Non-metabolic functions of phosphofructokinase-1 orchestrate tumor cellular invasion and genome maintenance under bevacizumab therapy.

BACKGROUND: Glioblastoma (GBM) is a highly lethal malignancy for which neoangiogenesis serves as a defining hallmark. The anti-VEGF antibody, bevacizumab, has been approved for the treatment of recurrent GBM, but resistance is universal. METHODS: We analyzed expression data of GBM patients treated with bevacizumab to discover potential resistance mechanisms. Patient-derived xenografts (PDXs) and cultures were interrogated for effects of phosphofructokinase-1, muscle isoform (PFKM) loss on tumor cell motility, migration, and invasion through genetic and pharmacologic targeting. RESULTS: We identified PFKM as a driver of bevacizumab resistance. PFKM functions dichotomize based on subcellular location: cytosolic PFKM interacted with KIF11, a tubular motor protein, to promote tumor invasion, whereas nuclear PFKM safeguarded genomic stability of tumor cells through interaction with NBS1. Leveraging differential transcriptional profiling, bupivacaine phenocopied genetic targeting of PFKM, and enhanced efficacy of bevacizumab in preclinical GBM models in vivo. CONCLUSION: PFKM drives novel molecular pathways in GBM, offering a translational path to a novel therapeutic paradigm.

Effect of Bladder Injection of OnabotulinumtoxinA on the Central Expression of Genes Associated with the Control of the Lower Urinary Tract: A Study in Normal Rats.

To investigate a possible central mechanism of action of Botulinum toxin A (BoNT/A) following injection in the bladder, complementary to the acknowledged peripheral bladder effect, we studied changes in the expression of neuropeptides and receptors involved in lower urinary tract function in the spinal cord (SC) and dorsal root ganglia (DRG) of normal rats following BoNT/A bladder injection. Thirty-six Sprague-Dawley rats, divided into three groups of n = 12, received bladder injections of 2U or 5U OnabotulinumtoxinA (BOTOX®), or saline. Six animals from each group were sacrificed on days 7 and 14. Expression of Tachykinin 1 (Tac1), capsaicin receptor (TRPV1), neuropeptide Y (NPY), proenkephalin (PENK) and muscarinic receptors M1, M2, M3, was evaluated in the bladder, L6-S1 DRG, and SC segments using real-time PCR and Western blotting. Real-time PCR revealed increased expression of NPY in all tissues except for SC, and increased TRPV1 and PENK expression in DRG and SC, whereas expression of Tac1, M1 and M2 was decreased. Less significant changes were noted in protein levels. These findings suggest that bladder injections of OnabotulinumtoxinA may be followed by changes in the expression of sensory, sympathetic and cholinergic bladder function regulators at the DRG/SC level.

SPT6-driven error-free DNA repair safeguards genomic stability of glioblastoma cancer stem-like cells.

Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility of DNA lesions to DNA repair machineries are crucial for the maintenance of genomic stability. Understanding the functional impact of chromatin remodeling on DNA repair in GSCs may lay the foundation for advancing the efficacy of radio-sensitizing therapies. Here, we present the results of a high-content siRNA microscopy screen, revealing the transcriptional elongation factor SPT6 to be critical for the genomic stability and self-renewal of GSCs. Mechanistically, SPT6 transcriptionally up-regulates BRCA1 and thereby drives an error-free DNA repair in GSCs. SPT6 loss impairs the self-renewal, genomic stability and tumor initiating capacity of GSCs. Collectively, our results provide mechanistic insights into how SPT6 regulates DNA repair and identify SPT6 as a putative therapeutic target in glioblastoma.

Retrograde transport of radiolabelled botulinum neurotoxin type A to the CNS after intradetrusor injection in rats.

