treatment. Mechanistically, we found that METTL3 regulated 5-FU sensitivity and apoptosis induction by modulating TRAP1 expression. Further investigations using m6A colorimetric ELISA, dot blot, MeRIP-qPCR and RNA stability assays demonstrated that METTL3 regulated TRAP1 mRNA stability in an m6A-dependent manner. Additionally, overexpression of TRAP1 mitigated the cytotoxic effects of 5-FU on CRC cells. In summary, our study uncovers the pivotal role of the METTL3/TRAP1 axis in modulating 5-FU chemosensitivity in CRC. These findings provide new insights into the mechanisms underlying CRC resistance to 5-FU and may offer potential targets for future therapeutic interventions.

Mitochondrial chaperon TNF-receptor- associated protein 1 as a novel apoptotic regulator conferring susceptibility to Pneumocystis jirovecii pneumonia.

Molecular chaperons stabilize protein folding and play a vital role in maintaining tissue homeostasis. To this intent, mitochondrial molecular chaperons may be involved in the regulation of oxidative phosphorylation and apoptosis during stress events such as infections. However, specific human infectious diseases relatable to defects in molecular chaperons have yet to be identified. To this end, we performed whole exome sequencing and functional immune assessment in a previously healthy Asian female, who experienced severe respiratory failure due to Pneumocystis jiroveci pneumonia and non-HIV-related CD4 lymphocytopenia. This revealed that a chaperon, the mitochondrial paralog of HSP90, TRAP1, may have been involved in the patient's susceptibility to an opportunistic infection. Two rare heterozygous variants in TRAP1, E93Q, and A64T were detected. The patient's peripheral blood mononuclear cells displayed diminished TRAP1 expression, but had increased active, cleaved caspase-3, caspase-7, and elevated IL-1β production. Transfection of A64T and E93Q variants in cell lines yielded decreased TRAP1 compared to transfected wildtype TRAP1 and re-capitulated the immunotypic phenotype of enhanced caspase-3 and caspase-7 activity. When infected with live P. jiroveci, the E93Q or A64T TRAP1 mutant expressing cells also exhibited reduced viability. Patient cells and cell lines transfected with the TRAP1 E93Q/A64T mutants had impaired respiration, glycolysis, and increased ROS production. Of note, co-expression of E93Q/A64T double mutants caused more functional aberration than either mutant singly. Taken together, our study uncovered a previously unrecognized role of TRAP1 in CD4+ lymphocytopenia, conferring susceptibility to opportunistic infections.

The Role of HSP90 and TRAP1 Targets on Treatment in Hepatocellular Carcinoma.

Hepatocellular Carcinoma (HCC) is the predominant form of liver cancer and arises due to dysregulation of the cell cycle control machinery. Heat Shock Protein 90 (HSP90) and mitochondrial HSP90, also referred to as TRAP1 are important critical chaperone target receptors for early diagnosis and targeting HCC. Both HSP90 and TRAP1 expression was found to be higher in HCC patients. Hence, the importance of HSP90 and TRAP1 inhibitors mechanism and mitochondrial targeted delivery of those inhibitors function is widely studied. This review also focuses on importance of protein-protein interactions of HSP90 and TRAP1 targets and association of its interacting proteins in various pathways of HCC. To further elucidate the mechanism, systems biology approaches and computational biology approach studies are well explored in the association of inhibition of herbal plant molecules with HSP90 and its mitochondrial type in HCC.

Gene Expression and Drug Sensitivity Analysis of Mitochondrial Chaperones Reveals That HSPD1 and TRAP1 Expression Correlates with Sensitivity to Inhibitors of DNA Replication and Mitosis.

