Sachiko Okabe

Telomestatin Impairs Glioma Stem Cell Survival and Growth through the Disruption of Telomeric G-Quadruplex and Inhibition of the Proto-oncogene, c-Myb

Purpose: Glioma stem cells (GSC) are a critical therapeutic target of glioblastoma multiforme (GBM). Experimental Design: The effects of a G-quadruplex ligand, telomestatin, were evaluated using patient-derived GSCs, non-stem tumor cells (non-GSC), and normal fetal neural precursors in vitro and in vivo. The molecular targets of telomestatin were determined by immunofluorescence in situ hybridization (iFISH) and cDNA microarray. The data were then validated by in vitro and in vivo functional assays, as well as by immunohistochemistry against 90 clinical samples. Results: Telomestatin impaired the maintenance of GSC stem cell state by inducing apoptosis in vitro and in vivo. The migration potential of GSCs was also impaired by telomestatin treatment. In contrast, both normal neural precursors and non-GSCs were relatively resistant to telomestatin. Treatment of GSC-derived mouse intracranial tumors reduced tumor sizes in vivo without a noticeable cell death in normal brains. iFISH revealed both telomeric and non-telomeric DNA damage by telomestatin in GSCs but not in non-GSCs. cDNA microarray identified a proto-oncogene, c-Myb, as a novel molecular target of telomestatin in GSCs, and pharmacodynamic analysis in telomestatin-treated tumor-bearing mouse brains showed a reduction of c-Myb in tumors in vivo. Knockdown of c-Myb phenocopied telomestatin-treated GSCs both in vitro and in vivo, and restoring c-Myb by overexpression partially rescued the phenotype. Finally, c-Myb expression was markedly elevated in surgical specimens of GBMs compared with normal tissues. Conclusions: These data indicate that telomestatin potently eradicates GSCs through telomere disruption and c-Myb inhibition, and this study suggests a novel GSC-directed therapeutic strategy for GBMs. Clin Cancer Res; 18(5); 1268–80. ©2012 AACR.

Pharmacological targeting of constitutively active truncated androgen receptor by nigericin and suppression of hormone-refractory prostate cancer cell growth.

In prostate cancer, blockade of androgen receptor (AR) signaling confers a therapeutic benefit. Nevertheless, this standard therapy allows relapse of hormone-refractory prostate cancer (HRPC) with a poor prognosis. HRPC cells often express variant ARs, such as point-mutated alleles and splicing isoforms, resulting in androgen-independent cell growth and resistance to antiandrogen (e.g., flutamide). However, a pharmacological strategy to block such aberrant ARs remains to be established. Here, we established a reporter system that monitors AR-mediated activation of a prostate-specific antigen (PSA) promoter. Our chemical library screening revealed that the antibiotic nigericin inhibits AR-mediated activation of the PSA promoter and PSA production in prostate cancer cells. Nigericin suppressed the androgen-dependent LNCaP cell growth even though the cells expressed a flutamide-resistant mutant AR. These effects were caused by AR suppression at the mRNA and post-translational levels. In HRPC 22Rv1 cells, which express the full-length AR and the constitutively active, truncated ARs lacking the carboxyl-terminal ligand-binding domain, small interfering RNA-mediated knockdown of both AR isoforms efficiently suppressed the androgen-independent cell growth, whereas knockdown of the full-length AR alone had no significant effect. It is noteworthy that nigericin was able to mimic the knockdown of both AR isoforms: it reduced the expression of the full-length and the truncated ARs, and it induced G1 cell-cycle arrest and apoptosis of 22Rv1 cells. These observations suggest that nigericin-like compounds that suppress AR expression at the mRNA level could be applied as new-type therapeutic agents that inhibit a broad spectrum of AR variants in HRPC.

Acyl-CoA synthetase as a cancer survival factor: its inhibition enhances the efficacy of etoposide

Lipid metabolism is often elevated in cancer cells and plays an important role in their growth and malignancy. Acyl‐CoA synthetase (ACS), which converts long‐chain fatty acids to acyl‐CoA, is overexpressed in various types of cancer. However, the role of ACS in cancer remains unknown. Here, we found that ACS enzyme activity is required for cancer cell survival. Namely, the ACS inhibitor Triacsin c induced massive apoptosis in glioma cells while this cell death was completely suppressed by overexpression of ACSL5, the Triacsin c–resistant ACS isozyme, but not by overexpression of a catalytically inactive ACSL5 mutant. ACS inhibition by Triacsin c markedly potentiated the Bax‐induced intrinsic apoptotic pathway by promoting cytochrome c release and subsequent caspase activation. These effects were abrogated by ACSL5 overexpression. Correspondingly, ACS inhibition synergistically potentiated the glioma cell death induced by etoposide, a well‐known activator of apoptosis. Furthermore, in a nude mouse xenograft model, Triacsin c at a non‐toxic dose enhanced the antitumor efficacy of a low‐dose chemotherapy with etoposide. These results indicate that ACS is an apoptosis suppressor and that ACS inhibition could be a rational strategy to amplify the antitumor effect of etoposide. (Cancer Sci 2009)

Inhibition of ATP citrate lyase induces triglyceride accumulation with altered fatty acid composition in cancer cells.

