Development of resistance to FGFR inhibition in urothelial carcinoma via multiple pathways in vitro.

Alterations of fibroblast growth factor receptors (FGFRs) are common in bladder and other cancers and result in disrupted signalling via several pathways. Therapeutics that target FGFRs have now entered the clinic, but, in common with many cancer therapies, resistance develops in most cases. To model this, we derived resistant sublines of two FGFR-driven bladder cancer cell lines by long-term culture with the FGFR inhibitor PD173074 and explored mechanisms using expression profiling and whole-exome sequencing. We identified several resistance-associated molecular profiles. These included HRAS mutation in one case and reversible mechanisms resembling a drug-tolerant persister phenotype in others. Upregulated IGF1R expression in one resistant derivative was associated with sensitivity to linsitinib and a profile with upregulation of a YAP/TAZ signature to sensitivity to the YAP inhibitor CA3 in another. However, upregulation of other potential therapeutic targets was not indicative of sensitivity. Overall, the heterogeneity in resistance mechanisms and commonality of the persister state present a considerable challenge for personalised therapy. Nevertheless, the reversibility of resistance may indicate a benefit from treatment interruptions or retreatment following disease relapse in some patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Frequent inactivating mutations of STAG2 in bladder cancer are associated with low tumour grade and stage and inversely related to chromosomal copy number changes.

Inactivating mutations of STAG2 have been reported at low frequency in several cancers. In glioblastoma, the function of STAG2 has been related to maintenance of euploidy via its role in the cohesin complex. In a screen of a large series of bladder tumours and cell lines, we found inactivating mutations (nonsense, frameshift and splicing) in 67 of 307 tumours (21.8%) and 6 of 47 cell lines. Thirteen missense mutations of unknown significance were also identified. Inactivating mutation was associated with low tumour stage (P = 0.001) and low grade (P = 0.0002). There was also a relationship with female patient gender (P = 0.042). Examination of copy number profiles revealed an inverse relationship of mutation with both fraction of genome altered and whole chromosome copy number changes. Immunohistochemistry showed that in the majority of cases with inactivating mutations, STAG2 protein expression was absent. Strikingly, we identified a relatively large subset of tumours (12%) with areas of both positive and negative immunoreactivity, in only four of which a potentially function-altering mutation was detected. Regions of differential expression were contiguous and showed similar morphological phenotype in all cases. Microdissected positive and negative areas from one tumour showed an inactivating mutation to be present only in the negative area, suggesting intra-tumoral sub-clonal genomic evolution. Our findings indicate that loss of STAG2 function plays a more important role in non-invasive than that in muscle-invasive bladder cancer and suggest that cohesin complex-independent functions are likely to be important in these cases.

Molecular profile of pure squamous cell carcinoma of the bladder identifies major roles for OSMR and YAP signalling.

Pure squamous cell carcinoma (SCC) is the most common pure variant form of bladder cancer, found in 2-5% of cases. It often presents late and is unresponsive to cisplatin-based chemotherapy. The molecular features of these tumours have not been elucidated in detail. We carried out whole-exome sequencing (WES), copy number, and transcriptome analysis of bladder SCC. Muscle-invasive bladder cancer (MIBC) samples with no evidence of squamous differentiation (non-SD) were used for comparison. To assess commonality of features with urothelial carcinoma with SD, we examined data from SD samples in The Cancer Genome Atlas (TCGA) study of MIBC. TP53 was the most commonly mutated gene in SCC (64%) followed by FAT1 (45%). Copy number analysis revealed complex changes in SCC, many differing from those in samples with SD. Gain of 5p and 7p was the most common feature, and focal regions on 5p included OSMR and RICTOR. In addition to 9p deletions, we found some samples with focal gain of 9p24 containing CD274 (PD-L1). Loss of 4q35 containing FAT1 was found in many samples such that all but one sample analysed by WES had FAT1 mutation or deletion. Expression features included upregulation of oncostatin M receptor (OSMR), metalloproteinases, metallothioneins, keratinisation genes, extracellular matrix components, inflammatory response genes, stem cell markers, and immune response modulators. Exploration of differentially expressed transcription factors identified BNC1 and TFAP2A, a gene repressed by PPARG, as the most upregulated factors. Known urothelial differentiation factors were downregulated along with 72 Kruppel-associated (KRAB) domain-containing zinc finger family protein (KZFP) genes. Novel therapies are urgently needed for these tumours. In addition to upregulated expression of EGFR, which has been suggested as a therapeutic target in basal/squamous bladder cancer, we identified expression signatures that indicate upregulated OSMR and YAP/TAZ signalling. Preclinical evaluation of the effects of inhibition of these pathways alone or in combination is merited.