Mitochondria-critical metabolic hubs in eukaryotic cells-are involved in a wide range of cellular functions, including differentiation, proliferation, and death. Mitochondria import most of their proteins from the cytosol in a linear form, after which they are folded by mitochondrial chaperones. However, despite extensive research, the extent to which the function of particular chaperones is essential for maintaining specific mitochondrial and cellular functions remains unknown. In particular, it is not known whether mitochondrial chaperones influence the sensitivity to drugs used in the treatment of cancers. By mining gene expression and drug sensitivity data for cancer cell lines from publicly available databases, we identified mitochondrial chaperones whose expression is associated with sensitivity to oncology drugs targeting particular cellular pathways in a cancer-type-dependent manner. Importantly, we found the expression of TRAP1 and HSPD1 to be associated with sensitivity to inhibitors of DNA replication and mitosis. We confirmed experimentally that the expression of HSPD1 is associated with an increased sensitivity of ovarian cancer cells to drugs targeting mitosis and a reduced sensitivity to drugs promoting apoptosis. Taken together, our results support a model in which particular mitochondrial pathways hinge upon specific mitochondrial chaperones and provide the basis for understanding selectivity in mitochondrial chaperone-substrate specificity.

Abstract 273: Synergistically suppressive effects by combination of glycolytic inhibitor with TRAP1-depletion in colorectal cancer

Abstract Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial homolog of HSP90 chaperones. It plays an important role in protection against oxidative stress and apoptosis by regulating reactive oxidative species (ROS). Recent studies have suggested TRAP1 as a regulator of metabolism and mitochondrial homeostasis in diverse pathological states, including cancer. To further elucidate the mechanistic role of TRAP1 in regulating tumor cell survival, we used gamitrinib-triphenylphosphonium (G-TPP) to inhibit TRAP1 signaling pathways in colon cancer. Inhibition of TRAP1 disrupted redox homeostasis and increased ER stress in colon cancer. To confirm the G-TPP effects of TRAP1 inhibition in colon cancer, we performed TRAP1 CRISPR/Cas9 to deplete TRAP1 expression in colon cancer cell line CT26. TRAP1-depleted cells were found to acquire several distinct phenotypes: induction of reactive oxidative species (ROS) level, reduction of cell growth, and lower pH value of culture medium. In addition, proteomic data showed that depletion of TRAP1 induced significant metabolism alteration in CT26 cells. Using seahorse assay to access mitochondrial respiration and glycolysis, we found that TRAP1-depleted cells were insensitive to Oxidative Phosphorylation (OXPHOS) inhibitor treatment and significantly increased extracellular acidification rate (ECAR) compared to parental cells. Furthermore, higher glucose consumption rate and higher lactic acid level of culture medium were observed in TRAP1-depleted cells. On the other hand, Glycolytic inhibitor (2DG) led to excessive ROS production, reduction of glucose uptake and exacerbated cell death in TRAP1-depleted cell compared to parental cells. Taken together, depletion of TRAP1 induces mitochondrial dysfunction and modulates metabolic switch toward aerobic glycolysis in TRAP1-depleted colon cancer cells. Dual inhibition of TRAP1 and glycolytic signaling may increase therapeutic efficiency through induction of excessive ROS production in colon cancer. Citation Format: Hong-Yuan Tsai, Miao-Hsueh Chen, Herath Lakmini Senavirathna, Jihye Yun, Sheng Pan, Ru Chen. Synergistically suppressive effects by combination of glycolytic inhibitor with TRAP1-depletion in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 273.

The Mitochondrial HSP90 Paralog TRAP1: Structural Dynamics, Interactome, Role in Metabolic Regulation, and Inhibitors

The HSP90 paralog TRAP1 was discovered more than 20 years ago; yet, a detailed understanding of the function of this mitochondrial molecular chaperone remains elusive. The dispensable nature of TRAP1 in vitro and in vivo further complicates an understanding of its role in mitochondrial biology. TRAP1 is more homologous to the bacterial HSP90, HtpG, than to eukaryotic HSP90. Lacking co-chaperones, the unique structural features of TRAP1 likely regulate its temperature-sensitive ATPase activity and shed light on the alternative mechanisms driving the chaperone’s nucleotide-dependent cycle in a defined environment whose physiological temperature approaches 50 °C. TRAP1 appears to be an important bioregulator of mitochondrial respiration, mediating the balance between oxidative phosphorylation and glycolysis, while at the same time promoting mitochondrial homeostasis and displaying cytoprotective activity. Inactivation/loss of TRAP1 has been observed in several neurodegenerative diseases while TRAP1 expression is reported to be elevated in multiple cancers and, as with HSP90, evidence of addiction to TRAP1 has been observed. In this review, we summarize what is currently known about this unique HSP90 paralog and why a better understanding of TRAP1 structure, function, and regulation is likely to enhance our understanding of the mechanistic basis of mitochondrial homeostasis.

