Inhibition of CDK8/19 Mediator kinase potentiates HER2-targeting drugs and bypasses resistance to these agents in vitro and in vivo.

Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with HER2-targeting antibodies (such as trastuzumab) or small-molecule kinase inhibitors (such as lapatinib). However, most patients with metastatic HER2+ BrCa have intrinsic resistance and nearly all eventually become resistant to HER2-targeting therapy. Resistance to HER2-targeting drugs frequently involves transcriptional reprogramming associated with constitutive activation of different signaling pathways. We have investigated the role of CDK8/19 Mediator kinase, a regulator of transcriptional reprogramming, in the response of HER2+ BrCa to HER2-targeting drugs. CDK8 was in the top 1% of all genes ranked by correlation with shorter relapse-free survival among treated HER2+ BrCa patients. Selective CDK8/19 inhibitors (senexin B and SNX631) showed synergistic interactions with lapatinib and trastuzumab in a panel of HER2+ BrCa cell lines, overcoming and preventing resistance to HER2-targeting drugs. The synergistic effects were mediated in part through the PI3K/AKT/mTOR pathway and reduced by PI3K inhibition. Combination of HER2- and CDK8/19-targeting agents inhibited STAT1 and STAT3 phosphorylation at S727 and up-regulated tumor suppressor BTG2. The growth of xenograft tumors formed by lapatinib-sensitive or -resistant HER2+ breast cancer cells was partially inhibited by SNX631 alone and strongly suppressed by the combination of SNX631 and lapatinib, overcoming lapatinib resistance. These effects were associated with decreased tumor cell proliferation and altered recruitment of stromal components to the xenograft tumors. These results suggest potential clinical benefit of combining HER2- and CDK8/19-targeting drugs in the treatment of metastatic HER2+ BrCa.

CDK8/19 Mediator kinases potentiate induction of transcription by NFκB.

The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to be mediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB include IL8, CXCL1, and CXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes.

Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities.

Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity. p21 and CDK8 also cooperate in the formation of internucleolar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy.

Mediator kinase inhibition reverses castration resistance of advanced prostate cancer.

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

TWIK-1 and TREK-1 are potassium channels contributing significantly to astrocyte passive conductance in rat hippocampal slices.

Expression of a linear current-voltage (I-V) relationship (passive) K(+) membrane conductance is a hallmark of mature hippocampal astrocytes. However, the molecular identifications of the K(+) channels underlying this passive conductance remain unknown. We provide the following evidence supporting significant contribution of the two-pore domain K(+) channel (K(2P)) isoforms, TWIK-1 and TREK-1, to this conductance. First, both passive astrocytes and the cloned rat TWIK-1 and TREK-1 channels expressed in CHO cells conduct significant amounts of Cs(+) currents, but vary in their relative P(Cs)/P(K) permeability, 0.43, 0.10, and 0.05, respectively. Second, quinine, which potently inhibited TWIK-1 (IC(50) = 85 microm) and TREK-1 (IC(50) = 41 microm) currents, also inhibited astrocytic passive conductance by 58% at a concentration of 200 microm. Third, a moderate sensitivity of passive conductance to low extracellular pH (6.0) supports a combined expression of acid-insensitive TREK-1, and to a lesser extent, acid-sensitive TWIK-1. Fourth, the astrocyte passive conductance showed low sensitivity to extracellular Ba(2+), and extracellular Ba(2+) blocked TWIK-1 channels at an IC(50) of 960 microm and had no effect on TREK-1 channels. Finally, an immunocytochemical study showed colocalization of TWIK-1 and TREK-1 proteins with the astrocytic markers GLAST and GFAP in rat hippocampal stratum radiatum. In contrast, another K(2P) isoform TASK-1 was mainly colocalized with the neuronal marker NeuN in hippocampal pyramidal neurons and was expressed at a much lower level in astrocytes. These results support TWIK-1 and TREK-1 as being the major components of the long-sought K(+) channels underlying the passive conductance of mature hippocampal astrocytes.

Thiopurine-induced mitotic catastrophe in Rad51d-deficient mammalian cells.

