The Recurrence Problem in Bladder Cancer. Urothelial carcinoma of the bladder (UCB) has the highest recurrence rate of any cancer. Approximately 70% of non-muscle-invasive cases recur after initial treatment, and 10-20% progress to muscle-invasive disease. Of those presenting with muscle-invasive UCB, half will relapse with metastatic disease. Understanding the molecular basis of recurrence is essential for improving outcomes.
This study uses RNA sequencing (RNA-seq), a high-throughput technique that simultaneously profiles gene expression and alternative splicing across the entire transcriptome. Compared to earlier microarray technology, RNA-seq can detect not only expression level changes but also abnormal patterns of alternative splicing -- a key mechanism by which cancer cells generate proteins with altered or dysregulated functions.
Study Goal. Deep RNA-seq at approximately 50x coverage was performed on tumor tissue and adjacent non-tumor tissue from a single recurrent, muscle-invasive, cisplatin-resistant bladder cancer patient. Key findings were then validated by quantitative PCR in 11 additional patients including 6 with recurrent/drug-resistant UCB and 5 newly diagnosed patients. The study represents the first comprehensive RNA-seq transcriptome analysis of recurrent, drug-resistant UCB.
Sequencing Scale. Approximately 62 million read pairs were generated from the tumor sample and 64 million from the non-tumor tissue, with about 89% and 83% uniquely mapped to the human genome (hg19) respectively. Sequencing depth averaged approximately 50x coverage of the human transcriptome (~113 million base pairs of unique exon sequence), providing comprehensive gene expression data.
Gene expression was quantified using Cufflinks, measuring Fragments Per Kilobase of exon per Million mapped reads (FPKM). Genes with FPKM greater than 1 were considered expressed -- 14,520 in tumor and 14,199 in non-tumor tissue, representing the majority of annotated human reference genes. A total of 1,879 significantly differentially expressed genes (DEGs) were identified between tumor and non-tumor tissue (FDR less than 0.01).
Alternative Splicing Analysis. Alternative splicing events were detected using the MISO (Mixture of Isoforms) probabilistic framework, which quantifies differential isoform usage between samples. Seven types of splicing events were cataloged: alternative 3' splice sites, alternative 5' splice sites, alternative first exons, mutually exclusive exons, retained introns, skipped exons, and tandem 3' UTRs. The analysis identified 462 differential splicing events from 390 unique genes, narrowed to 43 reliable events in 38 cancer-associated genes after stringent filtering.
VEGFA Upregulation in Bladder Cancer. Vascular endothelial growth factor A (VEGFA) was significantly upregulated in tumor tissue. This finding aligns with two prior studies using different technologies and confirms VEGFA as a commonly overexpressed gene in UCB. VEGFA promotes angiogenesis -- the formation of new blood vessels that sustain tumor growth.
Five candidate genes were validated by quantitative RT-PCR in the sequencing patient and 11 additional patients: CDH1 (E-cadherin), VEGFA, PTPRF, CLDN7, and MMP2. In the additional patients, CDH1, VEGFA, PTPRF, and CLDN7 were upregulated in six of eleven cancer samples, while MMP2 was downregulated in 10 of 11 samples -- suggesting MMP2 downregulation is a consistent feature of UCB.
Recurrence-Associated Gene Expression. CDH1, VEGFA, and PTPRF were upregulated in 66.7% (4 of 6) of recurrent UCB patients but only 40% (2 of 5) of newly diagnosed patients. This higher rate of upregulation in recurrent cases suggests these three genes may be specifically associated with the biology of recurrent disease and could serve as biomarkers to identify patients at high recurrence risk.
Known UCB diagnostic markers including KRT20 (Cytokeratin 20), BIRC5 (Survivin), CDH1, and PSCA were all found to be dysregulated, confirming the biological relevance of the sequencing data. Genes known to be commonly mutated in UCB (ARPC5/p16 and FGF2) showed no change in expression level, illustrating that genetic mutations and expression changes represent distinct layers of cancer dysregulation.