Aberrant HSP90 Expression in Lymphocytes and HSP90 Response to Anti-PD-1 Therapy in Lymphoma Patients.

HSP90 family of molecular chaperones has been shown to be implicated in various stages of tumor growth and development. Recent studies have highlighted the role of extracellular HSP90 in tumor immunology, however, the role that HSP90 plays in the regulation of immune responses and the impact of cancer immunotherapy, including immune checkpoint blockade, on HSP90 is still unclear. Here we assessed the surface and intracellular expression of constitutive cytosolic HSP90β isoform, mitochondrial HSP90 homolog TRAP1 and co-chaperone STIP1/HOP in T, NK, B and NKT cells derived from peripheral blood and bone marrow samples of patients with Hodgkin and B-cell Non-Hodgkin lymphomas. HSP90β and STIP1 were overexpressed in B lymphocytes, while TRAP1 expression was decreased in T, B, NK and NKT cells of lymphoma patients. HSP90 overexpression in B cells was not associated with malignant B cell clones, since no clonotypic B cells were detected by immunoglobulin heavy chain (IgH) gene rearrangements. PD-1 blockade was found to differently affect the intracellular and surface HSP90 in T, B, NK and NKT cells in patients with relapsed or refractory classical Hodgkin lymphoma. Modulating HSP90 was found to affect the NK cell degranulation response and IFNγ production in lymphoma patients. These findings provide the rationale to further explore HSP90 homologs for improving patient response to cancer immunotherapy.

Enhanced Expression of TRAP1 Protects Mitochondrial Function in Motor Neurons under Conditions of Oxidative Stress.

TNF-receptor associated protein (TRAP1) is a cytoprotective mitochondrial-specific member of the Hsp90 heat shock protein family of protein chaperones that has been shown to antagonise mitochondrial apoptosis and oxidative stress, regulate the mitochondrial permeability transition pore and control protein folding in mitochondria. Here we show that overexpression of TRAP1 protects motor neurons from mitochondrial dysfunction and death induced by exposure to oxidative stress conditions modelling amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease in which motor neurons degenerate, leading to muscle weakness and atrophy and death, typically within 3 years of diagnosis. In primary murine motor neurons, shRNA-mediated knockdown of TRAP1 expression results in mitochondrial dysfunction but does not further exacerbate damage induced by oxidative stress alone. Together, these results show that TRAP1 may be a potential therapeutic target for neurodegenerative diseases such as ALS, where mitochondrial dysfunction has been shown to be an early marker of pathogenesis.

Markers to sensibility and relapse on IMR-32 neuroblastoma cell line cultured in monolayer (2D) and neurosphere (3D) models cisplatin-treated

The complexity of different components of tumor stroma poses huge challenges for therapies targeting the neuroblastoma (NB) microenvironment. The present study aimed to evaluate platinum-based response in IMR-32 neuroblastoma cell line cultured in monolayer (2D) and neurosphere (3D) models. For this, we evaluated mRNA expression of heat shock proteins HSPA1A, HSPB1, TRAP1, HSPA1AL, HSPD1, and DNA damage repair gene ERCC1. After treatment, residual cells were grafted on CAM (chicken chorioallantoic membrane) to evaluate the growth capability and histological paraffin sections were made to assess Ki-67 and HER-2 proteins by immunofluorescence. Our results showed that cisplatin induces mRNA downregulation of Heat Shock Proteins and ERCC1 in IMR-32 cells cultured in 2D or 3D models. In addition, the cisplatin-treatment approach increased HER-2 expression in residual IMR-32 cells grafted on the CAM. Therefore, these insights provide many advances in neuroendocrine tumor biology and knowledge about cisplatin-response in neuroblastoma.

