Balram Sukhu

Regulation of Osteogenesis by Fetuin

Osteoporosis is a common problem of aging and results from a failure of homeostatic mechanisms to regulate osteogenesis and mineralization. Bovine and human forms of fetuin glycoprotein bind to the transforming growth factor (TGF)-β/BMP (bone morphogenic protein) cytokines and block their osteogenic activity in cell culture assays (Demetriou, M., Binkert, C., Sukhu, B., Tenenbaum, H. C., and Dennis, J. W. (1996) J. Biol. Chem. 271, 12755–12761). Fetuin is a prominent serum glycoprotein and a major noncollagenous component of mineralized bone in mammals. In this study, we show that recombinant fetuin and native serum protein have similar potency as inhibitors of osteogenesis in dexamethasone-treated rat bone marrow cell cultures (dex-RBMC). Recombinant bovine fetuin also bound to TGF-β1 and BMP-2 in vitro with kinetics similar to native fetuin. Although TGF-β1 is required for osteogenesis in dex-RBMC, the cytokine also inhibited osteogenesis at concentrations ≥10 pm. Titration of fetuin or anti-TGF-β1 antibodies into the bone marrow cultures in the presence of 10 pm TGF-β1 restored osteogenesis, whereas titrations of the same reagents into cultures with 0.3 pmadded TGF-β1 were inhibitory, confirming the biphasic nature of the TGF-β1 response. Suppression of osteogenesis by both TGF-β1 and the antagonist proteins required their presence within the first 6 days of culture, well before mineralization at 10–12 days. Northern analysis showed that both fetuin and high dose TGF-β1 suppressed expression of the bone-associated transcripts alkaline phosphatase, osteopontin, collagen type I, and bone sialoprotein. The suppression of osteogenesis by fetuin and by high dose TGF-β1 was accompanied by the differentiation of an alternate cell lineage with adipocyte characteristics. In summary, the biphasic osteogenic response to TGF-β1 suggests that overlapping gradients of TGF-β/BMP cytokines and fetuin regulate osteogenesis in remodeling bone.

Expression of tissue non-specific alkaline phosphatase stimulates differentiated behaviour in specific transformed cell populations.

Tissue non‐specific alkaline phosphatase (TN‐AP) is a membrane‐bound glycoprotein enzyme which is characterized by its phosphohydrolytic activity. This enzyme is distributed virtually in all mammalian tissues during embryonic development (it can be demonstrated as early as the 2‐cell stage) where its expression is stage specific. The expression of TN‐AP is frequently associated with cell differentiation and as such it has been used as a marker for this process. By employing a stable gene transfer and forced gene expression technique, previous findings suggested that TN‐AP expression might influence cellular proliferation and morphological differentiation. The focus of this study was to determine whether this was a cell‐specific effect or not.

Next-generation RNA Sequencing of Archival Formalin-fixed Paraffin-embedded Urothelial Bladder Cancer

UNLABELLED Molecular profiling of individual cancers is key to personalised medicine. While sequencing technologies have required stringent sample collection and handling, recent technical advances offer sequencing from tissues collected in routine practice and tissues already stored in archives. In this paper, we establish methods for whole-transcriptome RNA sequencing (RNA-seq) from formalin-fixed paraffin-embedded tissues. We obtain average RNA-seq reads of >100 million per sample using the Illumina HiSeq2000 platform. We find high concordance with results from matching fresh frozen samples (>0.8 Spearman correlation). For validation, we compared low- and high-grade bladder cancer transcriptomes in 49 tumour samples after transurethral resection of bladder tumour. We found 947 differentially expressed protein-coding genes. While high-grade lesions exhibited distinct intertumour transcriptome heterogeneity, the transcriptome of low-grade tumours was homogeneous. PATIENT SUMMARY In this report, we show that it is now possible to use universally available bladder cancer samples that have been fixed in formalin to perform high-quality transcriptome analysis. This ability will facilitate the development of transcriptome-wide tests based on gene expression correlated with clinical outcome.

