Tumor heterogeneity of fibroblast growth factor receptor 3 (FGFR3) mutations in invasive bladder cancer: implications for perioperative anti-FGFR3 treatment.

BACKGROUND: Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer. Preclinical studies show that anti-FGFR3 treatment slows down tumor growth, suggesting that this tyrosine kinase receptor is a candidate for personalized bladder cancer treatment, particularly in patients with mutated FGFR3. We addressed tumor heterogeneity in a large multicenter, multi-laboratory study, as this may have significant impact on therapeutic response. PATIENTS AND METHODS: We evaluated possible FGFR3 heterogeneity by the PCR-SNaPshot method in the superficial and deep compartments of tumors obtained by transurethral resection (TUR, n = 61) and in radical cystectomy (RC, n = 614) specimens and corresponding cancer-positive lymph nodes (LN+, n = 201). RESULTS: We found FGFR3 mutations in 13/34 (38%) T1 and 8/27 (30%) ≥T2-TUR samples, with 100% concordance between superficial and deeper parts in T1-TUR samples. Of eight FGFR3 mutant ≥T2-TUR samples, only 4 (50%) displayed the mutation in the deeper part. We found 67/614 (11%) FGFR3 mutations in RC specimens. FGFR3 mutation was associated with pN0 (P < 0.001) at RC. In 10/201 (5%) LN+, an FGFR3 mutation was found, all concordant with the corresponding RC specimen. In the remaining 191 cases, RC and LN+ were both wild type. CONCLUSIONS: FGFR3 mutation status seems promising to guide decision-making on adjuvant anti-FGFR3 therapy as it appeared homogeneous in RC and LN+. Based on the results of TUR, the deep part of the tumor needs to be assessed if neoadjuvant anti-FGFR3 treatment is considered. We conclude that studies on the heterogeneity of actionable molecular targets should precede clinical trials with these drugs in the perioperative setting.

PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.

BACKGROUND: Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. METHODS: We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. RESULTS: Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. CONCLUSIONS: Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.

Putative tumour suppressor gene necdin is hypermethylated and mutated in human cancer.

BACKGROUND: Necdin (NDN) expression is downregulated in telomerase-immortalised normal human urothelial cells. Telomerase-immortalised normal human urothelial cells have no detected genetic alterations. Accordingly, many of the genes whose expression is altered following immortalisation are those for which epigenetic silencing is reported. METHODS: NDN expression was examined in normal tissues and tumour cell lines by quantitative real-time PCR and immunoblotting. Immunohistochemistry was performed on urothelial carcinoma (UC). Urothelial carcinoma and UC cell lines were subject to HumanMethylation27 BeadChip Array-based methylation analyses. Mutation screening was performed. The functional significance of NDN expression was investigated using retroviral-mediated downregulation or overexpression. RESULTS: NDN protein was widely expressed in normal tissues. Loss of expression was observed in 38 out of 44 (86%) of UC cell lines and 19 out of 25 (76%) of non-UC cell lines. Loss of NDN protein was found in the majority of primary UC. Oncomine analysis demonstrated downregulation of expression in multiple tumour types. In UC, tumour-specific hypermethylation of NDN and key CpG sites where hypermethylation correlated with reduced expression were identified. Six novel mutations, including some of predicted functional significance, were identified in colorectal and ovarian cancer cell lines. Functional studies showed that NDN could suppress colony formation at low cell density and affect anchorage-independent growth and anoikis in vitro. CONCLUSION: NDN is a novel tumour suppressor candidate that is downregulated and hypermethylated or mutated in cancer.

Knockdown by shRNA identifies S249C mutant FGFR3 as a potential therapeutic target in bladder cancer.

More than 60% of low-grade non-invasive papillary urothelial cell carcinomas contain activating point mutations of fibroblast growth factor receptor 3 (FGFR3). The phenotypic consequences of constitutive activation of FGFR3 in bladder cancer have not been elucidated and further studies are required to confirm the consequences of inhibiting receptor activity in urothelial cells. We measured FGFR3 transcript levels and demonstrated that transcript levels were significantly more abundant in low-stage and grade tumours. We identified a tumour cell line, 97-7, expressing the most common FGFR3 mutation (S249C) at similar FGFR3 transcript levels to low-stage and grade tumours. In these cells, S249C FGFR3 protein formed stable homodimers and was constitutively phosphorylated. We used retrovirus-mediated delivery of shRNA to knockdown S249C FGFR3. This induced cell flattening, decreased cell proliferation and reduced clonogenicity on plastic and in soft agar. However, no effects of knockdown of wild-type FGFR3 were observed in telomerase immortalized normal human urothelial cells, indicating possible dependence of the tumour cell line on mutant FGFR3. Re-expression of S249C FGFR3 in shRNA-expressing 97-7 cells resulted in a reversal of phenotypic changes, confirming the specificity of the shRNA. These results indicate that targeted inhibition of S249C FGFR3 may represent a useful therapeutic approach in superficial bladder cancer.

DBC1 re-expression alters the expression of multiple components of the plasminogen pathway.

