Markus D. Siegelin

Broad targeting of resistance to apoptosis in cancer

Apoptosis or programmed cell death is natural way of removing aged cells from the body. Most of the anti-cancer therapies trigger apoptosis induction and related cell death networks to eliminate malignant cells. However, in cancer, de-regulated apoptotic signaling, particularly the activation of an anti-apoptotic systems, allows cancer cells to escape this program leading to uncontrolled proliferation resulting in tumor survival, therapeutic resistance and recurrence of cancer. This resistance is a complicated phenomenon that emanates from the interactions of various molecules and signaling pathways. In this comprehensive review we discuss the various factors contributing to apoptosis resistance in cancers. The key resistance targets that are discussed include (1) Bcl-2 and Mcl-1 proteins; (2) autophagy processes; (3) necrosis and necroptosis; (4) heat shock protein signaling; (5) the proteasome pathway; (6) epigenetic mechanisms; and (7) aberrant nuclear export signaling. The shortcomings of current therapeutic modalities are highlighted and a broad spectrum strategy using approaches including (a) gossypol; (b) epigallocatechin-3-gallate; (c) UMI-77 (d) triptolide and (e) selinexor that can be used to overcome cell death resistance is presented. This review provides a roadmap for the design of successful anti-cancer strategies that overcome resistance to apoptosis for better therapeutic outcome in patients with cancer.

Classifying ambiguous melanocytic lesions with FISH and correlation with clinical long-term follow up

Recently, initial studies describing the use of multicolor fluorescence in situ hybridization (FISH) for classifying melanocytic skin lesions have been published demonstrating a high sensitivity and specificity in discriminating melanomas from nevi. However, the majority of these studies included neither histologically ambiguous lesions nor a clinical long-term follow up. This study was undertaken to validate a special multicolor FISH test in histologically ambiguous melanocytic skin lesions with known clinical long-term follow up. FISH was scored by three independent pathologists in a series of 22 melanocytic skin lesions, including 12 ambiguous cases using four probes targeting chromosome 6p25, centromere 6, 6q23, and 11q13. The FISH results were compared with array comparative genomic hybridization data and correlated to the clinical long-term follow up (mean: 65 months). Pair-wise comparison between the interpretations of the observers showed a moderate to substantial agreement (κ 0.47–0.61). Comparing the FISH results with the clinical behavior reached an overall sensitivity of 60% and a specificity of 50% (χ2=0.25; P=0.61) for later development of metastases. Comparison of array comparative genomic hybridization data with FISH analyses did not yield significant results but array comparative genomic hybridization data demonstrated that melanocytic skin lesions with the development of metastases showed significantly more chromosomal aberrations (P<0.01) compared with melanocytic skin lesions without the development of metastases. The FISH technique with its present composition of locus-specific probes for RREB1/MYB and CCND1 did not achieve a clinically useful sensitivity and specificity. However, a reassessment of the probes and better standardization of the method may lead to a valuable diagnostic tool.

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.

Quercetin promotes degradation of survivin and thereby enhances death-receptor–mediated apoptosis in glioma cells

The flavonoid quercetin has been reported to inhibit the proliferation of cancer cells, whereas it has no effect on nonneoplastic cells. U87-MG, U251, A172, LN229, and U373 malignant glioma cells were treated with quercetin (50-200 microM). Quercetin did not cause cytotoxicity 24 h after treatment. Combining quercetin with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) strongly augmented TRAIL-mediated apoptosis in U87-MG, U251, A172, and LN229 glioma cells; U373 cells could not be sensitized by quercetin to TRAIL-mediated apoptosis. TRAIL-induced apoptosis was enhanced by quercetin-induced reduction of survivin protein levels. Upon treatment with quercetin, the protein level of survivin was strongly suppressed in U87-MG, U251, and A172 but not in U373 glioma cells. Quercetin exposure resulted in proteasomal degradation of survivin. TRAIL-quercetin-induced apoptosis was markedly reduced by overexpression of survivin. In addition, upon treatment with quercetin, downregulation of survivin was also regulated by the Akt pathway. Taken together, the results of the present study suggest that quercetin sensitizes glioma cells to death-receptor-mediated apoptosis by suppression of inhibitor of the apoptosis protein survivin.

