Naoto Miyajima

Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis.

Significance TNF receptor-associated protein (TRAP1) is found predominantly in mitochondria. A possible direct impact of TRAP1 on mitochondrial metabolism remains unexplored. We used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1 deficiency promotes increased mitochondrial respiration, fatty acid oxidation, tricarboxylic acid cycle intermediates, ATP and reactive oxygen species, while concomitantly suppressing glucose metabolism. TRAP1 deficiency also results in strikingly enhanced cell motility and invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor.

Tripartite Motif Protein 32 Facilitates Cell Growth and Migration via Degradation of Abl-Interactor 2

Tripartite motif protein 32 (TRIM32) mRNA has been reported to be highly expressed in human head and neck squamous cell carcinoma, but the involvement of TRIM32 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that TRIM32 binds to Abl-interactor 2 (Abi2), which is known as a tumor suppressor and a cell migration inhibitor, and we showed that TRIM32 mediates the ubiquitination of Abi2. Overexpression of TRIM32 promoted degradation of Abi2, resulting in enhancement of cell growth, transforming activity, and cell motility, whereas a dominant-negative mutant of TRIM32 lacking the RING domain inhibited the degradation of Abi2. In addition, we found that TRIM32 suppresses apoptosis induced by cis-diamminedichloroplatinum (II) in HEp2 cell lines. These findings suggest that TRIM32 is a novel oncogene that promotes tumor growth, metastasis, and resistance to anticancer drugs.

Asymmetric Hsp90 N Domain SUMOylation Recruits Aha1 and ATP-Competitive Inhibitors

The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90s function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs.

Peri-operative morbidity and mortality related to radical cystectomy: a multi-institutional retrospective study in Japan.

Study Type – Therapy (outcomes) Level of Evidence 2b Whats known on the subject? and What does the study add? Radical cystectomy remains associated with comparatively high perioperative morbidity and mortality, despite improvements in surgical techniques and perioperative care. At present, most studies on the complications associated with open radical cystectomy were derived from Western academic high‐volume centres, and data from Japan and other Asian countries were very limited. Using the modified Clavien grading system and 11 category grouping reported from MSKCC, we observed that 68% of patients experienced at least one complication within 90 days of surgery, and 17% of patients experienced major complications (90‐day mortality rate = 2%), which were compatible with reports from Western high‐volume centres. As far as we know, our report is the largest one regarding perioperative morbidity and mortality in Asian patients who underwent radical cystectomy.

TRIM24 mediates ligand-dependent activation of androgen receptor and is repressed by a bromodomain-containing protein, BRD7, in prostate cancer cells

The androgen receptor (AR) is a ligand-dependent transcription factor that belongs to the family of nuclear receptors, and its activity is regulated by numerous AR coregulators. AR plays an important role in prostate development and cancer. In this study, we found that TRIM24/transcriptional intermediary factor 1alpha (TIF1alpha), which is known as a ligand-dependent nuclear receptor co-regulator, interacts with AR and enhances transcriptional activity of AR by dihydrotestosterone in prostate cancer cells. We showed that TRIM24 functionally interacts with TIP60, which acts as a coactivator of AR and synergizes with TIP60 in the transactivation of AR. We also showed that TRIM24 binds to bromodomain containing 7 (BRD7), which can negatively regulate cell proliferation and growth. A luciferase assay indicated that BRD7 represses the AR transactivation activity upregulated by TRIM24. These findings indicate that TRIM24 regulates AR-mediated transcription in collaboration with TIP60 and BRD7.

The HSP90 Inhibitor Ganetespib Synergizes with the MET Kinase Inhibitor Crizotinib in both Crizotinib-Sensitive and -Resistant MET-Driven Tumor Models

The proto-oncogene MET is aberrantly activated via overexpression or mutation in numerous cancers, making it a prime anticancer molecular target. However, the clinical success of MET-directed tyrosine kinase inhibitors (TKI) has been limited due, in part, to mutations in the MET kinase domain that confer therapeutic resistance. Circumventing this problem remains a key challenge to improving durable responses in patients receiving MET-targeted therapy. MET is an HSP90-dependent kinase, and in this report we show that HSP90 preferentially interacts with and stabilizes activated MET, regardless of whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET-TKI show a preference for the inactive form of the kinase, and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET-TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in both TKI-sensitive and -resistant MET-driven tumor models. These data suggest that inclusion of an HSP90 inhibitor can partially restore TKI sensitivity to previously resistant MET mutants, and they provide the foundation for clinical evaluation of this therapeutic combination in patients with MET-driven cancers.

TRIM68 regulates ligand-dependent transcription of androgen receptor in prostate cancer cells.

