Hsiao-Hui Kuo

HSP70 colocalizes with PLK1 at the centrosome and disturbs spindle dynamics in cells arrested in mitosis by arsenic trioxide.

Heat shock protein 70 (HSP70) has been shown to be a substrate of Polo-like kinase 1 (PLK1), and it prevents cells arrested in mitosis by arsenic trioxide (ATO) from dying. Here, we report that HSP70 participates in ATO-induced spindle elongation, which interferes with mitosis progression. Our results demonstrate that HSP70 and PLK1 colocalize at the centrosome in ATO-arrested mitotic cells. HSP70 located at the centrosome was found to be phosphorylated by PLK1 at Ser631 and Ser633. Moreover, unlike wild-type HSP70 (HSP70wt) and its phosphomimetic mutant (HSP70SS631,633DD), a phosphorylation-resistant mutant of HSP70 (HSP70SS631,633AA) failed to localize at the centrosome. ATO-induced spindle elongation was abolished in cells overexpressing HSP70SS631,633AA. Conversely, mitotic spindles in cells ectopically expressing HSP70SS631,633DD were more resistant to nocodazole-induced depolymerization than in those expressing HSP70wt or HSP70SS631,633AA. In addition, inhibition of PLK1 significantly reduced HSP70 phosphorylation and induced early onset of apoptosis in ATO-arrested mitotic cells. Taken together, our results indicate that PLK1-mediated phosphorylation and centrosomal localization of HSP70 may interfere with spindle dynamics and prevent apoptosis of ATO-arrested mitotic cells.

Arsenic Trioxide Induces Abnormal Mitotic Spindles Through a PIP4KIIγ/Rho Pathway

Arsenite-induced spindle abnormalities result in mitotic cell apoptosis in several cancer cell lines, but how arsenite induces these effects is not known. Evidence to date has revealed that arsenite activates Rho guanosine triphosphatases (GTPases). Because Rho GTPases regulate spindle orientation, chromosome congression, and cytokinesis, we therefore examined the involvement of Rho GTPases and their modulators in arsenite-induced mitotic abnormalities. We demonstrated that arsenic trioxide (ATO) disrupted the positioning of bipolar mitotic spindles and induced centrosome and spindle abnormalities. ATO increased the level of the active guanosine triphosphate-bound form of Rho. Inhibition of Rho-associated protein kinases (ROCKs) by Y-27632 ameliorated ATO-induced spindle defects, mitotic arrest, and cell death. These results indicate that ATO may induce spindle abnormalities and mitotic cell death through a Rho/ROCK pathway. In addition, screening of a human kinase and phosphatase shRNA library to select genes that mediate ATO induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-biphosphate (PIP2) synthesis enzyme that belongs to the phosphatidylinositol phosphate kinase (PIPK) family. Sequestration of PIP2 by ectopic overexpression of the pleckstrin homology domain of phospholipase C-δ1 protected cells from ATO-induced cell death. Furthermore, depletion of PIP4KIIγ, but not other isoforms of the PIPK family, not only reduced Rho GTPase activation in ATO-treated cells but also alleviated ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. Thus, our results imply that ATO induces abnormalities in mitotic spindles through a PIP4KIIγ/Rho pathway, leading to apoptosis of mitotic cells.

Inhibition of the Heat Shock Response by PI103 Enhances the Cytotoxicity of Arsenic Trioxide

Heat shock factor 1 (HSF1) is a key regulator of the cytoprotective and anti-apoptotic heat shock response and can be activated by arsenite. Inhibition of HSF1 activation may therefore enhance the cytotoxicity of arsenic trioxide (ATO). We show that ATO induced HSF1 phosphorylation at serine 326 (S326) and induced HSF1-dependent expression of heat shock proteins (HSPs) 27 and 70 in cultured cells. HSF1 significantly reduced cell sensitivity to ATO by reducing apoptosis. Disruption of HSF1 function not only reduced ATO induction of HSP27 and 70 but also enhanced ATO cytotoxicity by elevating apoptosis. These results reveal that HSF1 activation and the resulting induction of HSPs may protect cells from ATO cytotoxicity. The diminished expression of HSPs and hypersensitivity to ATO in cells stably depleted of HSF1 was rescued by ectopic expression of wild-type HSF1 but not an S326A substitution mutant, indicating that phosphorylation at S326 was critical for the protective effect of HSF1. Simultaneous treatment of cells with ATO and PI103, an inhibitor of members of the phosphatidylinositol 3-kinase (PI3K) family, suppressed not only ATO-induced expression of an HSP70 promoter-reporter construct and endogenous HSP70 but also phosphorylation of HSF1 S326. PI103 considerably reduced HSF1 transactivation in ATO-treated cells but had only a limited effect on HSF1 nuclear translocation and DNA binding. Furthermore, PI103 enhanced ATO cytotoxicity in an HSF1-dependent manner. Thus, inhibition of S326 phosphorylation by PI103 blocks the transactivation of HSF1 and may consequently suppress ATO induction of the heat shock response and sensitize cells to ATO.