Arhgap21 Deficiency Results in Increase of Osteoblastic Lineage Cells in the Murine Bone Marrow Microenvironment.

ARHGAP21 is a member of the RhoGAP family of proteins involved in cell growth, differentiation, and adhesion. We have previously shown that the heterozygous Arhgap21 knockout mouse model (Arhgap21+/−) presents several alterations in the hematopoietic compartment, including increased frequency of hematopoietic stem and progenitor cells (HSPC) with impaired adhesion in vitro, increased mobilization to peripheral blood, and decreased engraftment after bone marrow transplantation. Although these HSPC functions strongly depend on their interactions with the components of the bone marrow (BM) niche, the role of ARHGAP21 in the marrow microenvironment has not yet been explored. In this study, we investigated the composition and function of the BM microenvironment in Arhgap21+/− mice. The BM of Arhgap21+/− mice presented a significant increase in the frequency of phenotypic osteoblastic lineage cells, with no differences in the frequencies of multipotent stromal cells or endothelial cells when compared to the BM of wild type mice. Arhgap21+/− BM cells had increased capacity of generating osteogenic colony-forming units (CFU-OB) in vitro and higher levels of osteocalcin were detected in the Arhgap21+/− BM supernatant. Increased expression of Col1a1, Ocn and decreased expression of Trap1 were observed after osteogenic differentiation of Arhgap21+/− BM cells. In addition, Arhgap21+/− mice recipients of normal BM cells showed decreased leucocyte numbers during transplantation recovery. Our data suggest participation of ARHGAP21 in the balanced composition of the BM microenvironment through the regulation of osteogenic differentiation.

Upregulation of HSPB1 heat shock gene and ERCC1 gene on serous ovarian cancer cell line in HIPEC in vitro model.

e17559 Background: Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with the presence of chemoresistance contributing to the poor prognosis. Approximately 80% of cases are diagnosed in stage III C and are treated with cytoreduction surgery followed by adjuvant chemotherapy. However, 70 percent of these patients have pelvic and peritoneal recurrences. Heat Shock Proteins are produced in response to pathophysiological stress and take part in several stages of carcinogenesis, acting primarily as anti-apoptotic agents. They are also implicated in resistance to chemotherapy in several types of tumors. In an attempt to improve oncological results, new therapeutic approaches such as intraperitoneal chemotherapy and HIPEC have been proposed in recent studies with gains in overall survival (OS). However, some questions have not yet been answered. Methods: in the study cultures of ovarian cancer cells were performed TOV-21G (clear cell carcinoma), SK-OV-3 (platinum-resistant serous carcinoma) and OV-90 (high-grade serous). Cell cytotoxicity (MTT) assay was performed. The ovarian cancer cells lines were treated with cisplatin in normothermia (37 degrees Celsius) and cisplatin in hyperthermia (41 degrees Celsius) and a control group treated with PBS saline solution at (37 degrees Celsius and 41 degrees Celsius) for 24 hours followed by new supplementation and a new 3-hours incubation. Clonogenic assay was performed. Then they were submitted to RNA extraction and reverse transcription. qRT-PCR was performed to compare the expression of TRAP1, HSPB1, HSPD1, HSPA1A, HSPA1L and ERCC1 in different treatments. Results: There was no statistical difference in relation to cytotoxicity between treatment with heated cisplatin compared to treatment with normothermia. It was not possible to evaluate the expression of the heat shock genes in the SK-OV3 lineage.The HSPB1, HSPD1, TRAP1 and ERCCC1 genes were positively regulated in OV-90 submitted to hyperthermia in relation to normothermia and there were no significant changes in expression in the TOV-21-G. Conclusions: In conclusion, we suggest that OV-90 Serous ovarian cancer cell line was more susceptibly at hyperthermia by cisplatin. The HSPA1A, HSPA1L, TRAP1 and HSPB1 heat shock genes and ERCC1 genes were upregulated in the heated cisplatin group and contribute to a poor prognosis related to resistance. The HSPB1 and ERCC1 genes had the greatest expression with 1000x higher.Thus, it is necessary to evaluate these genes in a clinical study of HIPEC.