Deleted in bladder cancer 1 (DBC1) is a candidate gene for the bladder tumour suppressor locus at 9q33.1. The function of the gene is currently unknown but a cross-species sequence comparison suggests an important role, as it is highly evolutionarily conserved. Here, we transfected a nonexpressing human bladder cancer cell line with a set of human DBC1 cDNA constructs. The effect on global expression patterns was assessed using cDNA microarrays. The cell clone with the lowest level of DBC1 expression showed induced expression of 26 genes including plasminogen activator inhibitor 2 (SERPINB5; 4.6-fold), heparin-binding EGF-like growth factor precursor (DTR; 4.2-fold), small proline-rich protein 2B (SPRR2B; 3.6-fold), metallothionein 1 isoforms (MT1B/MT1A/MT-1F; from 2.9- to 3.2-fold), tissue-type plasminogen activator precursor (PLAT; 2.8-fold) and urokinase-type plasminogen activator precursor (PLAU; 2.7-fold). In clustering analysis, both PLAT and PLAU clustered with the functionally related urokinase plasminogen activator surface receptor (PLAUR; 1.9-fold). Furthermore, 14 human bladder tumours were analysed by real-time quantitative PCR using gene-specific primers for selected (n=20) genes. The expression levels of SERPINB5, PLAU, PLAUR and MT1 correlated with the DBC1 levels, suggesting previously unknown involvement of DBC1 in the urokinase-plasminogen pathway.

Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway.

The CDKN2A locus is frequently inactivated in urothelial cell carcinoma (UCC), yet how this alteration contributes to bladder tumorigenesis is not known. Although most UCC express telomerase, inactivation of the p16/Rb pathway is generally required for in vitro immortalisation. This and the involvement of p16 in senescence of normal human urothelial cells (NHUC) suggest that CDKN2A deletion may aid bypass of senescence and allow immortalisation. CDKN2A encodes p16 and p14ARF and therefore inactivation of this locus can disrupt both the Rb and p53 tumour suppressor pathways. Retrovirus-mediated transduction was used to specifically modulate the p16/Rb and/or p53 tumour suppressor pathways in NHUC and to express human telomerase reverse transcriptase (hTERT). Expression of hTERT bypassed Rb and p53 pathway-dependent barriers to proliferation and immortalised NHUC. TERT-NHUC had normal karyotypes, were non-tumorigenic and unexpectedly retained CDKN2A. Thus, the phenotypic significance of inactivation of CDKN2A in UCC may not be solely related to bypass of senescence. Phenotypic assays in human urothelial cells have relied on cell strains derived from invasive tumours or NHUC immortalised by expression of SV40-large T. The production of genetically normal but immortal NHUC lines now provides a valuable platform for experiments to examine the timing and combination of events necessary for UCC tumorigenesis.

Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar.

The complete sequence of the Atlantic salmon (Salmo salar) mitochondrial genome has been determined. The entire sequence is 16665 base pairs (bp) in length, with a gene content (13 protein-coding, two ribosomal RNA [rRNA] and 22 transfer RNA [tRNA] genes) and order conforming to that observed in most other vertebrates. Base composition and codon usage have been detailed. Nucleotide and derived amino acid sequences of the 13 protein-coding genes from Atlantic salmon have been compared with their counterparts in rainbow trout. A putative structure for the origin of L-strand replication (O(L)) is proposed, and sequence features of the control region (D-loop) are described.

Application of magnetite and silica-magnetite composites to the isolation of genomic DNA.

Magnetite and silica-magnetite composites were used as adsorbents for the isolation of genomic DNA from maize kernels. Two methods are described for the preparation of silica-magnetite composites, both of which afford higher yields of genomic DNA than when using magnetite alone, or a commerically available kit. DNA isolated using silica-magnetite was suitable for use in further applications such as polymerase chain reaction amplification and enzyme digestion.

Inactivation of the Rb pathway and overexpression of both isoforms of E2F3 are obligate events in bladder tumours with 6p22 amplification.

E2F3 and CDKAL1 are candidate genes from the 6p22 region frequently amplified in bladder cancer. Expression of E2F3 isoforms (E2F3a and b) and CDKAL1 were examined and modulated in 6p22-amplified bladder cell lines. Eight lines with amplification showed overexpression of both E2F3 isoforms and CDKAL1. shRNA-mediated knockdown of CDKAL1 had no effect on proliferation. Knockdown of E2F3a or E2F3b alone induced antiproliferative effects, with the most significant effect on proliferation being observed when both isoforms were knocked down together. As E2Fs interact with the Rb tumour suppressor protein, Rb expression was analysed. There was a striking relationship between 6p22.3 amplification, E2F3 overexpression and lack of Rb expression. This was also examined in primary bladder tumours. Array-CGH detected 6p22.3 amplification in 8/91 invasive tumours. Five were studied in more detail. Four showed 13q14.2 loss (including RB1) and expressed no Rb protein. In the fifth, 13q was unaltered but the CDKN2A locus was deleted. This tumour was negative for p16 and positive for Rb protein. As p16 is a negative regulator of the Rb pathway, its loss represents an alternative mechanism for inactivation. Indeed, a phospho-specific Rb antibody showed much Rb protein in a hyperphosphorylated (inactive) form. We conclude that inactivation of the Rb pathway is required in addition to E2F3 overexpression in this subset of bladder tumours.