Takehiko Dohi

Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis

Evasion of apoptosis is a hallmark of cancer, but the molecular circuitries of this process are not understood. Here we show that survivin, a member of the inhibitor of apoptosis gene family that is overexpressed in cancer, exists in a novel mitochondrial pool in tumor cells. In response to cell death stimulation, mitochondrial survivin is rapidly discharged in the cytosol, where it prevents caspase activation and inhibits apoptosis. Selective targeting of survivin to mitochondria enhances colony formation in soft agar, accelerates tumor growth in immunocompromised animals, and abolishes tumor cell apoptosis in vivo. Therefore, mitochondrial survivin orchestrates a novel pathway of apoptosis inhibition, which contributes to tumor progression.

Regulation of Tumor Cell Mitochondrial Homeostasis by an Organelle-Specific Hsp90 Chaperone Network

Molecular chaperones, especially members of the heat shock protein 90 (Hsp90) family, are thought to promote tumor cell survival, but this function is not well understood. Here, we show that mitochondria of tumor cells, but not most normal tissues, contain Hsp90 and its related molecule, TRAP-1. These chaperones interact with Cyclophilin D, an immunophilin that induces mitochondrial cell death, and antagonize its function via protein folding/refolding mechanisms. Disabling this pathway using novel Hsp90 ATPase antagonists directed to mitochondria causes sudden collapse of mitochondrial function and selective tumor cell death. Therefore, Hsp90-directed chaperones are regulators of mitochondrial integrity, and their organelle-specific antagonists may provide a previously undescribed class of potent anticancer agents.

IAP regulation of metastasis

Inhibitor-of-Apoptosis (IAP) proteins contribute to tumor progression, but the requirements of this pathway are not understood. Here, we show that intermolecular cooperation between XIAP and survivin stimulates tumor cell invasion and promotes metastasis. This pathway is independent of IAP inhibition of cell death. Instead, a survivin-XIAP complex activates NF-kappaB, which in turn leads to increased fibronectin gene expression, signaling by beta1 integrins, and activation of cell motility kinases FAK and Src. Therefore, IAPs are direct metastasis genes, and their antagonists could provide antimetastatic therapies in patients with cancer.

Hsp60 regulation of tumor cell apoptosis

Molecular chaperones may promote cell survival, but how this process is regulated, especially in cancer, is not well understood. Using high throughput proteomics screening, we identified the cell cycle regulator and apoptosis inhibitor survivin as a novel protein associated with the molecular chaperone Hsp60. Acute ablation of Hsp60 by small interfering RNA destabilizes the mitochondrial pool of survivin, induces mitochondrial dysfunction, and activates caspase-dependent apoptosis. This response involves disruption of an Hsp60-p53 complex, which results in p53 stabilization, increased expression of pro-apoptotic Bax, and Bax-dependent apoptosis. In vivo, Hsp60 is abundantly expressed in primary human tumors, as compared with matched normal tissues, and small interfering RNA ablation of Hsp60 in normal cells is well tolerated and does not cause apoptosis. Therefore, Hsp60 orchestrates a broad cell survival program centered on stabilization of mitochondrial survivin and restraining of p53 function, and this process is selectively exploited in cancer. Hsp60 inhibitors may function as attractive anticancer agents by differentially inducing apoptosis in tumor cells.

Exploiting the mitochondrial unfolded protein response for cancer therapy in mice and human cells

Fine tuning of the protein folding environment in subcellular organelles, such as mitochondria, is important for adaptive homeostasis and may participate in human diseases, but the regulators of this process are still largely elusive. Here, we have shown that selective targeting of heat shock protein-90 (Hsp90) chaperones in mitochondria of human tumor cells triggered compensatory autophagy and an organelle unfolded protein response (UPR) centered on upregulation of CCAAT enhancer binding protein (C/EBP) transcription factors. In turn, this transcriptional UPR repressed NF-κB-dependent gene expression, enhanced tumor cell apoptosis initiated by death receptor ligation, and inhibited intracranial glioblastoma growth in mice without detectable toxicity. These data reveal what we believe to be a novel role of Hsp90 chaperones in the regulation of the protein-folding environment in mitochondria of tumor cells. Disabling this general adaptive pathway could potentially be used in treatment of genetically heterogeneous human tumors.

Heat Shock Protein 60 Regulation of the Mitochondrial Permeability Transition Pore in Tumor Cells

Mitochondrial apoptosis plays a critical role in tumor maintenance and dictates the response to therapy in vivo; however, the regulators of this process are still largely elusive. Here, we show that the molecular chaperone heat shock protein 60 (Hsp60) directly associates with cyclophilin D (CypD), a component of the mitochondrial permeability transition pore. This interaction occurs in a multichaperone complex comprising Hsp60, Hsp90, and tumor necrosis factor receptor-associated protein-1, selectively assembled in tumor but not in normal mitochondria. Genetic targeting of Hsp60 by siRNA triggers CypD-dependent mitochondrial permeability transition, caspase-dependent apoptosis, and suppression of intracranial glioblastoma growth in vivo. Therefore, Hsp60 is a novel regulator of mitochondrial permeability transition, contributing to a cytoprotective chaperone network that antagonizes CypD-dependent cell death in tumors.