HIF1\u03b1-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.

Deoxyribonuclease 1-Mediated Clearance of Circulating Chromatin Prevents From Immune Cell Activation and Pro-inflammatory Cytokine Production, a Phenomenon Amplified by Low Trap1 Activity: Consequences for Systemic Lupus Erythematosus.

Increased concentrations of circulating chromatin, especially oligo-nucleosomes, are observed in sepsis, cancer and some inflammatory autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, circulating nucleosomes mainly result from increased apoptosis and decreased clearance of apoptotic cells. Once released, nucleosomes behave both as an autoantigen and as a damage-associated molecular pattern (DAMP) by activating several immune cells, especially pro-inflammatory cells. Deoxyribonuclease 1 (DNase1) is a major serum nuclease whose activity is decreased in mouse and human lupus. Likewise, the mitochondrial chaperone tumor necrosis factor (TNF) receptor-associated protein-1 (Trap1) protects against oxidative stress, which is increased in SLE. Here, using wild type, DNase1-deficient and DNase1/Trap1-deficient mice, we demonstrate that DNase1 is a major serum nuclease involved in chromatin degradation, especially when the plasminogen system is activated. In vitro degradation assays show that chromatin digestion is strongly impaired in serum from DNase1/Trap1-deficient mice as compared to wild type mice. In vivo, after injection of purified chromatin, clearance of circulating chromatin is delayed in DNase1/Trap1-deficient mice in comparison to wild type mice. Since defective chromatin clearance may lead to chromatin deposition in tissues and subsequent immune cell activation, spleen cells were stimulated in vitro with chromatin. Splenocytes were activated by chromatin, as shown by interleukin (IL)-12 secretion and CD69 up-regulation. Moreover, cell activation was exacerbated when Trap1 is deficient. Importantly, we also show that cytokines involved in lupus pathogenesis down-regulate Trap1 expression in splenocytes. Therefore, combined low activities of both DNase1 and Trap1 lead to an impaired degradation of chromatin in vitro, delayed chromatin clearance in vivo and enhanced activation of immune cells. This situation may be encountered especially, but not exclusively, in SLE by the negative action of cytokines on Trap1 expression.

TRAP1 Regulates Wnt/β-Catenin Pathway through LRP5/6 Receptors Expression Modulation.

Wnt/β-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 60–70% of human colorectal carcinomas (CRCs) and favors stem cells maintenance, modulating the Wnt/β-Catenin pathway and preventing β-Catenin phosphorylation/degradation. The role of TRAP1 in the regulation of Wnt/β-Catenin signaling was further investigated in human CRC cell lines, patient-derived spheroids, and CRC specimens. TRAP1 relevance in the activation of Wnt/β-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 β-Catenin, LRP5 and LRP6. Altogether, these data suggest that Wnt/β-Catenin signaling is modulated at multiple levels by TRAP1.

Mitochondrial chaperone, TRAP1 modulates mitochondrial dynamics and promotes tumor metastasis

Mitochondria play a central role in regulating cellular energy metabolism. However, the present understanding of mitochondria has changed from its unipotent functions to pluripotent and insists on understanding the role of mitochondria not only in regulating the life and death of cells, but in pathological conditions such as cancer. Unlike other cellular organelles, subtle alterations in mitochondrial organization may significantly influence the balance between metabolic networks and cellular behavior. Therefore, the delicate balance between the fusion and fission dynamics of mitochondrion can indicate cell fate. Here, we present mitochondrial chaperone TRAP1 influence on mitochondrial architecture and its correlation with tumor growth and metastasis. We show that TRAP1 overexpression (TRAP1 OE) promotes mitochondrial fission, whereas, TRAP1 knockdown (TRAP1 KD) promotes mitochondrial fusion. Interestingly, TRAP1 OE or KD had a negligible effect on mitochondrial integrity. However, TRAP1 OE cells exhibited enhanced proliferative potential, while TRAP1 KD cells showing increased doubling time. Further, TRAP1 dependent mitochondrial dynamic alterations appeared to be unique since mitochondrial localization of TRAP1 is a mandate for dynamic changes. The expression patterns of fusion and fission genes have failed to correlate with TRAP1 expression, indicating a possibility that the dynamic changes can be independent of these genes. In agreement with enhanced proliferative potential, TRAP1 OE cells also exhibited enhanced migration in vitro and tumor metastasis in vivo. Further, TRAP1 OE cells showed altered homing properties, which may challenge site-specific anticancer treatments. Our findings unravel the TRAP1 role in tumor metastasis, which is in addition to altered energy metabolism.

