Karen J. Aitken

Rodents have the capacity for spontaneous bladder regeneration and bladder smooth muscle cell (BSMC) migration following a subtotal cystectomy (STC). YAP/WWTR1 and BDNF (Brain-derived neurotrophic factor) play crucial roles in development and regeneration. During partial bladder outlet obstruction (PBO), excessive YAP/WWTR1 signaling and BDNF expression increases BSMC hypertrophy and dysfunction. YAP/WWTR1 and expression of BDNF and CYR61 were examined in models of regeneration and wound repair. Live cell microscopy was utilized in an ex vivo model of STC to visualize cell movement and division. In Sprague-Dawley female rats, STC was performed by resection of the bladder dome sparing the trigone, followed by closure of the bladder. Smooth muscle migration and downstream effects on signaling and expression were also examined after scratch wound of BSMC with inhibitors of YAP and BDNF signaling. Sham, PBO and incision (cystotomy) were comparators for the STC model. Scratch wound in vitro increased SMC migration and expression of BDNF, CTGF and CYR61 in a YAP/WWTR1-dependent manner. Inhibition of YAP/WWTR1 and BDNF signaling reduced scratch-induced migration. BDNF and CYR61 expression was elevated during STC and PBO. STC induces discrete genes associated with endogenous de novo cell regeneration downstream of YAP/WWTR1 activation.

Partial bladder outlet obstruction due to prostate hyperplasia or posterior urethral valves, is a widespread cause of urinary dysfunction, patient discomfort and also responsible for immense health care costs. Even after removal or relief of obstruction, the functional and pathologic aspects of obstruction remain as a chronic obstructive bladder disease (COBD). Epigenetic changes, such as DNA methylation, contribute to the persistent character of many chronic diseases, and may be altered in COBD. We tested whether candidate genes and pathways and the pathophysiology of COBD were affected by a hypomethylating agent, decitabine (DAC). COBD was created in female Sprague-Dawley rats by surgical ligation of the urethra for 6 weeks, followed by removal of the suture. Sham ligations were performed by passing the suture behind the urethra. After removal of the obstruction or sham removal, animals were randomized to DAC treatment (1 mg/kg/3-times/week intraperitoneally) or vehicle (normal saline). Bladder function was non-invasively tested using metabolic cages, both one day prior to de-obstruction at 6 weeks and prior to sacrifice at 10 weeks. Residual volume and bladder mass were measured for each bladder. Bladders were examined by immunostaining as well as qPCR. The effects of DNA methyltransferase (DNMT)-3A knockout or overexpression on smooth muscle cell (SMC) function and phenotype were also examined in bladder SMC and ex vivo culture. Residual volumes of the DAC treated group were not significantly different from the NS group. Compared to COBD NS, COBD DAC treatment helped preserve micturition volume with a significant recovery of the voiding efficiency (ratio of the maximum voided volume/maximum bladder capacity) by one third (Fig. 1, p > 0.05). Brain-derived neurotrophic factor (BDNF) variants 1 and 5 were upregulated by COBD and significantly reduced by DAC treatment. Deposition of collagen in the COBD bladder was reduced by DAC, but gross hypertrophy remained. In bladder SMC, DNMT3A overexpression led to a loss of contractile function and phenotype. In bladders, persistently altered by COBD, inhibition of DNA-methylation enhances functional recovery, unlike treatment during partial obstruction, which exacerbates obstructive pathology. The underlying mechanisms may relate to the gene expression changes in BDNF and their effects on signaling in the bladder.

Neuroblastoma (NB), a tumor of the primitive neural crest, despite aggressive treatment portends a poor long-term survival for patients with advanced high stage NB. New treatment strategies are required. We investigated coordinated targeting of essential homeostatic regulatory factors involved in cancer progression, histone deacetylases (HDACs) and carbonic anhydrases (CAs). We evaluated the antitumor potential of the HDAC inhibitor (HDACi), pyridylmethyl-N-{4-[(2-aminophenyl)-carbamoyl]-benzyl}-carbamate (MS-275) in combination with a pan CA inhibitor, acetazolamide (AZ) on NB SH-SY5Y, SK-N-SH and SK-N-BE(2) cells. The key observation was that the combination AZ + MS-275 significantly inhibited growth, induced cell cycle arrest and apoptosis, and reduced migration capacity of NB cell line SH-SY5Y. In addition, this combination significantly inhibited tumor growth in vivo, in a pre-clinical xenograft model. Evidence was obtained for a marked reduction in tumorigenicity and in the expression of mitotic, proliferative, HIF-1α and CAIX. NB xenografts of SH-SY5Y showed a significant increase in apoptosis. MS-275 alone at nanomolar concentrations significantly reduced the putative cancer stem cell (CSC) fraction of NB cell lines, SH-SY5Y and SK-N-BE(2), in reference to NT2/D1, a teratocarcinoma cell line, exhibiting a strong stem cell like phenotype in vitro. Whereas stemness genes (OCT4, SOX2 and Nanog) were found to be significantly downregulated after MS-275 treatment, this was further enhanced by AZ co-treatment. The significant reduction in initial tumorigenicity and subsequent abrogation upon serial xenografting suggests potential elimination of the NB CSC fraction. The significant potentiation of MS-275 by AZ is a promising therapeutic approach and one amenable for administration to patients given their current clinical utility.