Thiopurines are part of a clinical regimen used for the treatment of autoimmune disorders and childhood acute lymphoblastic leukemia. However, despite these successes, there are also unintended consequences such as therapy-induced cancer in long-term survivors. Therefore, a better understanding of cellular responses to thiopurines will lead to improved and personalized treatment strategies. RAD51D is an important component of homologous recombination (HR), and our previous work established that mammalian cells defective for RAD51D are more sensitive to the thiopurine 6-thioguanine (6TG) and have dramatically increased numbers of multinucleated cells and chromosome instability. 6TG is capable of being incorporated into telomeres, and interestingly, RAD51D contributes to telomere maintenance, although the precise function of RAD51D at the telomeres remains unclear. We sought here to investigate: (1) the activity of RAD51D at telomeres, (2) the contribution of RAD51D to protect against 6TG-induced telomere damage, and (3) the fates of Rad51d-deficient cells following 6TG treatment. These results demonstrate that RAD51D is required for maintaining the telomeric 3' overhangs. As measured by γ-H2AX induction and foci formation, 6TG induced DNA damage in Rad51d-proficient and Rad51d-deficient cells. However, the extent of γ-H2AX telomere localization following 6TG treatment was higher in Rad51d-deficient cells than in Rad51d-proficient cells. Using live-cell imaging of 6TG-treated Rad51d-deficient cells, two predominant forms of mitotic catastrophe were found to contribute to the formation of multinucleated cells, failed division and restitution. Collectively, these findings provide a unique window into the role of the RAD51D HR protein during thiopurine induction of mitotic catastrophe. Environ. Mol. Mutagen. 59:38-48, 2018. © 2017 Wiley Periodicals, Inc.

Cellular Model of p21-Induced Senescence.

Cellular senescence is a unique process of normal physiology, from embryonic development to aging, also known for its association with a broad range of pathological conditions. Therefore a reliable model of cellular senescence remains an indispensable tool for the investigation of senescence-associated changes and human disease. Here we describe a model of HT1080 fibrosarcoma cells with an inducible senescence phenotype. These cells are equipped with the lac repressor and exogenous p21 under the control of a lac repressor regulated promoter. The senescent phenotype is induced in these cells by isopropyl-ß-D-thiogalactopyranoside (IPTG)-inducible expression of senescence-associated cell cycle inhibitor p21Waf1/Cip1/Sdi1.

Differential display: a critical analysis.

Differential display (DD) is a well-established analytical tool for measuring gene expression that is still popular due to its documented success and ability to identify novel genes not yet available for analysis by more powerful microarray hybridization. For a comprehensive analysis of all mRNAs in a given cell, it is statistically predicted that at least 240 different DD primer combinations are required. This prediction, however, has never been empirically tested. Using far more primer combinations than that predicted to evaluate 90% of the mRNAs in a cell, plus other modifications, we identified and confirmed the induction of five mRNAs by hydrogen peroxide in HA-1 hamster cells. However, five other known oxidant-inducible mRNAs were not identified by DD. Filter microarray hybridization did not result in the identification of any additional species modulated twofold or greater but previous two-dimensional protein gel electrophoresis identified 15 induced protein species. We conclude that the current statistical prediction for comprehensive analysis of all the mRNAs in a given cell is inaccurate, at least in our hands, and further conclude that DD is a useful but less than comprehensive method for assessing changes in mRNA levels.

The contribution of TWIK-1 channels to astrocyte K(+) current is limited by retention in intracellular compartments.

TWIK-1 two-pore domain K(+) channels are expressed abundantly in astrocytes. In the present study, we examined the extent to which TWIK-1 contributes to the linear current-voltage (I-V) relationship (passive) K(+) membrane conductance, a dominant electrophysiological feature of mature hippocampal astrocytes. Astrocytes from TWIK-1 knockout mice have a more negative resting potential than those from wild type animals and a reduction in both inward rectification and Cs(+) permeability. Nevertheless, the overall whole-cell passive conductance is not altered significantly in TWIK-1 knockout astrocytes. The expression of Kir4.1 and TREK-1, two other major astrocytic K(+) channels, or of other two-pore K(+) channels is not altered in TWIK-1 knockout mice, suggesting that the mild effect of TWIK-1 knockout does not result from compensation by these channels. Fractionation experiments showed that TWIK-1 is primarily localized in intracellular cytoplasmic fractions (55%) and mildly hydrophobic internal compartment fractions (41%), with only 5% in fractions containing plasma membranes. Our study revealed that TWIK-1 proteins are mainly located in the intracellular compartments of hippocampal astrocyte under physiological condition, therefore a minimal contribution of TWIK-1 channels to whole-cell currents is likely attributable to a relatively low level presence of channels in the plasma membrane.

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells.

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIß, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIß and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G 2. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.

Tamoxifen mediated estrogen receptor activation protects against early impairment of hippocampal neuron excitability in an oxygen/glucose deprivation brain slice ischemia model.