Mitochondrial chaperone, TRAP1 as a potential pharmacological target to combat cancer metabolism

The stress response forms the most ancient defense system in living cells. Heat shock proteins (Hsps) are highly conserved across species and play major roles in mounting the stress response. The emerging information now suggests that Hsp90 family of chaperones display additional cellular roles contributing to diseases like cancer. For this reason, pharmacological targeting of Hsp90 has emerged as a novel antitumor strategy. However, its mitochondrial homologue TRAP1 has not been implicated in cancer with conclusive mechanistic insights. Since understanding the mutational spectrum of cancer cells indicates the outcome of the disease as well as treatment response, we examined mutational spectrum of TRAP1. Our in silico analyses of TRAP1 SNPs and CNVs correlated with the aggressive cancer phenotypes, and are found to be predominant over Hsp90 itself. The increased CNVs have been correlated with increased expression of TRAP1 in metastatic cancer cells, increased ATP production, and decreased oxygen consumption rate of mitochondria. Examining TRAP1 knockdown as well as over expression in metastatic cancer cells furthered our understanding that TRAP1 likely to facilitate the altered energy metabolism in the functional compromise of mitochondrial OXPHOS. Interestingly, the increased ATP levels in the TRAP1 background are found to be independent of glucose oxidation. Our results suggest TRAP1 role in triggering the alternate energy metabolism in cancer cells. Since targeting tumor metabolism is considered as an alternate strategy to combat cancer, we propose pharmacological targeting of TRAP1 to target alternate energy metabolism.

P11.15 Exosomal MicroRNA expression profiling altered by high TRAP1 expression in glioblastoma

Abstract BACKGROUND Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial molecular chaperone, which regulates the steady state transition between cellular aerobic glycolysis and oxidative phosphorylation. In contrast to normal brain, Glioblastoma multiforme (GBM) shows increased TRAP1 expression. However, the mechanisms responsible for metabolic switch mediated by TRAP1 have not been fully elucidated. Exosomes have been recently implicated in an emerging hallmark of cancer, the cancer cell metabolism. In this study, we studied whether the high expression of TRAP1 can affect the expression profile of miRNAs in the exosomes released by GBM, thus affecting the cell metabolism in the tumor microenvironment. MATERIAL AND METHODS We examined the expression profile of miRNAs in exosomes secreted by T98G GBM cell lines or T98G GBM cell lines depleted of TRAP1. The cancer-secreted exosomes were isolated by ultracentrifugation of the culture supernatant, and total RNAs, including miRNAs, were extracted from the isolated exosomes and subjected to miRNA microarray, and 3 miRNAs were further validated using qPCR. Target genes of differentially expressed miRNAs were the intersection predicted with 3 databases(Targetscan,microRNAorg,PITA). GO analysis and KEGG analysis were applied to determine the roles of these target genes. RESULTS Expression profiling of exosomal miRNA revealed significant downregulation of 237 miRNAs and upregulation of 134 miRNAs after deletion of TRAP1 in T98G GBM cell lines, while 3 of the upregulated miRNAs were further validated using qPCR. We found that these 3 miRNAs (hsa-miR-30e-5p,hsa-miR-1291,hsa-miR-300) showed a correlation with tumor cell glycolysis through literature searching and cross-searching of three microRNA databases. CONCLUSION We demonstrate high expression of TRAP1 can affect the expression profile of miRNAs in the exosomes released by GBM, that might reprogram the metabolic machinery following their uptake by other cells in the tumor microenvironment.