High-resolution analysis of genomic alteration on chromosome arm 8p in urothelial carcinoma.

Loss of chromosome arm 8p, sometimes in combination with amplification of proximal 8p, is found in urothelial carcinoma (UC) and other epithelial cancers and is associated with more advanced tumor stage. We carried out array comparative genomic hybridization on 174 UC and 33 UC cell lines to examine breakpoints and copy number. This was followed by a detailed analysis of the cell lines using fluorescence in situ hybridization (FISH) and, in some cases, M-FISH, to refine breakpoints and determine translocation partners, heterozygosity analysis, and analysis of expression of selected genes. We showed an overall pattern of 8p loss with reduced heterozygosity and reduced gene expression. Amplification was seen in some samples and shown in the cell line JMSU1 to correlate with overexpression of ZNF703, ERLIN2, PROSC, GPR124, and BRF2. Apart from the centromere, no single breakpoint was overrepresented, and we postulate that frequent complex changes without consistent breakpoints reflect the need for alterations of combinations of genes. The region around 2 Mb, which was homozygously deleted in one cell line and includes the gene ARHGEF10 and the micro-RNA hsa-mir-596, is one candidate tumor suppressor gene region.

Pericyte Insulin Receptors Modulate Retinal Vascular Remodeling and Endothelial Angiopoietin Signaling.

Pericytes regulate vascular development, stability, and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFRß-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited perivenous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in perivenous and periarterial plexi, and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the perivenous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease.

Stage-stratified molecular profiling of non-muscle-invasive bladder cancer enhances biological, clinical, and therapeutic insight.

Understanding the molecular determinants that underpin the clinical heterogeneity of non-muscle-invasive bladder cancer (NMIBC) is essential for prognostication and therapy development. Stage T1 disease in particular presents a high risk of progression and requires improved understanding. We present a detailed multi-omics study containing gene expression, copy number, and mutational profiles that show relationships to immune infiltration, disease recurrence, and progression to muscle invasion. We compare expression and genomic subtypes derived from all NMIBCs with those derived from the individual disease stages Ta and T1. We show that sufficient molecular heterogeneity exists within the separate stages to allow subclassification and that this is more clinically meaningful for stage T1 disease than that derived from all NMIBCs. This provides improved biological understanding and identifies subtypes of T1 tumors that may benefit from chemo- or immunotherapy.

Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer.

The phosphatidylinositol 3-kinase (PI3K) pathway is a critical signal transduction pathway that regulates multiple cellular functions. Aberrant activation of this pathway has been identified in a wide range of cancers. Several pathway components including AKT, PI3K and mTOR represent potential therapeutic targets and many small molecule inhibitors are in development or early clinical trials. The complex regulation of the pathway, together with the multiple mechanisms by which it can be activated, make this a highly challenging pathway to target. For successful inhibition, detailed molecular information on individual tumours will be required and it is already clear that different tumour types show distinct combinations of alterations. Recent results have identified alterations in pathway components PIK3CA, PTEN, AKT1 and TSC1 in bladder cancer, some of which are significantly related to tumour phenotype and clinical behaviour. Co-existence of alterations to several PI3K pathway genes in some bladder tumours indicates that these proteins may have functions that are not related solely to the known canonical pathway.

Bladder tumour-derived somatic TSC1 missense mutations cause loss of function via distinct mechanisms.

More than 50% of transitional cell carcinomas of the bladder show loss of heterozygosity of a region spanning the TSC1 locus at 9q34 and mutations of TSC1 have been identified in 14.5% of tumours. These comprise nonsense mutations, splicing mutations, small deletions and missense mutations. Missense mutations are only rarely found in the germline in TSC disease. Therefore, we have examined six somatic missense mutations found in bladder cancer to determine whether these result in loss of function. We describe loss of function via distinct mechanisms. Five mutations caused mutually exclusive defects at mRNA and protein levels. Of these, two mutations caused pre-mRNA splicing errors that were predicted to result in premature protein truncation and three resulted in markedly reduced stability of exogenous TSC1 protein. Primary tumours with aberrant TSC1 pre-mRNA splicing were confirmed as negative for TSC1 expression by immunohistochemistry. Expression was also significantly reduced in a tumour with a TSC1 missense mutation resulting in diminished protein half-life. A single TSC1 missense mutation identified in a tumour with retained heterozygosity of the TSC1 region on chromosome 9 caused an apparently TSC2- and mTOR-independent localization defect of the mutant protein. We conclude that although TSC1 missense mutations do not play a major role in causation of TSC disease, they represent a significant proportion of somatic loss of function mutations in bladder cancer.