MP65-11 RNA SEQUENCING IDENTIFIES 3 DIFFERENT MOLECULAR GRADES AND IMMUNE CHECKPOINT CASCADES WITH DISTINCT CLINICAL BEHAVIOUR IN NON MUSCLE INVASIVE BLADDER CANCER

INTRODUCTION AND OBJECTIVES: Non muscle invasive bladder cancer (NMIBC) has a highly variable clinical behaviour not adequately predicted by their histological grade and clinical parameters. Some are indolent; others quickly progress to muscle-invasive disease. The discrepancy between phenotype and genotype is compounded further by interobserver variability in pathological grading. There is an unmet need to improve the prediction of NMIBCs behavior. METHODS: Whole transcriptomic (WT) analysis of 178 bladder tumours (158 NMIBC and 20 MIBC or metastatic) was performed from formalin fixed paraffin embedded (FFPE) tissues incorporating messenger RNA expression, splice variants, gene fusion, mutation detection and immune checkpoint inhibitor cascades. CTLA, PD-1, LAG3, TIM3, TIGIT and B7 were compiled as an index with all major genes in the cascade included. These datawere integrated and tested for correlations with both pathological grading and clinical outcomes. Conventional pathological grading for bothWHO 1973 (grade 1, 2 and 3) and 2004 (low grade-LG vs high grade-HG) classifications was reviewed by 3 different expert uro-pathologists and kappa statistic for interobserver variability was calculated. For validation we used an independent RNASequencing dataset (n1⁄4209, Hedegaard et al. 2016 Cancer Cell). RESULTS: Unsupervised clustering of data from RNA-Seq distinguished three molecular subtypes of NMIBC termed Molecular Grade Related Index (MGRI) 1, MGRI2 and MGRI3. MGRI1 comprised of almost exclusively LG tumours,whileMGRI3clusteredwithHGMIBC tumours.After assessment byexpert pathologists, kappa for interobserver variability in 1973 WHO histological grading was only 0.40 and 0.78 for the 2004 classification. FGFR3 mutations, FGFR3::TACC3 fusion events and MGRI1 genes were strongly associated with components of xenobiotic metabolism (p1⁄42.51x10 ) signallingsystems, inparticular,GTPase regulation (p1⁄40.002), respiratory cycle genes (p1⁄40.004) and a HOX cluster (p1⁄40.005). MGRI independently predicted progression to MIBC (n1⁄4138, HR1⁄42.96, 95%CI1⁄41.70-5.13, p1⁄41.20x10). Five year progression-free survival in a combined data set (n1⁄4347)differedsignificantly forMGRI1(100%)vsMGRI2(92.2%)vsMGRI3 (73.5%,p1⁄41.99x10,Grays test). PD-1 ImmuneCheckpointCascade,was an independent predictor of progression (OR 2.85, p<0.05). CONCLUSIONS: RNA sequencing delineates three molecular classes of NMIBC with different risks of progression to muscle invasion, compared to conventional histologic grading.

MP65-09 MOLECULAR TUMOUR GRADING AND OUTCOME PROGNOSTICATION OF NON MUSCLE INVASIVE BLADDER CANCER BASED ON WHOLE TRANSCRIPTOME ANALYSIS

resulting in regulation of transcription and cellular processes. HDAC inhibition has been shown to induce genes coding for MHC class I and class II molecules and immunologic activation molecules. Tissue microarray analysis of bladder cancer cell lines has demonstrated high HDAC expression in 40-60% of urothelial tumors. Our objective was to investigate the use of HDAC inhibitors on bladder tumors to stimulate immune mediated tumor cell destruction. METHODS: In vitro human bladder cancer cell lines were cultured with exposure to various HDAC inhibitors and HLA matched human T cells. The murine urothelial cell line, MB49 was also cultured with HDAC inhibitors and activated murine splenocytes. MTT assay was performed to measure cell viability. We transduced the murine tumor line to carry Firefly luciferase to allow for bioluminescence monitoring post implantation and then implanted the cells intra-vesically using a 24Fr angiocatheter delivery method. Following implantation bioluminescence signals were monitored using IVIS Lumina imaging system. Our treatment arm consisted of a single intra-vesical instillation of CI-994 for 60 minutes on post-implantation Day 6 with intraperitoneal injection of PD-1 blockade, appropriate controls were also performed. Total flux was monitored by IVIS in the treatment and control arms. RESULTS: In Vitro analysis showed a statistically significant decrease in viable tumor cells when treated with a short course of CI994 and then exposed to activated human T cells (p 0.0025). There was an even greater response when the T cells had received PD-1 blockade (p 0.0003). MTT assay analysis of MB49 yielded similar positive results with CI-994 treatment and T cell exposure. In vivo mice who received intravesical CI-994 in combination with intraperitoneal PD-1 blockade showed a more immediate and durable response than mice in the control arms. The average bioluminescence signal for our combination treatment arm indicates minimal to no retained tumor burden. CONCLUSIONS: HDACs are widely expressed in urothelial cancer and inhibition of the selective HDAC inhibitor CI-994 has shown promising results in vitro and in an in vivo orthotopic model at reducing tumor burden.