Down-regulation of STIP1 regulate apoptosis and invasion of glioma cells via TRAP1/AKT signaling pathway

BackgroundIn recent years, many studies have confirmed that STIP1 (phosphorylation-induced protein 1) is involved in the development and progression of various tumors. However, its potential role in glioma progression and the underlying mechanisms of glioma development remain unclear. MethodsWe analyzed the expression of STIP1 in 35 human glioma tissue specimens of different grades, using 6 normal brain tissues for comparison. We transfected U87 and U251 cell lines with small interfering RNA (siRNA) to downregulate STIP1, and set up a negative control group and a blank group for comparison. The MTT assay was used to detect cell proliferation, and cell cycle progression and apoptosis were analyzed through flow cytometry. Transwell experiments were employed to detect the invasion and migration of STIP1-depleted and control U87 and U251 cells and western blotting was used to detect the expression of TRAP1/Akt pathway proteins. In addition, immunohistochemical analysis was used to reveal differences in expression and localization between transplanted tumor specimens of each group. ResultsWe observed a high expression of STIP1 in glioblastoma, MTT assay revealed a decreased cell proliferation rate in the STIP1-downregulated cells. Cell cycle analysis revealed an increased proportion of cells in G1 phase, as well as an increase in apoptosis, upon STIP1 downregulation. Western blotting showed that TRAP1, pAkt, and MMP2 expression was decreased upon STIP1 downregulation. In addition, TRAP1, ki-67, and MMP2 displayed a decreased expression in vivo. ConclusionsSTIP1 is highly expressed in glioblastoma compared to normal brain tissues. Downregulation of STIP1 in glioma cells reduces cell proliferation rate and invasion and increases cell apoptosis.

PRC2 Inactivation Induces Resistance to Chemotherapy-Induced Apoptosis By Upregulating the TRAP1 Mitochondrial Chaperone in T-ALL

The tendency of mitochondria to undergo or resist BCL2-controlled apoptosis (so-called mitochondrial priming) is a powerful predictor of response to cytotoxic chemotherapy. Fully exploiting this finding will require unraveling the molecular genetics underlying phenotypic variability in mitochondrial priming.We analyzed pre-treatment T-ALL clinical specimens from a cohort of 47 patients (enriched for treatment failure, but with sufficient controls) treated on the COG AALL0434 or DFCI 05001 clinical trials using BH3 profiling analysis to assess mitochondrial apoptotic priming. We found that there was a strong association between resistance to mitochondrial apoptosis and a poor response to induction chemotherapy (P = 0.008). Furthermore, mitochondrial apoptosis resistance predicted significantly inferior event-free survival (65% vs. 91% at 5 years; P = 0.0376).To define the molecular determinants of this mitochondrial apoptosis resistance, we performed targeted exon sequencing and array CGH copy number analysis. This revealed that loss-of-function mutations in the polycomb repressive complex 2 (PRC2) core subunits (EZH2, EED or SUZ12) were associated with mitochondrial apoptosis resistance (P = 0.007) in clinical specimens. PRC2 is a chromatin modifying complex best known for its role in transcriptional repression, which functions as a tumor suppressor in T-ALL, but whether PRC2 regulates mitochondrial apoptosis is unknown.Using shRNA knockdown in human T-ALL cells, we found that depletion of PRC2 subunits in T-ALL cells induced mitochondrial apoptosis resistance, as assessed by BH3 profiling analysis (P < 0.001). PRC2 inactivation also induced resistance to chemotherapy-induced apoptosis (P < 0.0001), and increased T-ALL fitness following treatment with the antileukemic drug vincristine (P = 0.0001). Apoptosis resistance upon inactivation of EZH2 (a PRC2 catalytic subunit) was reversed by transduction of wild-type EZH2, but not by an EZH2 point mutant with impaired methyltransferase activity, indicating that this effect is mediated by the enzymatic activity of PRC2. In normal mouse thymocytes, heterozygous deletion of the PRC2 subunits Ezh2 or Eed was sufficient to induce apoptosis resistance in non-transformed double-negative T-cell progenitors (P < 0.010), indicating that apoptosis resistance can arise prior to oncogenic transformation.The best-known regulators of mitochondrial apoptosis are BCL2-family genes, but RNA-seq analysis of shRNA knockdown of the PRC2 subunits in a T-ALL cell line revealed that PRC2 did not regulate expression of any of the known BCL2 family members. Instead, PRC2 loss led to upregulation of TRAP1, a mitochondrially localized chaperone of the HSP90 family. TRAP1 upregulation was necessary for induction of apoptosis resistance following PRC2 inactivation, because shRNA knockdown of TRAP1 in the human CCRF-CEM cell line completely blocked induction of apoptosis resistance following PRC2 inactivation (P < 0.0001). Moreover, pharmacologic TRAP1 inhibition synergized with the antileukemic drugs dexamethasone and doxorubicin (combination index = 0.37 and 0.42, respectively).To define how PRC2 regulates TRAP1, we performed ChIP-seq analysis, which revealed that TRAP1 regulation by PRC2 is indirect. Combined ChIP-seq and RNA-seq analysis revealed a number of direct targets of PRC2, all of which were tested for their ability to upregulate TRAP1 and induce apoptosis resistance. This showed that the LIM domain transcription factor CRIP2 is a direct target of PRC2 that is necessary and sufficient for regulation of TRAP1, and for induction of apoptosis resistance downstream of PRC2 inactivation. To confirm the relevance of our findings, we used the EZH2 inhibitor GSK126 to inhibit enzymatic activity of PRC2, which revealed that EZH2 normally represses CRIP2 and TRAP1 expression in primary patient-derived xenografts. Finally, we found that increased TRAP1 expression correlates with treatment failure in T-ALL clinical specimens (P = 0.028).Taken together, our findings support a model in which loss of PRC2 induces transcriptional upregulation of its direct target CRIP2, which subsequently activates expression of TRAP1, leading to resistance to chemotherapy-induced mitochondrial apoptosis. DisclosuresAries:Pfizer: Employment. Teachey:Amgen: Consultancy; La Roche: Consultancy. Letai:AstraZeneca: Consultancy, Other: Lab research report; AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership.

