A PI3-kinase-mediated negative feedback regulates neuronal excitability.

Use-dependent downregulation of neuronal activity (negative feedback) can act as a homeostatic mechanism to maintain neuronal activity at a particular specified value. Disruption of this negative feedback might lead to neurological pathologies, such as epilepsy, but the precise mechanisms by which this feedback can occur remain incompletely understood. At one glutamatergic synapse, the Drosophila neuromuscular junction, a mutation in the group II metabotropic glutamate receptor gene (DmGluRA) increased motor neuron excitability by disrupting an autocrine, glutamate-mediated negative feedback. We show that DmGluRA mutations increase neuronal excitability by preventing PI3 kinase (PI3K) activation and consequently hyperactivating the transcription factor Foxo. Furthermore, glutamate application increases levels of phospho-Akt, a product of PI3K signaling, within motor nerve terminals in a DmGluRA-dependent manner. Finally, we show that PI3K increases both axon diameter and synapse number via the Tor/S6 kinase pathway, but not Foxo. In humans, PI3K and group II mGluRs are implicated in epilepsy, neurofibromatosis, autism, schizophrenia, and other neurological disorders; however, neither the link between group II mGluRs and PI3K, nor the role of PI3K-dependent regulation of Foxo in the control of neuronal excitability, had been previously reported. Our work suggests that some of the deficits in these neurological disorders might result from disruption of glutamate-mediated homeostasis of neuronal excitability.

Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo.

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.

Defective Replication Stress Response Is Inherently Linked to the Cancer Stem Cell Phenotype.

Extensive spontaneous DNA damage from oncogene-induced replication stress is ubiquitous in precancerous lesions. While this damage induces differentiation, senescence, or apoptosis in normal cells, defects in DNA replication stress response (RSR) allow cells to continue proliferating, ultimately leading to early tumorigenesis. Using systems-level approaches, we developed a replication stress response defect gene signature that predicted risk of cancer development from hyperplastic lesions. Intriguingly, we found that replication stress response defects rewire non-malignant cells into a cancer stem cell (CSC)-like state, and analysis of CSCs indicated that they inherently harbor replication stress response defects. High-throughput drug screening to elucidate molecules required for survival of replication stress response defective cells identified a dependence on MEK/ERK signaling. Inhibition of this signaling cascade restored oncogene-induced senescence through a p53-independent MDM2/p21 axis. Moreover, MEK/ERK inhibition also depleted CSC populations. Together, these findings provide insights into the role of replication stress response defects in CSCs and an actionable pathway for therapeutic targeting.

MicroPET/CT Imaging of AXL Downregulation by HSP90 Inhibition in Triple-Negative Breast Cancer.

AXL receptor tyrosine kinase is overexpressed in a number of solid tumor types including triple-negative breast cancer (TNBC). AXL is considered an important regulator of epithelial-to-mesenchymal transition (EMT) and a potential therapeutic target for TNBC. In this work, we used microPET/CT with 64Cu-labeled anti-human AXL antibody (64Cu-anti-hAXL) to noninvasively interrogate the degradation of AXL in vivo in response to 17-allylamino-17-demethoxygeldanamycin (17-AAG), a potent inhibitor of HSP90. 17-AAG treatment caused significant decline in AXL expression in orthotopic TNBC MDA-MB-231 tumors, inhibited EMT, and delayed tumor growth in vivo, resulting in significant reduction in tumor uptake of 64Cu-anti-hAXL as clearly visualized by microPET/CT. Our data indicate that 64Cu-anti-hAXL can be useful for monitoring anti-AXL therapies and for assessing inhibition of HSP90 molecular chaperone using AXL as a molecular surrogate.

Improved prediction of PARP inhibitor response and identification of synergizing agents through use of a novel gene expression signature generation algorithm.