Pretreatment of ovarectomized rats with estrogen shows long-term protection via activation of the estrogen receptor (ER). However, it remains unknown whether activation of the ER can provide protection against early neuronal damage when given acutely. We simulated ischemic conditions by applying oxygen and glucose deprived (OGD) solution to acute male rat hippocampal slices and examined the neuronal electrophysiological changes. Pyramidal neurons and interneurons showed a time-dependent membrane potential depolarization and reduction in evoked action potential frequency and amplitude over a 10 to 15 min OGD exposure. These changes were largely suppressed by 10 microM TAM. The TAM effect was neuron-specific as the OGD-induced astrocytic membrane potential depolarization was not altered. The TAM effect was mediated through ER activation because it could be simulated by 17beta-estradiol and was completely inhibited by the ER inhibitor ICI 182, 780, and is therefore an example of TAM's selective estrogen receptor modulator (SERM) action. We further show that TAM's effects on OGD-induced impairment of neuronal excitability was largely due to activation of neuroprotective BK channels, as the TAM effect was markedly attenuated by the BK channel inhibitor paxilline at 10 microM. TAM also significantly reduced the frequency and amplitude of AMPA receptor mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons which is an early consequence of OGD. Altogether, this study demonstrates that both 17beta-estradiol and TAM attenuate neuronal excitability impairment early on in a simulated ischemia model via ER activation mediated potentiation of BK K(+) channels and reduction in enhanced neuronal AMPA/NMDA receptor-mediated excitotoxicity.

Resveratrol attenuates early pyramidal neuron excitability impairment and death in acute rat hippocampal slices caused by oxygen-glucose deprivation.

Accumulating evidence indicates that the polyphenol resveratrol (trans-3, 5, 4"-trihydroxystibene, RVT) potently protects against cerebral ischemia neuronal damage due to its oxygen free radicals scavenging and antioxidant properties. However, it is unknown whether RVT can attenuate ischemia-induced early impairment of neuronal excitability. To address this question, we simulated ischemic conditions by applying oxygen-glucose deprivation (OGD) to acute rat hippocampal slices and examined the effect of RVT on OGD-induced pyramidal neuron excitability impairment using whole-cell patch clamp recording. 100 microM RVT largely inhibited the 15 min OGD-induced progressive membrane potential (Vm) depolarization and the reduction in evoked action potential frequency and amplitude in pyramidal neurons. In a parallel neuronal viability study using TO-PRO-3 iodide staining, 20 min OGD induced irreversible CA1 pyramidal neuronal death which was significantly reduced by 100 microM RVT. No similar effects were found with PQQ treatment, an antioxidant also showing potent neuroprotection in the rat rMCAO ischemia model. This suggests that antioxidant action per se, is unlikely accounting for the observed early effects of RVT. RVT also markedly reduced the frequency and amplitude of AMPA mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons, which is also an early consequence of OGD. RVT effects on neuronal excitability were inhibited by the large-conductance potassium channel (BK channel) inhibitor paxilline. Together, these studies demonstrate that RVT attenuates OGD-induced neuronal impairment occurring early in the simulated ischemia slice model by enhancing the activation of BK channel and reducing the OGD-enhanced AMPA/NMDA receptor mediated neuronal EPSCs.

Development of GLAST(+) astrocytes and NG2(+) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive.

Glia show marked heterogeneity in terms of electrophysiology in the developing brain, and two major types can be identified based on GFAP or NG2 expression. However, it remains to be determined if such an electrophysiological diversity holds for the adult brain and how GFAP and NG2 lineage glia are associated with different electrophysiological phenotypes during the course of development. To address these fundamental questions, we performed in situ whole cell recording from morphologically identified glia from the rat hippocampal CA1 region from postnatal (P) days 1-106 and double-stained postrecorded cells with GLAST and NG2 antibodies. We found glia express mostly voltage-gated outward K(+) currents and also have inward Na(+) currents in the newborn (P1-P3), but these are no longer present after P22. They consist equally of GLAST(+) and NG2(+) cells in the newborn, but are mainly NG2(+) in juvenile animals (P4-P21). Glia showing voltage-gated outward and inward K(+) currents are also present at P1, peak at P5 and decline to a stationary level of approximately 10% in the adult. They are GLAST(+) astrocytes from newborn to juvenile but NG2(+) glia in the adult. Electrophysiologically passive glia first appear at P4 and increase to 91% in adults, of which 85% are GLAST(+). These results indicate that glial electrophysiological diversity occurs predominantly in the developing brain. While most glia in the NG2 lineage preserve a certain amount of voltage-gated ion conductances, mature GLAST(+) astrocytes are electrophysiologically passive.