Inhibition of AKT enhances mitotic cell apoptosis induced by arsenic trioxide

Accumulated evidence has revealed a tight link between arsenic trioxide (ATO)-induced apoptosis and mitotic arrest in cancer cells. AKT, a serine/threonine kinase frequently over-activated in diverse tumors, plays critical roles in stimulating cell cycle progression, abrogating cell cycle checkpoints, suppressing apoptosis, and regulating mitotic spindle assembly. Inhibition of AKT may therefore enhance ATO cytotoxicity and thus its clinical utility. We show that AKT was activated by ATO in HeLa-S3 cells. Inhibition of AKT by inhibitors of the phosphatidyl inositol 3-kinase/AKT pathway significantly enhanced cell sensitivity to ATO by elevating mitotic cell apoptosis. Ectopic expression of the constitutively active AKT1 had no effect on ATO-induced spindle abnormalities but reduced kinetochore localization of BUBR1 and MAD2 and accelerated mitosis exit, prevented mitotic cell apoptosis, and enhanced the formation of micro- or multi-nuclei in ATO-treated cells. These results indicate that AKT1 activation may prevent apoptosis of ATO-arrested mitotic cells by attenuating the function of the spindle checkpoint and therefore allowing the formation of micro- or multi-nuclei in surviving daughter cells. In addition, AKT1 activation upregulated the expression of aurora kinase B (AURKB) and survivin, and depletion of AURKB or survivin reversed the resistance of AKT1-activated cells to ATO-induced apoptosis. Thus, AKT1 activation suppresses ATO-induced mitotic cell apoptosis, despite the presence of numerous spindle abnormalities, probably by upregulating AURKB and survivin and attenuating spindle checkpoint function. Inhibition of AKT therefore effectively sensitizes cancer cells to ATO by enhancing mitotic cell apoptosis.

Novel 2-substituted quinolin-4-yl-benzenesulfonate derivatives: synthesis, antiproliferative activity, and inhibition of cellular tubulin polymerization.

A series of 2‐substituted quinolin‐4‐yl‐benzenesulfonate derivatives were synthesized for the purpose of evaluating antiproliferative activity. Structure–activity relationships of the newly synthesized compounds against human lymphoblastic leukemia and various solid tumor cell growths in culture are discussed. Of these derivatives, 2‐phenyl‐6‐pyrrolidinyl‐4‐quinoline sulfonate analogues 10 f, 10 g, and 10 k, and 4′‐nitrophenyl sulfonate 10 m exhibit superior cytotoxicity over other sulfonates. The antiproliferative activities of these compounds correlate well with their abilities to induce mitotic arrest and apoptosis. Mechanistic studies indicate that they target the vinblastine binding site of tubulin and inhibit cellular tubulin polymerization. Hence, these compounds induce the formation of aberrant mitotic spindles and mitotic arrest, resulting in intensive apoptosis. The tested compounds were shown to be poor substrates for membrane multidrug resistance transporters. The present studies suggest that these newly synthesized compounds are promising tubulin polymerization inhibitors and are worthy of further investigation as antitumor agents.

Derivatives of 6-cinnamamido-quinoline-4-carboxamide impair lysosome function and induce apoptosis

Autophagy is a lysosomal degradative process that protects cancer cells from multiple types of stress. In this study, we synthesized a series of derivatives of 6-cinnamamido-quinoline-4-carboxamide (CiQ), and investigated their effects on the proliferation and autophagy of cancer cells in vitro. These derivatives effectively inhibited the proliferation of a broad spectrum of cancer cell lines. Further study revealed that CiQ derivatives may induce autophagy and result in disruption of autophagy propagation. Consequently, these derivatives triggered massive apoptosis, as evidenced by caspase-9 activation and PARP cleavage. Blockage of autophagy by depletion of autophagy related gene ATG5 or BECN1 considerably alleviated CiQ-induced cell death, indicating that autophagy may mediate CiQ-induced cell death. Furthermore, treatment with CiQ derivatives increased lysosome membrane permeability (LMP) and enhanced accumulation of ubiquitinated proteins, which collectively indicate impaired lysosome function. In addition, treatment of cells with CiQ derivatives activated extracellular signal-regulated kinase (ERK); abrogation of ERK activation, either by treating cells with U0126, an inhibitor of mitogen-activated protein/ERK kinase 1 (MEK1), or by ectopically overexpressing a dominant-negative MEK1, significantly reduced CiQ derivative-induced LMP, LC3 and p62 accumulation, and cytotoxicity. These results indicate that CiQ derivatives activate ERK and disrupt lysosome function, thereby altering autophagic flux and resulting in apoptotic cell death.

HSP70 regulates the function of mitotic centrosomes.