Integrated genomic and transcriptional analysis of the in vitro evolution of telomerase-immortalized urothelial cells (TERT-NHUC).

Much progress has been made in identifying the molecular genetic alterations that occur in bladder cancer. However, in many cases the genes targeted by these alterations are not known. Telomerase immortalized human urothelial cells (TERT-NHUC) are a useful resource for in vitro studies of genes involved in urothelial transformation. When cultured under standard conditions they remain genetically stable but when cultured under low-density conditions they exhibit genetic instability and acquire chromosomal alterations. TERT-NHUC from three donors were cultured at low plating density and examined at four time-points during a culture period of 600 days. Analyses included population doubling kinetics, array-based CGH (aCGH), chromosome counts, fluorescence in situ hybridization (FISH), mutation analysis, Affymetrix gene expression analysis, Western blotting for p16, anchorage-independent growth and tumorigenicity assays. Alterations acquired during continued culture of TERT-NHUC at low density (TERT-NHUC-L) included some observed in urothelial carcinoma (UC) cell lines and primary UC. Examination of gene expression in TERT-NHUC with distinct acquired genetic aberrations may pinpoint genes targeted by these alterations. Data from an aCGH study of UC cell lines and primary tumors were examined for changes in chromosomal regions that also showed alterations in TERT-NHUC-L. Loss of a region on 2q including BOK was identified in UC cell lines and primary tumors. DNER and FRAS1 were identified as potential candidate genes, whose expression is altered independently of the acquisition of any genetic event.

Papillary and muscle invasive bladder tumors with distinct genomic stability profiles have different DNA repair fidelity and KU DNA-binding activities.

Low-grade noninvasive papillary bladder tumors are genetically stable whereas muscle invasive bladder tumors display high levels of chromosomal aberrations. As cells deficient for nonhomologous end-joining (NHEJ) pathway components display increased genomic instability, we sought to determine the NHEJ repair characteristics of bladder tumors and correlate this with tumor stage and grade. A panel of 13 human bladder tumors of defined stage and grade were investigated for chromosomal aberrations by comparative genomic hybridization and for NHEJ repair fidelity and function. Repair assays were conducted with extracts made directly from bladder tumor specimens to avoid culture-induced phenotypic alterations and selection bias as only a minority of bladder tumors grow in culture. Four noninvasive bladder tumors (pTaG2), which were genetically stable, repaired a partially incompatible double-strand break (DSB) by NHEJ-dependent annealing of termini and fill-in of overhangs with minimal loss of nucleotides. In contrast, four muscle invasive bladder cancers (pT2-3G3), which displayed gross chromosomal rearrangements, repaired DSBs in an error-prone manner involving extensive resection and microhomology association. Four minimally invasive bladder cancers (pT1G3) had characteristics of both repair types. Error-prone repair in bladder tumors correlated with reduced KU DNA-binding and loss of TP53 function. In conclusion, there were distinct differences in DSB repair between noninvasive papillary tumors and higher stage/grade invasive cancers. End-joining fidelity correlated with stage and was increasingly error-prone as tumors became more invasive and KU binding activity reduced; these changes may underlie the different genomic profiles of these tumors.

Elevated circulating amyloid concentrations in obesity and diabetes promote vascular dysfunction.

Diabetes, obesity, and Alzheimer's disease (AD) are associated with vascular complications and impaired nitric oxide (NO) production. Furthermore, increased ß-site amyloid precursor protein-cleaving (APP-cleaving) enzyme 1 (BACE1), APP, and ß-amyloid (Aß) are linked with vascular disease development and increased BACE1 and Aß accompany hyperglycemia and hyperlipidemia. However, the causal relationship between obesity and diabetes, increased Aß, and vascular dysfunction is unclear. We report that diet-induced obesity (DIO) in mice increased plasma and vascular Aß42 that correlated with decreased NO bioavailability, endothelial dysfunction, and increased blood pressure. Genetic or pharmacological reduction of BACE1 activity and Aß42 prevented and reversed, respectively, these outcomes. In contrast, expression of human mutant APP in mice or Aß42 infusion into control diet-fed mice to mimic obese levels impaired NO production, vascular relaxation, and raised blood pressure. In humans, increased plasma Aß42 correlated with diabetes and endothelial dysfunction. Mechanistically, higher Aß42 reduced endothelial NO synthase (eNOS), cyclic GMP (cGMP), and protein kinase G (PKG) activity independently of diet, whereas endothelin-1 was increased by diet and Aß42. Lowering Aß42 reversed the DIO deficit in the eNOS/cGMP/PKG pathway and decreased endothelin-1. Our findings suggest that BACE1 inhibitors may have therapeutic value in the treatment of vascular disease associated with diabetes.