De novo lipogenesis is activated in most cancers and several lipogenic enzymes have been implicated as therapeutic targets. Here, we demonstrate a novel function of the lipogenic enzyme, ATP citrate lyase (ACLY), in lipid metabolism in cancer cells. ACLY depletion by small interfering RNAs caused growth suppression and/or apoptosis in a subset of cancer cell lines. To investigate the effect of ACLY inhibition on lipid metabolism, metabolome and transcriptome analysis was performed. ACLY depletion blocks the fatty acid chain elongation from C16 to C18 in triglyceride (TG), but not in other lipid classes. Meanwhile, wild‐type ACLY overexpression enhanced fatty acid elongation of TG, whereas an inactive mutant ACLY did not change it. ACLY depletion‐mediated blockade of fatty acid elongation was coincident with downregulation of long‐chain fatty acid elongase ELOVL6, which resides in endoplasmic reticulum (ER). Paradoxically, ACLY depletion‐mediated growth suppression was associated with TG accumulation. ACLY depletion downregulated the expression of carnitine palmitoyltransferase 1A, which is a mitochondrial fatty acid transporter. Consistent with this finding, metabolome analysis revealed that ACLY positively regulates the carnitine system, which plays as an essential cofactor for fatty acid transport across mitochondrial membrane. AICAR, an activator of mitochondrial fatty acid oxidation (FAO), significantly reduced ACLY depletion‐mediated TG accumulation. These data indicate that inhibition of ACLY might affect both fatty acid elongation in ER and FAO in mitochondria, thereby explaining the TG accumulation with altered fatty acid composition. This phenotype may be a hallmark of growth suppression mediated by ACLY inhibition.

Inhibition of ATP Citrate Lyase Induces an Anticancer Effect via Reactive Oxygen Species AMPK as a Predictive Biomarker for Therapeutic Impact

De novo lipogenesis is activated in most cancers. Inhibition of ATP citrate lyase (ACLY), the enzyme that catalyzes the first step of de novo lipogenesis, leads to growth suppression and apoptosis in a subset of human cancer cells. Herein, we found that ACLY depletion increases the level of intracellular reactive oxygen species (ROS), whereas addition of an antioxidant reduced ROS and attenuated the anticancer effect. ACLY depletion or exogenous hydrogen peroxide induces phosphorylation of AMP-activated protein kinase (p-AMPK), a crucial regulator of lipid metabolism, independently of energy status. Analysis of various cancer cell lines revealed that cancer cells with a higher susceptibility to ACLY depletion have lower levels of basal ROS and p-AMPK. Mitochondrial-deficient ρ(0) cells retained high levels of ROS and p-AMPK and were resistant to ACLY depletion, whereas the replenishment of normal mitochondrial DNA reduced the levels of ROS and p-AMPK and restored the sensitivity to ACLY depletion, indicating that low basal levels of mitochondrial ROS are critical for the anticancer effect of ACLY depletion. Finally, p-AMPK levels were significantly correlated to the levels of oxidative DNA damage in colon cancer tissues, suggesting that p-AMPK reflects cellular ROS levels inxa0vitro and inxa0vivo. Together, these data suggest that ACLY inhibition exerts an anticancer effect via increased ROS, and p-AMPK could be a predictive biomarker for its therapeutic outcome.

G-quadruplex ligand-induced DNA damage response coupled with telomere dysfunction and replication stress in glioma stem cells.

Glioblastoma (GBM) is an invariably fatal brain tumor in which a small subpopulation of self-renewable glioma stem cells (GSCs) contributes to tumor propagation and relapse. Targeting GSCs could therefore have a significant clinical impact for GBM. Telomestatin is a naturally-occurring compound that preferentially impairs GSC growth by perturbing transcription and inducing a DNA damage response. Telomestatin stabilizes G-quadruplexes (G4s), which are guanine-rich four-strand nucleic acid structures observed inxa0vitro and inxa0vivo. However, the mechanism underlying the GSC-selective nature of the DNA damage response remains unknown. Here we demonstrate that GSCs are more susceptible to telomestatin-induced telomere dysfunction and replication stress when compared with GSC-derived non-stem glioma cells (NSGCs). Telomestatin induced dissociation of the telomere-capping protein TRF2 from telomeres, leading to telomeric DNA damage in GSCs-but not in NSGCs. BIBR1532, a telomerase catalytic inhibitor, did not preferentially inhibit GSC growth, suggesting that telomestatin promotes telomere dysfunction in a telomerase-independent manner. GSCs and NSGCs had comparable levels of G4s in their nuclei, and both responded to telomestatin with phosphorylation of RPA2 at Ser33-a hallmark of replication stress. However, activation of the checkpoint kinase Chk1, induction of a DNA damage response, and subsequent growth inhibition occurred only in telomestatin-treated GSCs. These observations suggest that telomestatin impairs GSC growth through removal of TRF2 from telomeres and potent activation of the replication stress response pathway. Therefore, a novel G4-directed therapeutic strategy could specifically target cancer stem cells in GBM.