To establish a functional bipolar mitotic spindle, the centrosome expands and matures, acquiring enhanced activities for microtubule (MT) nucleation and assembly at the onset of mitosis. However, the regulatory mechanisms of centrosome maturation and MT assembly from the matured centrosome are largely unknown. In this study, we showed that heat shock protein (HSP) 70 considerably accumulates at the mitotic centrosome during prometaphase to metaphase and is required for bipolar spindle assembly. Inhibition or depletion of HSP70 impaired the function of mitotic centrosome and disrupted MT nucleation and polymerization from the spindle pole, and may thus result in formation of abnormal mitotic spindles. In addition, HSP70 may associate with NEDD1 and γ-tubulin, two pericentriolar material (PCM) components essential for centrosome maturation and MT nucleation. Loss of HSP70 function disrupted the interaction between NEDD1 and γ-tubulin, and reduced their accumulation at the mitotic centrosome. Our results thus demonstrate a role for HSP70 in regulating centrosome integrity during mitosis, and indicate that HSP70 is required for the maintenance of a functional mitotic centrosome that supports the assembly of a bipolar mitotic spindle.

Abstract LB-083: Regulation of the mitotic centrosome by heat shock protein 70

A functional centrosome is essential for the assembly of a bipolar mitotic spindle and accurate chromosome segregation. Loss of centrosome integrity frequently occurs in transformed cells or in tumor tissues. Pathways maintaining the quality control of proteins are known to regulate the biogenesis and duplication of the centrosome. In this study, we explored the role of heat shock protein 70 (HSP70), an essential molecular chaperone for protein quality control, on maintaining the centrosome integrity during mitosis by treating cells with a HSP70 inhibitor or transducing cells with HSP70-specific shRNA. The effects of these treatments on mitosis progression, mitotic spindle assembly, and cell viability were investigated. Our results showed that inhibition or depletion of HSP70 disrupted microtubule nucleation and polymerization, induced abnormal mitotic spindles, and interfered with mitosis progression. In addition, HSP70 accumulated at the spindle pole and co-localized with γ-tubulin and pericentrin during mitosis. Loss of centrosomal HSP70 impeded the recruitment of pericentriolar components essential for centrosome maturation. These results indicate that HSP70 is required for the maintenance of a functional mitotic centrosome to support the assembly a bipolar mitotic spindle. Citation Format: Chieh-Ting Fang, Hsiao-Hui Kuo, Ling-Huei Yih. Regulation of the mitotic centrosome by heat shock protein 70. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-083. doi:10.1158/1538-7445.AM2015-LB-083

Abstract 1593: GSK3β mediated arsenite induction of spindle abnormalities

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Arsenite induces centrosome amplification and spindle abnormalities, leading to chromosome missegregation and consequently resulting in mitotic cell apoptosis or aneuploidy. However, the underlying mechanism of how arsenite disrupts mitotic spindles is not known. In this study, we showed that CGL2 cells treated with arsenic trioxide (ATO) were arrested at mitotic stage in company with the formation of substantial spindle abnormalities and significant reduction of acetylated- and detyrosinated-tubulin, characteristics of decreased MT stability. Immunofluorescence staining of EB1, a plus-end microtubule-binding protein, revealed discrete EB1 spots distributing along microtubule (MT) nucleated from the two spindle poles in untreated mitotic cells. However, it showed a disorganized pattern in ATO-arrested mitotic cells with the spot-like EB1 clusters splashing all over the cells. In addition, immunoblotting and immunofluorescence staining revealed that the mitosis-specific inhibitory phospho-GSK3 was diminished in ATO-arrested mitotic cells. Pharmacological inhibition of GSK3 could prevent ATO induction of spindle abnormalities, mitotic arrest, and cell death. Since GSK3β has been reported to reduce MT stability by phosphorylating several microtubule-associated proteins, our current results indicated that GSK3β may mediate ATO induction of spindle abnormalities. Citation Format: Tz-Chi Lin, Ren-Meei Chen, Hsiao-Hui Kuo, Ling-Huei Yih. GSK3β mediated arsenite induction of spindle abnormalities. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1593. doi:10.1158/1538-7445.AM2014-1593

Abstract 1304: HSF1 facilitates cell transformation by enhancing the expression of extracellular proteins.

The expression level or the activity of heat shock factor-1 (HSF1), a master transcriptional regulator of the heat shock response, is elevated in cancer cell lines and tumor tissues. Loss of HSF1 function has been demonstrated to prevent oncogenesis. We thus examined whether and how HSF1 induces cell transformation using anchorage-independent growth as a surrogate for transformation phenotype. CGL2 cells overexpressing HSF1 (CGL2-wtHSF1) were more proficient to form colonies in soft agar than the parental control cells (CGL2-Neo). Cells stably depleted of HSF1 (CGL2-shHSF1), on the other hand, were unable to form colonies in soft agar. The diminished ability of CGL2-shHSF1 cells to grow in soft agar was rescued by ectopic expression of wild-type HSF1 but not an S326A substitution mutant or a C-terminal (transactivation domain) deletion mutant, indicating that phosphorylation at S326 and/or the transactivation activity of HSF1 was required for its ability to enhance anchorage-independent growth. In addition, conditioned medium collected from HSF1-overexpressing cells could induce anchorage-independent growth of HSF1-depleted cells. These results indicated that HSF1 might enhance cell transformation by transactivating the expression of particular extracellular proteins. Citation Format: Hsiao-Hui Kuo, Ling-Huei Yih. HSF1 facilitates cell transformation by enhancing the expression of extracellular proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1304. doi:10.1158/1538-7445.AM2013-1304