Prospective Early Clinical, Radiological, and Kinematic Pedobarographic Analysis Following Subtalar Arthroereises for Paediatric Pes Planovalgus.

Introduction Arthroereises implants mechanically block eversion and limit subtalar motion. They are used in children with pes planovalgus in order to correct the valgus deformity. In this study, we aimed to objectively assess children with flatfoot before and after the insertion of the Kalix II implant, clinically, radiologically and by kinematic pedobarographic analysis. Materials and methods Six children (12 feet) were treated by the insertion of the Kalix II implant (Integra LifeSciences, Plainsboro, NJ). Patients completed the Manchester Oxford Foot Questionnaire (MOXFQ) preoperatively and at six months post operatively. Radiological outcome was assessed by lateral (L) and anterior posterior (AP) foot weight-bearing radiographs taken pre operatively and post operatively. Pedobarographic data was obtained pre operatively and at six months post operatively using a 1 meter RS Scan Footscan (RSscan International, Olen, Belgium) pedobarograph. In addition, patients underwent gait analysis pre and post operatively. Results Mean age was 11.05 +/-3.24 years (range 6.2 to 15.5 years). In all cases, screw removal was carried out at between 15 to 18 months post insertion. The mean pre op MOXFQ score was 55.3 +/-9.68 which reduced to 34.3 +/-15.66 post operatively with a p value < 0.00001 which was statistically significant. Mean Meary's angle preop was -15.21+/-5.51 degrees which corrected to -7.57+/-4.62 post op with a p value=0.00001. The mean calcaneal pitch before surgery was 11.96+/-3.8 which increased to 14.98+/-3.85 with a p value =0.00067. The first MTH: fifth MTH peak pressure ratio pre operatively was 4.53+/-2.78 which was found to reduce significantly post operatively to 1.35+/-0.97 (p=0.04), indicating a lateral shift of the foot pressures. Conclusion There were statistically significant improvements in the patient-reported MOXFQ, radiological improvements, and pedobarographic changes, indicating a lateral shift of the foot pressures. There were no complications.

Radiosensitization In Vivo by Histone Deacetylase Inhibition with No Increase in Early Normal Tissue Radiation Toxicity.

As the population ages, more elderly patients require radiotherapy-based treatment for their pelvic malignancies, including muscle-invasive bladder cancer, as they are unfit for major surgery. Therefore, there is an urgent need to find radiosensitizing agents minimally toxic to normal tissues, including bowel and bladder, for such patients. We developed methods to determine normal tissue toxicity severity in intestine and bladder in vivo, using novel radiotherapy techniques on a small animal radiation research platform (SARRP). The effects of panobinostat on in vivo tumor growth delay were evaluated using subcutaneous xenografts in athymic nude mice. Panobinostat concentration levels in xenografts, plasma, and normal tissues were measured in CD1-nude mice. CD1-nude mice were treated with drug/irradiation combinations to assess acute normal tissue effects in small intestine using the intestinal crypt assay, and later effects in small and large intestine at 11 weeks by stool assessment and at 12 weeks by histologic examination. In vitro effects of panobinostat were assessed by qPCR and of panobinostat, TMP195, and mocetinostat by clonogenic assay, and Western blot analysis. Panobinostat resulted in growth delay in RT112 bladder cancer xenografts but did not significantly increase acute (3.75 days) or 12 weeks' normal tissue radiation toxicity. Radiosensitization by panobinostat was effective in hypoxic bladder cancer cells and associated with class I HDAC inhibition, and protein downregulation of HDAC2 and MRE11. Pan-HDAC inhibition is a promising strategy for radiosensitization, but more selective agents may be more useful radiosensitizers clinically, resulting in fewer systemic side effects. Mol Cancer Ther; 17(2); 381-92. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

E3 Ligase cIAP2 Mediates Downregulation of MRE11 and Radiosensitization in Response to HDAC Inhibition in Bladder Cancer.

The MRE11/RAD50/NBS1 (MRN) complex mediates DNA repair pathways, including double-strand breaks induced by radiotherapy. Meiotic recombination 11 homolog (MRE11) is downregulated by histone deacetylase inhibition (HDACi), resulting in reduced levels of DNA repair in bladder cancer cells and radiosensitization. In this study, we show that the mechanism of this downregulation is posttranslational and identify a C-terminally truncated MRE11, which is formed after HDAC inhibition as full-length MRE11 is downregulated. Truncated MRE11 was stabilized by proteasome inhibition, exhibited a decreased half-life after treatment with panobinostat, and therefore represents a newly identified intermediate induced and degraded in response to HDAC inhibition. The E3 ligase cellular inhibitor of apoptosis protein 2 (cIAP2) was upregulated in response to HDAC inhibition and was validated as a new MRE11 binding partner whose upregulation had similar effects to HDAC inhibition. cIAP2 overexpression resulted in downregulation and altered ubiquitination patterns of MRE11 and mediated radiosensitization in response to HDAC inhibition. These results highlight cIAP2 as a player in the DNA damage response as a posttranscriptional regulator of MRE11 and identify cIAP2 as a potential target for biomarker discovery or chemoradiation strategies in bladder cancer. Cancer Res; 77(11); 3027-39. ©2017 AACR.