Senescence from glioma stem cell differentiation promotes tumor growth.

Glioblastoma (GBM) is a lethal brain tumor composed of heterogeneous cellular populations including glioma stem cells (GSCs) and differentiated non-stem glioma cells (NSGCs). While GSCs are involved in tumor initiation and propagation, NSGCs role remains elusive. Here, we demonstrate that NSGCs undergo senescence and secrete pro-angiogenic proteins, boosting the GSC-derived tumor formation in vivo. We used a GSC model that maintains stemness in neurospheres, but loses the stemness and differentiates into NSGCs upon serum stimulation. These NSGCs downregulated telomerase, shortened telomeres, and eventually became senescent. The senescent NSGCs released pro-angiogenic proteins, including vascular endothelial growth factors and senescence-associated interleukins, such as IL-6 and IL-8. Conditioned medium from senescent NSGCs promoted proliferation of brain microvascular endothelial cells, and mixed implantation of GSCs and senescent NSGCs into mice enhanced the tumorigenic potential of GSCs. The senescent NSGCs seem to be clinically relevant, because both clinical samples and xenografts of GBM contained tumor cells that expressed the senescence markers. Our data suggest that senescent NSGCs promote malignant progression of GBM in part via paracrine effects of the secreted proteins.

Design and synthesis of a berberine dimer: a fluorescent ligand with high affinity towards G-quadruplexes.

G-quadruplexes (G4) are thought to be important factors for telomerase inhibition and transcriptional/translational modulations. Bioinformatic analyses imply that the human genome and mRNA contain a multitude of G4-forming sequences; however, their analysis requires selective and detectable ligands. Given that two molecules of fluorescent berberine (BBR) coordinate to telomeric G4 in their co-crystals, we designed hydrocarbon-linked BBR-analogue dimers because we expected the alignment of two BBR chromophores would avoid Watson-Crick base pair intercalation, which should result in high selectivity towards G4. An alkene-cis-C2 BBR dimer showed the highest affinity (Kd ≤2.6u2005nM) and selectivity (ca. 900-fold vs. duplex) towards G4. The intrinsic light-up fluorescence properties of this BBR dimer, derived from its conformational switching by G4, allowed a selective visualization of various G4 in the gel without using additional bulky fluorescence dyes, which, combined with the observed lack of conformational change of the ligand, suggested future applications in in vitro detection systems.

Targeting glioma stem cells in vivo by a G-quadruplex-stabilizing synthetic macrocyclic hexaoxazole

G-quadruplex (G4) is a higher-order nucleic acid structure that is formed by guanine-rich sequences. G4 stabilization by small-molecule compounds called G4 ligands often causes cytotoxicity, although the potential medicinal impact of this effect has not been fully established. Here we demonstrate that a synthetic G4 ligand, Y2H2-6M(4)-oxazole telomestatin derivative (6OTD), limits the growth of intractable glioblastoma (grade IV glioma) and glioma stem cells (GSCs). Experiments involving a human cancer cell line panel and mouse xenografts revealed that 6OTD exhibits antitumor activity against glioblastoma. 6OTD inhibited the growth of GSCs more potently than it did the growth of differentiated non-stem glioma cells (NSGCs). 6OTD caused DNA damage, G1 cell cycle arrest, and apoptosis in GSCs but not in NSGCs. These DNA damage foci tended to colocalize with telomeres, which contain repetitive G4-forming sequences. Compared with temozolomide, a clinical DNA-alkylating agent against glioma, 6OTD required lower concentrations to exert anti-cancer effects and preferentially affected GSCs and telomeres. 6OTD suppressed the intracranial growth of GSC-derived tumors in a mouse xenograft model. These observations indicate that 6OTD targets GSCs through G4 stabilization and promotion of DNA damage responses. Therefore, G4s are promising therapeutic targets for glioblastoma.

JBIR-120: a new growth inhibitor of hormone-refractory prostate cancer cells.

Prostate cancer is a common nondermatological cancer in older adult men. Androgen receptor (AR) signaling has a central role in prostate cancer cell growth and survival,1 and therefore, androgen ablation therapy is recognized as a standard regimen for the treatment of advanced and metastatic prostate cancers.2 However, most patients who undergo androgen ablation progress from being androgen-dependent to developing hormone-refractory prostate cancer within 2 years after initiating therapy. Although the recurrent tumors are often resistant to standard AR-targeting agents, which cause deprivation of androgens or block androgen–AR interaction, AR-mediated signaling still has a key role in the development and maintenance of hormone-refractory prostate cancers.3, 4, 5 Thus, identifying new therapeutic agents targeting the AR signaling pathway may possibly control the occurrence of hormone-refractory prostate cancers. Mashima et al.6 earlier reported that nigericin can block AR-mediated signaling in hormone-refractory prostate cancer cells. In the current study, we discovered a novel compound—JBIR-120 (1)—extracted from the culture broth of a new Streptomyces strain, RI104-LiC104. This paper describes the isolation, structure elucidation and biological activity of 1.