OBJECTIVE: To investigate the potential distribution of radiolabelled botulinum neurotoxin type A (BoNT/A) in the CNS after bladder injection in normal rats, by using the gamma-emitting radionuclide technetium-99 m ((99m) Tc). MATERIALS AND METHODS: BoNT/A was radiolabelled by pretreatment with 2-iminothiolane and incubation with (99m) Tc-gluconate. The labelled toxin (99m) Tc-BoNT/A was purified using size exclusion HPLC. Twenty-four female Wistar rats were evenly injected in the bladder wall with either (99m) Tc-ΒοΝΤ/Α (n = 12) or free (99m) Tc (n = 12). Four rats from each group were killed at 1, 3 and 6 h after injection, respectively. The bladder, L6-S1 spinal cord segment and L6-S1 dorsal root ganglia (DRG) were harvested and their radioactivity counted in a gamma scintillation detector. Results were calculated as % injected dose (I.D.) per gram of tissue. The paired t-test was used for comparison of means of (99m) Tc-ΒοΝΤ/Α radioactivity vs free (99m) Tc in the tissues of interest. RESULTS: Radiolabelled BoNT/A had a high radiochemical stability of 70% after 24 h. Gradual accumulation of (99m) Tc-ΒοΝΤ/Α was observed in the DRG up to 6 h after injection (P = 0.04 and P = 0.029 compared with 1 h and 3 h, respectively), while no accumulation was detected for free (99m) Tc. Consequently, (99m) Tc-ΒοΝΤ/Α radioactivity in the DRG was higher than free (99m) Tc radioactivity (3.18 ± 0.67% I.D./g vs 0.19 ± 0.10% I.D./g [P = 0.002] 6 h after injection). Values for (99m) Tc-ΒοΝΤ/Α radioactivity in the spinal cord were higher than those for free (99m) Tc, but not significantly. The bladder retained higher dosages of (99m) Tc-ΒοΝΤ/Α than free (99m) Tc at all time points. CONCLUSIONS: Significant accumulation of the radiolabelled toxin in the lumbosacral DRG, together with a less significant uptake in the respective spinal cord segment as opposed to free radioactivity provide first evidence of the retrograde transport of BoNT/A to the CNS after bladder injection in rats.

A novel mechanism of TGFbeta-induced actin reorganization mediated by Smad proteins and Rho GTPases.

In previous studies, we have demonstrated that RhoA/B-dependent signaling regulates TGFbeta-induced rapid actin reorganization in Swiss 3T3 fibroblasts. Here we report that TGFbeta regulates long-term actin remodeling by increasing the steady-state mRNA levels of the RhoB gene in mouse Swiss 3T3 fibroblasts and human hepatoma HepG2 cells. We show that this regulation is specific for the RhoB gene and is facilitated by enhanced activity of the RhoB promoter. Adenovirus-mediated gene transfer of Smad2 and Smad3 in Swiss 3T3 fibroblasts induced transcription of the endogenous RhoB gene but not the RhoA gene. Interestingly, in JEG-3 choriocarcinoma cells that lack endogenous Smad3, TGFbeta-induced transcriptional up-regulation of the RhoB gene was not observed, but it was restored by adenoviral Smad3 overexpression. In addition, Smad2 and Smad3 triggered activation of RhoA and RhoB GTPases and long-term actin reorganization in Swiss 3T3 fibroblasts. Finally, Smad3, and to a lesser extent Smad2, induced transcription of the alpha-smooth muscle actin (alpha-SMA) gene, and enhanced the incorporation of alpha-SMA into microfilaments in Swiss 3T3 fibroblasts. These data reveal a novel mechanism of cross-talk between the classical TGFbeta/Smad pathway and Rho GTPases, regulating the rapid and the long-term actin reorganization that may control the fibroblast-myofibroblast differentiation program.

Neurosteroids as endogenous inhibitors of neuronal cell apoptosis in aging.

The neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester DHEAS, and allopregnanolone (Allo) are produced in the adrenals and the brain. Their production rate and levels in serum, brain, and adrenals decrease gradually with advancing age. The decline of their levels was associated with age-related neuronal dysfunction and degeneration, most probably because these steroids protect central nervous system (CNS) neurons against noxious agents. Indeed, DHEA(S) protects rat hippocampal neurons against NMDA-induced excitotoxicity, whereas Allo ameliorates NMDA-induced excitotoxicity in human neurons. These steroids exert also a protective role on the sympathetic nervous system. Indeed, DHEA, DHEAS, and Allo protect chromaffin cells and the sympathoadrenal PC12 cells (an established model for the study of neuronal cell apoptosis and survival) against serum deprivation-induced apoptosis. Their effects are time- and dose-dependent with EC(50) 1.8, 1.1, and 1.5 nM, respectively. The prosurvival effect of DHEA(S) appears to be NMDA-, GABA(A)- sigma1-, or estrogen receptor-independent, and is mediated by G-protein-coupled-specific membrane binding sites. It involves the antiapoptotic Bcl-2 proteins, and the activation of prosurvival transcription factors CREB and NF-kappaB, upstream effectors of the antiapoptotic Bcl-2 protein expression, as well as prosurvival kinase PKCalpha/beta, a posttranslational activator of Bcl-2. Furthermore, they directly stimulate biosynthesis and release of neuroprotective catecholamines, exerting a direct transcriptional effect on tyrosine hydroxylase, and regulating actin depolymerization and submembrane actin filament disassembly, a fast-response cellular system regulating trafficking of catecholamine vesicles. These findings suggest that neurosteroids may act as endogenous neuroprotective factors. The decline of neurosteroid levels during aging may leave the brain unprotected against neurotoxic challenges.

LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta.

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.