Development of gap junctions in hippocampal astrocytes: evidence that whole cell electrophysiological phenotype is an intrinsic property of the individual cell.

Gap junction communication between astrocytes is prevalent and has been proposed to be involved in several astrocyte functions. One such proposal involves gap junctions in potassium spatial buffering. However, little is known about the developmental time course of gap junction coupling and how much the syncytium affects whole cell measurements of ion currents. Our previous work described three types of hippocampal astrocyte, each with a distinct electrophysiological profile when recorded in whole cell voltage-clamp mode. In the current study we correlated post-whole cell recording immunohistochemistry for GLAST and the spread of injected dye from the recorded cell with the measured electrophysiological phenotype to quantify cell coupling of astrocytes and the type of astrocyte coupled, in the rat hippocampus. We found that passive astrocytes, which predominate after 3 wk postnatally, have much lower membrane resistances (Rm) and are more frequently dye coupled and to more cells, than outwardly and variably rectifying astrocytes that predominate in early postnatal development. Dye coupling in GLAST(+) cells was first detected in the first postnatal week and the degree of coupling peaked before the complete transition to the low Rm, passive electrophysiological type. Also, the degree of dye coupling did not correlate with the passive electrophysiological phenotype. Passive cells were also detected after pretreatment with a gap junction inhibitor. Further evidence that cell coupling does not contribute to the mature astrocyte electrophysiological phenotype came from recording of excised membrane patches, which predominantly corresponded to the ion channel expression profiles of their cells of origin. These findings imply that in the hippocampus, interastrocyte cell coupling likely contributes little to the overall whole cell current profile of diverse glia, and the electrophysiological passivity reflects the intrinsic ion channel expression of the mature astrocyte.

Pharmacological comparison of swelling-activated excitatory amino acid release and Cl- currents in cultured rat astrocytes.

Ubiquitously expressed volume-regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling-activated release of EAAs as D-[3H]aspartate efflux, and VRAC-mediated Cl- currents by whole-cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling-activated EAA release pathway and Cl- currents. The expression of candidate Cl- channels was confirmed by RT-PCR. The maxi Cl- channel (p-VDAC) blocker Gd3+, the ClC-2 inhibitor Cd2+, and the MDR-1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium-sensitive Cl- channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling-activated Cl- currents. The phorbol ester PDBu, which blocks ClC-3-mediated Cl- currents, had no effect on VRAC currents and up-regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume-dependent EAA release is largely mediated by the VRAC, and (ii) the ClC-2, ClC-3, ClC-4, ClC-5, VDAC, CaCC, MDR-1 and CFTR gene products do not contribute to EAA permeability.

Electrophysiologically "complex" glial cells freshly isolated from the hippocampus are immunopositive for the chondroitin sulfate proteoglycan NG2.

We have recently described a subgroup of isolated glial fibrillary acidic protein-positive (GFAP+) hippocampal astrocytes that predominantly express outwardly rectifying currents (which we term "ORAs" for outwardly rectifying astrocytes), which are similar to the currents already described for hippocampal GFAP- "complex glia." We now report that post-recording staining of cells that were first selected as "complex" by morphology and then confirmed by their electrophysiological characteristics were NG2+ approximately 90% of the time. Also, the morphology of freshly isolated NG2+ cells differs from that of isolated GFAP+ ORAs in having a smaller and round cell body with thinner processes, which usually are collapsed back onto the soma. Upon detailed examination, NG2+ cells were found to differ quantitatively in some electrophysiological characteristics from GFAP+ ORAs. The outward, transient K+ currents (IKa) in the NG2+ cells showed a slower decay than the IKa in ORAs, and their density decreased in NG2+ cells from older animals. The other two major cation currents, the voltage-activated Na+ and outwardly delayed rectifier K+ currents, were similar in NG2+ cells and ORAs. To further distinguish isolated complex cells from outwardly rectifying GFAP+ astrocytes, we performed immunocytochemistry for glial markers in fixed, freshly isolated rat hippocampal glia. NG2+ cells were negative for GFAP and also for the astrocytic glutamate transporters GLT-1 and GLAST. Thus the isolated hippocampal NG2+ glial cells, though having an electrophysiological phenotype similar to that of ORAs, are an immunologically and morphologically distinct glial cell population and most likely represent NG2+ cells in situ.