Deoxycytidine kinase expression underpins response to gemcitabine in bladder cancer.

PURPOSE: In a recent phase II clinical trial, low-dose (100 mg/m(2)) gemcitabine showed promise as a radiosensitizer in bladder cancer, but underlying mechanisms lack elucidation. Here, we investigated the mechanism of radiosensitization by low-dose gemcitabine in bladder cancer cell lines. EXPERIMENTAL DESIGN: Four bladder cancer cell lines were screened for radiosensitization by low-dose gemcitabine using clonogenic assay, and gemcitabine-resistant RT112gem and CALgem cells created by exposure to increasing gemcitabine doses. Four key gemcitabine-regulatory genes were knocked down by transient siRNA. Nude mice carrying CALgem subcutaneous xenografts were exposed to 100 mg/kg gemcitabine ± ionizing radiation (IR) and response assessed by tumor growth delay. RESULTS: Gemcitabine was cytotoxic in the low nanomolar range (10-40 nmol/L) in four bladder cancer cell lines and radiosensitized all four lines. Sensitizer enhancement ratios at 10% survival were: RT112 1.42, CAL29 1.55, T24 1.63, and VMCUB1 1.47. Transient siRNA knockdown of deoxycytidine kinase (dCK) significantly reduced radiosensitization by gemcitabine (P = 0.02). RT112gem and CALgem cells displayed robust decreases of dCK mRNA and protein levels; reexpression of dCK restored gemcitabine sensitivity. However, CALgem xenografts responded better to combination gemcitabine/IR than either treatment alone (P < 0.001) with dCK strongly expressed in the tumor vasculature and stroma. CONCLUSIONS: Gemcitabine resistance in bladder cancer cell lines was associated with decreased dCK expression, but gemcitabine-resistant xenografts were responsive to combination low-dose gemcitabine/IR. We propose that dCK activity in tumor vasculature renders it gemcitabine sensitive, which is sufficient to invoke a tumor response and permit tumor cell kill in gemcitabine-resistant tumors.

Post-transcriptional regulation of MRE11 expression in muscle-invasive bladder tumours.

Predictive assays are needed to help optimise treatment in muscle-invasive bladder cancer, where patients can be treated by either cystectomy or radical radiotherapy. Our finding that low tumour MRE11 expression is predictive of poor response to radiotherapy but not cystectomy was recently independently validated. Here we investigated further the mechanism underlying low MRE11 expression seen in poorly-responding patients. MRE11 RNA and protein levels were measured in 88 bladder tumour patient samples, by real-time PCR and immunohistochemistry respectively, and a panel of eight bladder cancer cell lines was screened for MRE11, RAD50 and NBS1 mRNA and protein expression. There was no correlation between bladder tumour MRE11 protein and RNA scores (Spearman's rho 0.064, p=0.65), suggesting MRE11 is controlled post-transcriptionally, a pattern confirmed in eight bladder cancer cell lines. In contrast, NBS1 and RAD50 mRNA and protein levels were correlated (p=0.01 and p=0.03, respectively), suggesting primary regulation at the level of transcription. MRE11 protein levels were correlated with NBS1 and RAD50 mRNA and protein levels, implicating MRN complex formation as an important determinant of MRE11 expression, driven by RAD50 and NBS1 expression. Our findings of the post-transcriptional nature of the control of MRE11 imply that any predictive assays used in patients need to be performed at the protein level rather than the mRNA level.

Radiosensitisation of bladder cancer cells by panobinostat is modulated by Ku80 expression.

BACKGROUND AND PURPOSE: In muscle-invasive bladder cancer there is an urgent need to identify relatively non-toxic radiosensitising agents for use in elderly patients. Histone deacetylase inhibitors radiosensitise tumour cells but not normal cells in vitro and variously downregulate DNA damage signalling, homologous recombination (HR) and non-homologous end-joining (NHEJ) repair proteins. We investigated panobinostat (PAN) as a potential radiosensitiser in bladder cancer cells. MATERIALS AND METHODS: Clonogenic assays were performed in RT112 bladder cancer cells, and RT112 cells stably knocked down for RAD51 or Ku80 by shRNAi. Resolution of γH2AX foci was determined by immunofluorescence confocal microscopy, cell cycle progression by FACS analysis and protein expression by western blotting. RESULTS: PAN had a greater radiosensitising effect in Ku80KD than RT112 or RAD51KD cells; enhancement ratios 1.35 for Ku80KD at 10nM (IC(20) for Ku80KD) and 1.31 for RT112 and RAD51KD at 25 nM (IC(40) for both). PAN downregulated MRE11, NBS1 and RAD51, but not Ku70 and Ku80, increased γH2AX foci formation in a dose-dependent manner and delayed γH2AX foci repair after ionising radiation. CONCLUSIONS: PAN acts as a radiosensitiser in bladder cancer cell lines, and appears to target HR rather than NHEJ. As muscle-invasive bladder tumours have reduced Ku-DNA binding, PAN could be particularly useful as a radiosensitiser in bladder cancer.