Host-pathogen interactions can induce epigenetic changes in the host directly, as well as indirectly through secreted factors. Previously, uropathogenic Escherichia coli (UPEC) was shown to increase DNA methyltransferase activity and expression, which was associated with methylation-dependent alterations in the urothelial expression of CDKN2A. Here, we showed that paracrine factors from infected cells alter expression of another epigenetic writer, EZH2, coordinate with proliferation. Urothelial cells were inoculated with UPEC, UPEC derivatives, or vehicle (mock infection) at low moi, washed, then maintained in media with Gentamycin. Urothelial conditioned media (CM) and extracellular vesicles (EV) were isolated after the inoculations and used to treat naïve urothelial cells. EZH2 increased with UPEC infection, inoculation-induced CM, and inoculation-induced EV vs. parallel stimulation derived from mock-inoculated urothelial cells. We found that infection also increased proliferation at one day post-infection, which was blocked by the EZH2 inhibitor UNC1999. Inhibition of demethylation at H3K27me3 had the opposite effect and augmented proliferation. CONCLUSION: Uropathogen-induced paracrine factors act epigenetically by altering expression of EZH2, which plays a key role in early host cell proliferative responses to infection.

Bronchial carcinoids (BC) are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive. The AC form of BC possesses a higher degree of invasiveness and metastatic potential. It is important to understand how the BC tumor initiating cells (TIC) or cancer stem cells (CSC) in BC account for this phenotype. In order to functionally characterize the suspected BC stem cell fraction, BC cell lines (H727 and H720) were grown under a non-adhesive condition to form 3D spheroids favouring expansion of the TIC. The third generation of spheroids was assessed for clonogenicity, tumorigenicity (xenograft model), stem cell and hypoxia markers, drug resistance, karyotype and microarray analysis compared to parental cells. We previously showed that a pan-carbonic anhydrase inhibitor, acetazolamide (AZ) significantly potentiated the anti-tumor effects of sulforophane (SFN, a natural isothiocyanate with HDACi activity) on these lung carcinoid cell lines in vitro and subcutaneous xenografts. Here we developed an orthotopic lung model to study tumor progression and metastatic behaviour of the spheroid forming BC cells. Orthotopically grown tumors were treated with AZ, SFN and the AZ+SFN combination using previous doses within clinically available ranges. Results demonstrated that the spheroid grown BC cells showed increased clonogenic, tumorigenic, and drug resistance potential, with enhanced expression of stem cell, carbonic anhydrase and hypoxia markers as compared with the control non-spheroid cells, all suggesting selection for the TIC or CSC fraction. As in the subcutaneous model the combination of AZ with SFN was more effective than either single agent alone in inhibiting orthotopic tumor growth and survival. Taken together the evidence indicates that the novel AZ+SFN combination is likely targeting the TIC/CSC population. This offers increased value in terms of the management of BC tumor progression and possible metastasis in BC patients presenting with more aggressive variants. Citation Format: Narges Baluch, Reza Bayat Mokhtari, Tina Homayouni, Pedram Akbari, Fatemeh Heshmati, Syed S Islam, Sushil Kumar, Karen Aitken, Carrie Fitzpatrick, Herman Yeger. The combination of Acetazolamide and Sulforophane targets the tumor initiating cells in human bronchial carcinoids. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C28.

Previous molecular studies showed that the mTOR inhibitor rapamycin prevents bladder smooth muscle hypertrophy in vitro. We investigated the effect of rapamycin treatment in vivo on bladder smooth muscle hypertrophy in a rat model of partial bladder outlet obstruction. A total of 48 female Sprague-Dawley® rats underwent partial bladder outlet obstruction and received daily subcutaneous injections of rapamycin (1 mg/kg) or vehicle commencing 2 weeks postoperatively. A total of 36 rats underwent sham surgery and received rapamycin or vehicle. Rats were sacrificed 3, 6 and 12 weeks after surgery. Before sacrifice, voiding was observed in a metabolic cage for 24 hours. Bladder-to-body weight in gm bladder weight per kg body weight and post-void residual urine were assessed. We evaluated Col1a1, Col3a1, Eln and Mmp7 mRNA expression and histology. Two-factor ANOVA and the post hoc t test were applied. Bladder outlet obstruction caused a significant increase in bladder weight in all obstructed groups. Three weeks postoperatively (1 week of treatment) there was no difference in the bladder-to-body weight ratio in the obstructed group. However, at 6 and 12 weeks (4 and 10 weeks of treatment, respectively) the bladder-to-body weight ratio of rats with obstruction plus rapamycin was significantly lower than that of rats with obstruction plus vehicle. Post-void residual urine volume after 6 and 12 weeks of obstruction was lower in obstructed rats with rapamycin compared to that in obstructed rats with vehicle. Rapamycin decreased the obstruction induced expression of Col1a1, Col3a1, Eln and Mmp7. Rapamycin prevents mechanically induced hypertrophy in cardiovascular smooth muscle. In vivo mTOR inhibition may attenuate obstruction induced detrusor hypertrophy and help preserve bladder function.

We used polarized light imaging and optical coherence tomography to quantify anisotropy in normal ex-vivo rat bladder during distension. The anisotropy change is due to micro-structural remodelling during distension rather than thickness change.