The flavonoid kaempferol sensitizes human glioma cells to TRAIL-mediated apoptosis by proteasomal degradation of survivin

Resistance to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2L) limits its potential as a drug for cancer therapy. Here, we report that kaempferol, a bioactive plant flavonoid, sensitizes U251 and U87 glioma cells to TRAIL-mediated apoptosis. In contrast, U373 cells are not affected by kaempferol treatment. Treatment of kaempferol alone for 24 h did not induce apoptosis in the cell lines. We provide evidence that TRAIL-induced apoptosis is partially driven by kaempferol-mediated reduction of survivin protein levels. On kaempferol treatment, proteasomal degradation of survivin was observed. Inhibition of proteasomal degradation with MG132 in kaempferol-treated cells restored survivin protein levels in both glial cell lines. Consequently, overexpression of survivin attenuated TRAIL-kaempferol–induced apoptosis. In addition, we show that kaempferol mediates down-regulation of phosphorylated Akt, thereby further reducing survivin protein level. Furthermore, the blockage of the serine/threonine kinase Akt activity by kaempferol is important for inhibition of survivin because active phosphorylated Akt enhances the stability of survivin. However, we also show that the combined treatment of TRAIL and kaempferol induces cleavage (activation) of caspase-8, thereby exerting a proapoptotic effect independent of survivin known not to inhibit caspase-8 activation. Other effects induced by kaempferol were suppression of X-linked inhibitor of apoptosis proteins as the antiapoptotic members of the Bcl-2 family, Bcl-2, Bcl-xL, and Mcl-1 in a concentration-dependent manner. In summary, we showed that suppression of survivin is an essential mechanism in TRAIL-kaempferol–mediated apoptosis. [Mol Cancer Ther 2008;7(11):3566–74]

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.

COX-2 expression in malignant melanoma: a novel prognostic marker?

Several studies illustrated considerably elevated levels of cyclooxygenase-2 (COX-2) protein in various types of human cancer including malignant melanoma. Recently, it was reported that COX-2 is strongly expressed in malignant melanoma and may be correlated with the development and progression of disease. In contrast, other groups did not detect COX-2 protein in primary melanoma cells but did in infiltrating inflammatory cells or metastases. However, there are no reports about patterns or alterations of COX-2 expression in melanoma cells during disease progression or of a correlation between COX-2 expression and overall survival. The aim of this study was to investigate whether there is a correlation between expression of COX-2 protein and disease prognosis in malignant melanoma. We therefore analyzed the expression of COX-2 protein by immunohistochemistry in 101 primary malignant melanomas and 28 metastases and correlated our data with Breslow tumor thickness, Clark levels, different melanoma subtypes, metastases, and overall survival. We detected a strong COX-2 expression in 95% of all primary melanomas, primarily restricted to melanoma cells as shown by various immunohistochemical methods. Levels of COX-2 expression in primary melanoma and corresponding metastases remained stable. A significant correlation between immunohistochemical staining intensity and tumor thickness was demonstrated. Furthermore, Kaplan–Meier curves illustrated a significant correlation between staining intensity and disease-specific survival. Our findings emphasize that the COX-2 protein might be a novel prognostic marker. Owing to its strong expression in melanoma cells it might also be a reasonable therapeutic target.