The androgen receptor (AR) is a transcription factor belonging to the family of nuclear receptors that mediate the action of androgen. AR plays an important role in normal development of the prostate, as well as in the progression of prostate cancer. AR is regulated by several posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. In this study, we found that the putative E3 ubiquitin ligase TRIM68, which is preferentially expressed in prostate cancer cells, interacts with AR and enhances transcriptional activity of the AR in the presence of dihydrotestosterone. We also found that TRIM68 functionally interacts with TIP60 and p300, which act as coactivators of AR, and synergizes in the transactivation of AR. Overexpression of TRIM68 in prostate cancer cells caused an increase in secretion of prostate-specific antigen (PSA), one of the most reliable diagnostic markers for prostate cancer, whereas knockdown of TRIM68 attenuated the secretion of PSA and inhibited cell growth and colony-forming ability. Moreover, we showed that TRIM68 expression is significantly up-regulated in human prostate cancers compared with the expression in adjacent normal tissues. These results indicate that TRIM68 functions as a cofactor for AR-mediated transcription and is likely to be a novel diagnostic tool and a potentially therapeutic target for prostate cancer.

TRIM36 interacts with the kinetochore protein CENP-H and delays cell cycle progression.

The tripartite motif-containing protein (TRIM) family is defined by the presence of a common domain structure composed of a RING finger, a B-box, and a coiled-coil motif. TRIM family proteins are involved in a broad range of biological processes and, consistently, their alterations result in diverse pathological conditions such as genetic diseases, viral infection, and cancer development. In this study, we found by using yeast two-hybrid screening that TRIM36 has a ubiquitin ligase activity and interacts with centromere protein-H, one of the kinetochore proteins. We also found by immunofluorescence analysis that TRIM36 colocalizes with alpha-tubulin, one of the microtubule proteins. Moreover, we found that overexpression of TRIM36 decelerates the cell cycle and attenuates cell growth. These results indicate that TRIM36 is potentially associated with chromosome segregation and that an excess of TRIM36 may cause chromosomal instability.

Combined inhibition of Wee1 and Hsp90 activates intrinsic apoptosis in cancer cells

Heat shock protein 90 (Hsp90) is an essential, evolutionarily conserved molecular chaperone. Cancer cells rely on Hsp90 to chaperone mutated and/or activated oncoproteins, and its involvement in numerous signaling pathways makes it an attractive target for drug development. Surprisingly, however, the impact of Hsp90 inhibitors on cancer cells is frequently cytostatic in nature, and efforts to enhance the antitumor activity of Hsp90 inhibitors in the clinic remain a significant challenge. In agreement with previous data obtained using Wee1 siRNA, we show that dual pharmacologic inhibition of Wee1 tyrosine kinase and Hsp90 causes cancer cells to undergo apoptosis in vitro and in vivo. Gene expression profiling revealed that induction of the intrinsic apoptotic pathway by this drug combination coincided with transcriptional downregulation of Survivin and Wee1, an outcome not seen in cells treated separately with either agent. At the translational level, expression of these two proteins, as well as activated Akt, was completely abrogated. These data support the hypothesis that Wee1 inhibition sensitizes cancer cells to Hsp90 inhibitors; they establish combined Wee1/Hsp90 inhibition as a novel therapeutic strategy; and they provide a mechanistic rationale for enhancing the pro-apoptotic activity of Hsp90 inhibitors.

Ubiquitin-Conjugating Enzyme UBE2Q2 Suppresses Cell Proliferation and Is Down-Regulated in Recurrent Head and Neck Cancer

The ubiquitin-proteasome system has a crucial role in maintaining and regulating cellular homeostasis including carcinogenesis. UBE2Q2, also designated Ubci, is one of the ubiquitin-conjugating enzymes (E2), and it has been reported that mRNA of UBE2Q2 is highly expressed in human head and neck squamous cell carcinoma, particularly hypopharyngeal carcinoma. However, the involvement of UBE2Q2 in carcinogenesis has not been fully elucidated. Most cases of head and neck carcinoma are treated with cis-diamminedichloroplatinum (II; CDDP) or docetaxel, which are the most effective chemotherapeutic agents against squamous cell carcinomas. Nevertheless, some head and neck cancers develop resistance to these drugs, although the causes and mechanisms remain unknown. In this study, we found high expression levels of UBE2Q2 in human head and neck carcinoma cell lines and cancer tissues by using an anti-UBE2Q2 antibody at the protein level. We also found that the expression level of UBE2Q2 is decreased in cell lines and cancer tissues that have resistance to CDDP or docetaxel and in cancer tissues treated with CDDP or docetaxel. Furthermore, we found that overexpression of UBE2Q2 affects cell proliferation and anchorage-independent cell growth. These findings suggest that UBE2Q2 is a novel oncosuppressor that inhibits tumor growth and is related to the resistance to anticarcinoma agents and that UBE2Q2 likely functions as a novel diagnostic tool and a potentially therapeutic target for head and neck squamous cell carcinoma. (Mol Cancer Res 2009;7(9):1553–62)