Activated Checkpoint Kinase 2 Provides a Survival Signal for Tumor Cells

Tumor cells often become resistant to DNA damage-based therapy; however, the underlying mechanisms are not yet understood. Here, we show that tumor cells exposed to DNA damage counteract cell death by releasing the antiapoptotic protein, survivin, from mitochondria. This is independent of p53, and requires activated checkpoint kinase 2 (Chk2), a putative tumor suppressor. Molecular or genetic targeting of Chk2 prevents the release of survivin from mitochondria, enhances DNA damage-induced tumor cell apoptosis, and inhibits the growth of resistant in vivo tumors. Therefore, activated Chk2 circumvents its own tumor-suppressive functions by promoting tumor cell survival. Inhibiting Chk2 in combination with DNA-damaging agents may provide a rational approach for treating resistant tumors.

miR-296 regulation of a cell polarity–cell plasticity module controls tumor progression

The expression of small, non-coding RNA or microRNAs (miR), is frequently deregulated in human cancer, but how these pathways affect disease progression is still largely elusive. Here, we report on a miR, miR-296, which is progressively lost during tumor progression and correlates with metastatic disease in colorectal, breast, lung, gastric, parathyroid, liver and bile ducts cancers. Functionally, miR-296 controls a global cell motility gene signature in epithelial cells by transcriptionally repressing the cell polarity–cell plasticity module, Scribble (Scrib). In turn, loss of miR-296 causes aberrantly increased and mislocalized Scrib in human tumors, resulting in exaggerated random cell migration and tumor cell invasiveness. Re-expression of miR-296 in MDA-MB231 cells inhibits tumor growth in vivo. Finally, miR-296 or Scrib levels predict tumor relapse in hepatocellular carcinoma patients. These data identify miR-296 as a global repressor of tumorigenicity and uncover a previously unexplored exploitation of Scrib in tumor progression in humans.

Control of Tumor Bioenergetics and Survival Stress Signaling by Mitochondrial HSP90s

Tumors successfully adapt to constantly changing intra- and extracellular environments, but the wirings of this process are still largely elusive. Here, we show that heat-shock-protein-90-directed protein folding in mitochondria, but not cytosol, maintains energy production in tumor cells. Interference with this process activates a signaling network that involves phosphorylation of nutrient-sensing AMP-activated kinase, inhibition of rapamycin-sensitive mTOR complex 1, induction of autophagy, and expression of an endoplasmic reticulum unfolded protein response. This signaling network confers a survival and proliferative advantage to genetically disparate tumors, and correlates with worse outcome in lung cancer patients. Therefore, mitochondrial heat shock protein 90s are adaptive regulators of tumor bioenergetics and tractable targets for cancer therapy.

Preclinical Characterization of Mitochondria-Targeted Small Molecule Hsp90 Inhibitors, Gamitrinibs, in Advanced Prostate Cancer

Purpose: This study aimed to characterize the preclinical activity of the first class of combinatorial, mitochondria-targeted, small molecule heat shock protein-90 (Hsp90) inhibitors, gamitrinibs, in models of hormone-refractory, drug-resistant, localized, and bone metastatic prostate cancer in vivo. Experimental Design: Mitochondrial permeability transition, apoptosis, and changes in metabolic activity were examined by time-lapse videomicroscopy, multiparametric flow cytometry, MTT, and analysis of isolated mitochondria. Drug-resistant prostate cancer cells were generated by chronic exposure of hormone-refractory PC3 cells to the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG). The effect of gamitrinibs on s.c. or intratibial prostate cancer growth was studied in xenograft models. Bone metastatic tumor growth and bone parameters were quantified by micro–computed tomography imaging. Results: In the NCI 60-cell line screening, gamitrinibs were active against all tumor cell types tested, and efficiently killed metastatic, hormone-refractory, and multidrug-resistant prostate cancer cells characterized by overexpression of the ATP binding cassette transporter P-glycoprotein. Mechanistically, gamitrinibs, but not 17-AAG, induced acute mitochondrial dysfunction in prostate cancer cells with loss of organelle membrane potential, release of cytochrome c, and caspase activity, independently of proapoptotic Bcl-2 proteins Bax and Bak. Systemic administration of gamitrinibs to mice was well tolerated, and inhibited s.c. or bone metastatic prostate cancer growth in vivo. Conclusions: Gamitrinibs have preclinical activity and favorable safety in models of drug-resistant and bone metastatic prostate cancer in vivo. Clin Cancer Res; 16(19); 4779–88. ©2010 AACR.