Epigenetic Silencing of Death Receptor 4 Mediates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Resistance in Gliomas

Purpose: To identify and characterize epigenetically regulated genes able to predict sensitivity or resistance to currently tested chemotherapeutic agents in glioma therapy. Experimental Design: We used methylation-sensitive BeadArray technology to identify novel epigenetically regulated genes associated with apoptosis and with potential therapeutic targets in glioma therapy. To elucidate the functional consequences of promoter methylation in the identified target death receptor 4 (DR4), we investigated tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–mediated and anti-DR4–mediated apoptosis in glioma cell lines (U373 and A172) with loss of DR4 and one glioma cell line (LN18) with robust DR4 expression. Results: In human astrocytic tumors, we detected DR4 promoter hypermethylation in 60% (n = 5) of diffuse astrocytomas WHO grade 2, in 75% (n = 8) of anaplastic astrocytomas WHO grade 3, and in 70% of glioblastomas WHO grade 4 (n = 33). DR4 is a cell surface protein restricted to glioma cells and is targeted by TRAIL. Glioma cell lines U373 and A172 harbored heavily methylated DR4 promoters, and 5-aza-2-deoxycytidine–mediated demethylation reconstituted DR4 expression in these cell lines. Functional knockdown of DR4 by DR4-specific small interfering RNA in TRAIL-sensitive glioma cell line LN18 significantly mitigated apoptosis induced by an agonistic anti-DR4 antibody. 5-Aza-2-deoxycytidine–mediated demethylation resulted in a functional reconstitution of DR4 on the cell surface of TRAIL-resistant glioma cell line U373 and sensitized U373 to TRAIL-mediated apoptosis. Suppression of DR4 by small interfering RNA in demethylated U373 successfully reestablished the TRAIL-resistant phenotype of U373. Conclusions: DR4 promoter methylation is frequent in human astrocytic gliomas, and epigenetic silencing of DR4 mediates resistance to TRAIL/DR4-based glioma therapies. (Clin Cancer Res 2009;15(17):5457–65)

Adaptive mitochondrial reprogramming and resistance to PI3K therapy

BACKGROUND Small molecule inhibitors of phosphatidylinositol-3 kinase (PI3K) have been developed as molecular therapy for cancer, but their efficacy in the clinic is modest, hampered by resistance mechanisms. METHODS We studied the effect of PI3K therapy in patient-derived tumor organotypic cultures (from five patient samples), three glioblastoma (GBM) tumor cell lines, and an intracranial model of glioblastoma in immunocompromised mice (n = 4-5 mice per group). Mechanisms of therapy-induced tumor reprogramming were investigated in a global metabolomics screening, analysis of mitochondrial bioenergetics and cell death, and modulation of protein phosphorylation. A high-throughput drug screening was used to identify novel preclinical combination therapies with PI3K inhibitors, and combination synergy experiments were performed. All statistical methods were two-sided. RESULTS PI3K therapy induces global metabolic reprogramming in tumors and promotes the recruitment of an active pool of the Ser/Thr kinase, Akt2 to mitochondria. In turn, mitochondrial Akt2 phosphorylates Ser31 in cyclophilin D (CypD), a regulator of organelle functions. Akt2-phosphorylated CypD supports mitochondrial bioenergetics and opposes tumor cell death, conferring resistance to PI3K therapy. The combination of a small-molecule antagonist of CypD protein folding currently in preclinical development, Gamitrinib, plus PI3K inhibitors (PI3Ki) reverses this adaptive response, produces synergistic anticancer activity by inducing mitochondrial apoptosis, and extends animal survival in a GBM model (vehicle: median survival = 28.5 days; Gamitrinib+PI3Ki: median survival = 40 days, P = .003), compared with single-agent treatment (PI3Ki: median survival = 32 days, P = .02; Gamitrinib: median survival = 35 days, P = .008 by two-sided unpaired t test). CONCLUSIONS Small-molecule PI3K antagonists promote drug resistance by repurposing mitochondrial functions in bioenergetics and cell survival. Novel combination therapies that target mitochondrial adaptation can dramatically improve on the efficacy of PI3K therapy in the clinic.