Despite rapid advancement in generation of large-scale microarray gene expression datasets, robust multigene expression signatures that are capable of guiding the use of specific therapies have not been routinely implemented into clinical care. We have developed an iterative resampling analysis to predict sensitivity algorithm to generate gene expression sensitivity profiles that predict patient responses to specific therapies. The resultant signatures have a robust capacity to accurately predict drug sensitivity as well as the identification of synergistic combinations. Here, we apply this approach to predict response to PARP inhibitors, and show it can greatly outperforms current clinical biomarkers, including BRCA1/2 mutation status, accurately identifying PARP inhibitor-sensitive cancer cell lines, primary patient-derived tumor cells, and patient-derived xenografts. These signatures were also capable of predicting patient response, as shown by applying a cisplatin sensitivity signature to ovarian cancer patients. We additionally demonstrate how these drug-sensitivity signatures can be applied to identify novel synergizing agents to improve drug efficacy. Tailoring therapeutic interventions to improve patient prognosis is of utmost importance, and our drug sensitivity prediction signatures may prove highly beneficial for patient management.

mTOR Inhibitors Suppress Homologous Recombination Repair and Synergize with PARP Inhibitors via Regulating SUV39H1 in BRCA-Proficient Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is a highly heterogeneous disease and has the worst outcome among all subtypes of breast cancers. Although PARP inhibitors represent a promising treatment in TNBC with BRCA1/BRCA2 mutations, there is great interest in identifying drug combinations that can extend the use of PARP inhibitors to a majority of TNBC patients with wild-type BRCA1/BRCA2 Here we explored whether mTOR inhibitors, through modulating homologous recombination (HR) repair, would provide therapeutic benefit in combination with PARP inhibitors in preclinical models of BRCA-proficient TNBC. EXPERIMENTAL DESIGN: We have studied the effects of mTOR inhibitors on HR repair following DNA double-strand breaks (DSB). We further demonstrated the in vitro and in vivo activities of combined treatment of mTOR inhibitors with PARP inhibitors in BRCA-proficient TNBC. Moreover, microarray analysis and rescue experiments were used to investigate the molecular mechanisms of action. RESULTS: We found that mTOR inhibitors significantly suppressed HR repair in two BRCA-proficient TNBC cell lines. mTOR inhibitors and PARP inhibitors in combination exhibited strong synergism against these TNBC cell lines. In TNBC xenografts, we observed enhanced efficacy of everolimus in combination with talazoparib (BMN673) compared with either drug alone. We further identified through microarray analysis and by rescue assays that mTOR inhibitors suppressed HR repair and synergized with PARP inhibitors through regulating the expression of SUV39H1 in BRCA-proficient TNBCs. CONCLUSIONS: Collectively, these findings strongly suggest that combining mTOR inhibitors and PARP inhibitors would be an effective therapeutic approach to treat BRCA-proficient TNBC patients.

TUSC4 functions as a tumor suppressor by regulating BRCA1 stability.

BRCA1 expression is lost frequently in breast cancers in which it promotes malignant development. In the present study, we performed a global expression analysis of breast cancer cells in which the tumor-suppressor candidate gene TUSC4 was silenced to gain insights into its function. TUSC4 silencing affected genes involved in cell cycle and cell death, which have broad reaching influence on cancer development. Most importantly, we found a cluster pattern of gene-expression profiles in TUSC4-silenced cells that defined a homologous recombination (HR) repair defect signature. Mechanistic investigations indicated that TUSC4 protein could physically interact with the E3 ligase Herc2, which prevents BRCA1 degradation through the ubiquitination pathway. TUSC4 silencing enhanced BRCA1 polyubiquitination, leading to its degradation and a marked reduction in HR repair efficiency. Notably, ectopic expression of TUSC4 suppressed the proliferation, invasion, and colony formation of breast cancer cells in vitro and tumorigenesis in vivo. Furthermore, TUSC4 silencing was sufficient to transform normal mammary epithelial cells and to enhance sensitivity to PARP inhibitors. Our results provide a set of genetic and biologic proofs that TUSC4 functions as a bona fide tumor suppressor by regulating the protein stability and function of BRCA1 in breast cancer.