Genomic Subtypes of Non-invasive Bladder Cancer with Distinct Metabolic Profile and Female Gender Bias in KDM6A Mutation Frequency.

Bladder cancer incurs a higher lifetime treatment cost than other cancers due to frequent recurrence of non-invasive disease. Improved prognostic biomarkers and localized therapy are needed for this large patient group. We defined two major genomic subtypes of primary stage Ta tumors. One of these was characterized by loss of 9q including TSC1, increased KI67 labeling index, upregulated glycolysis, DNA repair, mTORC1 signaling, features of the unfolded protein response, and altered cholesterol homeostasis. Comparison with muscle-invasive bladder cancer mutation profiles revealed lower overall mutation rates and more frequent mutations in RHOB and chromatin modifier genes. More mutations in the histone lysine demethylase KDM6A were present in non-invasive tumors from females than males.

Novel tumor subgroups of urothelial carcinoma of the bladder defined by integrated genomic analysis.

PURPOSE: There is a need for improved subclassification of urothelial carcinoma (UC) at diagnosis. A major aim of this study was to search for novel genomic subgroups. EXPERIMENTAL DESIGN: We assessed 160 tumors for genome-wide copy number alterations and mutation in genes implicated in UC. These comprised all tumor grades and stages and included 49 high-grade stage T1 (T1G3) tumors. RESULTS: Our findings point to the existence of genomic subclasses of the "gold-standard" grade/stage groups. The T1G3 tumors separated into 3 major subgroups that differed with respect to the type and number of copy number events and to FGFR3 and TP53 mutation status. We also identified novel regions of copy number alteration, uncovered relationships between molecular events, and elucidated relationships between molecular events and clinico-pathologic features. FGFR3 mutant tumors were more chromosomally stable than their wild-type counterparts and a mutually exclusive relationship between FGFR3 mutation and overrepresentation of 8q was observed in non-muscle-invasive tumors. In muscle-invasive (MI) tumors, metastasis was positively associated with losses of regions on 10q (including PTEN), 16q and 22q, and gains on 10p, 11q, 12p, 19p, and 19q. Concomitant copy number alterations positively associated with TP53 mutation in MI tumors were losses on 16p, 2q, 4q, 11p, 10q, 13q, 14q, 16q, and 19p, and gains on 1p, 8q, 10q, and 12q. Significant complexity was revealed in events affecting chromosome 9. CONCLUSIONS: These findings may lead to improved biologic understanding and the development of prognostic biomarkers. Novel regions of copy number alteration may reveal potential therapeutic targets.

Spectrum of phosphatidylinositol 3-kinase pathway gene alterations in bladder cancer.

PURPOSE: The phosphatidylinositol 3-kinase (PI3K) pathway can be activated by alterations affecting several pathway components. For rational application of targeted therapies, detailed understanding of tumor biology and approaches to predict efficacy in individual tumors are required. Our aim was to assess the frequency and distribution of pathway alterations in bladder cancer. EXPERIMENTAL DESIGN: We examined the pathway components (PIK3CA, PTEN, TSC1, RHEB, and LKB1) and putative upstream regulators (FGFR3 and RAS genes) for mutation, allelic loss, copy number alteration, and expression in bladder tumors and cell lines. RESULTS: No mutations were found in RHEB and only a single mutation in LKB1. PIK3CA mutations were detected in 25% of tumors and 26% of cell lines with a significant excess of helical domain mutations (E542K and E545K). There was over-representation but not amplification of the gene. Loss of heterozygosity of the PTEN region and homozygous deletion were found in 12% and 1.4% of tumors, and reduced expression in 49%. Forty-six percent of cell lines showed alterations that implicated PTEN. Sixteen percent of tumors and 11% of cell lines showed TSC1 mutation, and 9q loss of heterozygosity was common (57%). Pathway alterations were independently distributed, suggesting that the mutation of two pathway members may have additive or synergistic effects through noncanonical functions. CONCLUSIONS: PI3K pathway alterations are common in bladder cancer. The lack of redundancy of alterations suggests that single-agent PI3K-targeted therapy may not be successful in these cancers. This study provides a well-characterized series of cell lines for use in preclinical studies of targeted agents.