Imatinib radiosensitizes bladder cancer by targeting homologous recombination.

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611-20. ©2012 AACR.

Depletion of the type 1 IGF receptor delays repair of radiation-induced DNA double strand breaks.

BACKGROUND AND PURPOSE: IGF-1R depletion sensitizes prostate cancer cells to ionizing radiation and DNA-damaging cytotoxic drugs. This study investigated the hypothesis that IGF-1R regulates DNA double strand break (DSB) repair. METHODS: We tested effects of IGF-1R siRNA transfection on the repair of radiation-induced DSBs by immunoblotting and immunofluorescence for γH2AX, and pulsed-field gel electrophoresis. Homologous recombination (HR) was quantified by reporter assays, and cell cycle distribution by flow cytometry. RESULTS: We confirmed that IGF-1R depletion sensitized DU145 and PC3 prostate cancer cells to ionizing radiation. DU145 control transfectants resolved radiation-induced DSBs within 24 h, while IGF-1R depleted cells contained 30-40% unrepaired breaks at 24 h. IGF-1R depletion induced significant reduction in DSB repair by HR, although the magnitude of the repair defect suggests additional contributory factors. Radiation-induced G2-M arrest was attenuated by IGF-1R depletion, potentially suppressing cell cycle-dependent processes required for HR. In contrast, IGF-1R depletion induced only minor radiosensitization in LNCaP cells, and did not influence repair. Cell cycle profiles were similar to DU145, so were unlikely to account for differences in repair responses. CONCLUSIONS: These data indicate a role for IGF-1R in DSB repair, at least in part via HR, and support use of IGF-1R inhibitors with DNA damaging cancer treatments.

Modulation of Drosophila retinal epithelial integrity by the adhesion proteins capricious and tartan.

BACKGROUND: The development of the Drosophila eye imaginal disc requires complex epithelial rearrangements. Cells of the morphogenetic furrow are apically constricted and this leads to a physical indentation in the epithelium. Posterior to the furrow, cells start to rearrange into distinct clusters and eventually form a precisely patterned array of ommatidia. These morphogenetic processes include regulated changes of adhesion between cells. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that two transmembrane adhesion proteins, Capricious and Tartan, have dynamic and complementary expression patterns in the eye imaginal disc. We also describe novel null mutations in capricious and double null mutations in capricious and tartan. We report that they have redundant functions in regulating the architecture of the morphogenetic furrow and ommatidial spacing. CONCLUSIONS/SIGNIFICANCE: We conclude that Capricious and Tartan contribute to the adhesive properties of the cells in the morphogenetic furrow and that this regulated adhesion participates in the control of spacing ommatidial clusters.

The EGFR ligands Spitz and Keren act cooperatively in the Drosophila eye.

The EGFR signalling cascade is responsible for coordinating a wide variety of events during Drosophila eye development. It remains something of a mystery how it is that cells are able to interpret the signal so as to choose the appropriate response from the battery of possibilities: division, differentiation, cell shape change and so on. Since the cascade is essentially linear below the receptor, different cellular responses cannot be regulated by alternative signal transduction pathways. The main diversity lies upstream, in the multiple activating ligands. Spitz, Gurken and Vein have been long studied, but little is known about the physiological functions of the fourth ligand, Keren, although various roles have been predicted based on the differences between mutants in the known ligands and those of the receptor. Here, we have isolated a mutant in the keren gene, and demonstrate that Keren does indeed participate in EGFR signalling in the eye, where it acts redundantly with Spitz to control R8 spacing, cell clustering and survival. Thus, specificity cannot be determined by ligand choice, and must instead be a consequence of cell-intrinsic factors, although we speculate that there may be some quantitative differences in signalling elicited by the two ligands.

Cell-specific manipulation of second messengers; a toolbox for integrative physiology in Drosophila.

Every living cell must detect, and respond appropriately to, external signals. The functions of intracellular second messengers, such as guanosine 3'5'-cyclic monophosphate (cGMP), adenosine 3'5'-cyclic monophosphate (cAMP), and intracellular calcium, are thus intensively studied. However, artifact-free manipulation of these messengers is problematic, and simple pharmacology may not allow selective intervention in distinct cell types in a real, complex tissue. We have devised a method by which second messenger levels can be manipulated in cells of choice using the GAL4/UAS system. By placing different receptors (rat atrial natriuretic peptide [ANP] receptor and Drosophila serotonin receptors [5HT(Dro7) and 5HT(Dro1A)]) under UAS control, they can be targeted to arbitrary defined populations of cells in any tissue of the fly, and second messenger levels can be manipulated simply by adding the natural ligand. The potential of the system is illustrated in the Drosophila renal (Malpighian) tubule, where each receptor was shown to stimulate fluid secretion, to act through its cognate second messenger, and to be blocked by appropriate pharmacological antagonists. The results uncovered a new role for cGMP signaling in tubule and also demonstrate the utility of the tubule as a possible in vivo test bed for novel receptors, ligands, or agonists/antagonists.