Study of the mechanism underlying hsa-miR338-3p downregulation to promote fibrosis of the synovial tissue in osteoarthritis patients

Osteoarthritis (OA) is a degenerative joint disease characterized by the degradation of joint cartilage, the formation of osteophyma at joint margins, and synovial changes. Whereas lesions of the joint cartilage were the key point of the research and treatment of osteoarthritis before, a recent study showed that the synovium plays a crucial role in the pathological progress of OA. The inflammatory environment in the joints of OA patients always results in the overactivation of fibroblast-like synoviocytes (FLSs), which produce a multitude of inflammatory factors and media, not only leading to the degradation and injury of the cartilage tissue and promoting the development of osteoarthritis but also resulting in synovial fibrosis and joint stiffness. Therefore, the synovium has attracted increasing attention in the research of OA, and the study of the mechanism of activation of FLSs and the fibrosis of joint synovium may shed new light on OA treatment. By using high-throughput screening, we have identified that hsa-miR338-3p is significantly downregulated in the synovial tissue and joint effusion from OA patients. A functional study showed that overexpression of hsa-miR338-3p in the FLSs inhibited the TGF-β1-induced overactivation of the TGF-β/Smad fibrosis regulation pathway by suppressing TRAP-1 expression and thus reducing the TGF-β1-induced activation of the FLSs and the expression of vimentin and collagen I, two fibrosis markers. Meanwhile, a mechanism study also showed that the upregulation of hsa-miR338-3p reduced Smad2/3 phosphorylation by suppressing TRAP-1 and thus inhibited the TGF-β/Smad pathway and TIMP1, a downstream protein. The present study, for the first time, illustrates the role of hsa-miR338-3p in synovial fibrosis in OA patients and the related mechanism, which is of importance to the treatment of OA and its complications by targeting the FLSs and synovial tissue. Hsa-miR338-3p not only has the potential to be a target for the gene therapy of OA but also has the potential to be a new marker for the diagnosis of clinical progression in OA patients.