Cell Adhesion Molecules Pathway Is Most Significantly Altered. Gene ontology and KEGG pathway enrichment analysis of the 1,879 DEGs identified the cell adhesion molecules (CAMs) pathway as the most significantly enriched (FDR = 2.67 x 10^-8, fold enrichment 3.02). This finding was consistent with previous reports on UCB and cancers of the kidney and testis, suggesting CAM pathway dysregulation is a consistent molecular feature of urological malignancies.
Additional significantly enriched pathways included focal adhesion and extracellular matrix (ECM)-receptor interaction. Dysregulation of the CAMs pathway and ECM receptors enables cancer cells to escape the primary tumor mass, invade adjacent tissues, and metastasize -- processes directly relevant to the muscle-invasive and recurrent nature of this tumor.
Immune and Inflammatory Pathways Broadly Dysregulated. The most significantly enriched Gene Ontology biological process terms included immune response (fold enrichment 2.30, corrected p = 4.69 x 10^-18), cell adhesion (fold enrichment 2.15, corrected p = 1.84 x 10^-14), and inflammatory response. This widespread immune pathway dysregulation is consistent with modern understanding of the tumor microenvironment's role in cancer progression and chemotherapy response.
Extensive Alternative Splicing in Bladder Cancer. Among the 462 differential splicing events detected, skipped exons were the most common pattern (50%), followed by retained introns (22%) and alternative 5' and 3' splice sites. Skipped exons are particularly important because they can truncate functional domains of proteins, generating isoforms with altered or gain-of-function activity that contributes to cancer cell behavior.
CD44 as a Cancer-Specific Marker. The CD44 transmembrane glycoprotein, which participates in cell division, survival, migration, and adhesion, showed cancer-specific inclusion of variant exons v8, v9, and v10 in the tumor tissue but not in the non-tumor tissue. The CD44E variant (exon v8-10 expressed) has previously been reported as a prognostic predictor and indicator of disease extent in urothelial cancer. Increased exon inclusion of CD44 was seen in 50% of recurrent patients versus 20% of newly diagnosed patients in validation cohorts.
PDGFA (platelet-derived growth factor alpha), not previously reported in bladder cancer, showed cancer-specific retention of exon 6, producing the long PDGFAA isoform in tumor tissue versus the short PDGFAB isoform in normal tissue. The long isoform attaches to the extracellular matrix while the short form diffuses freely. PDGFA exon inclusion was seen in 83% of recurrent UCB samples but only 40% of newly diagnosed cases, suggesting the PDGFAA isoform may drive or mark recurrent disease.
NUMB (64% of UCB patients), LPHN2 (73% of UCB patients), MACF1, and ADD3 also showed cancer-associated differential splicing events validated across the patient cohort. Several of these splicing events had been reported in lung and other cancers but were identified here for the first time in bladder cancer, expanding the known landscape of bladder cancer-relevant splicing alterations.
Molecular Basis of Cisplatin Resistance. Since the tumor analyzed was cisplatin-resistant, the researchers investigated expression changes in genes associated with drug resistance. Genes involved in DNA repair and apoptosis pathways were found to be dysregulated, suggesting that enhanced DNA damage repair combined with suppression of apoptotic cell death may underlie the chemoresistance phenotype of this tumor.
The MACF1 splicing event -- increased inclusion of exon 8 in tumor tissue -- was previously reported in non-small cell lung cancer and breast cancer. This event is thought to alter Wnt signaling through MACF1's interactions with Axin, beta-catenin, GSK3B, and APC -- molecules with well-established roles in tumorigenesis. Finding the same splicing event in bladder cancer suggests it may be a broadly conserved cancer-associated splicing change.
Limitations and Future Directions. This is a single-patient deep sequencing study, which limits the generalizability of the transcriptome findings. However, key differentially expressed genes and splicing events were validated in 11 additional patients, providing initial evidence of their broader relevance. Future studies with larger cohorts are needed to determine which of these molecular features are most consistently associated with recurrence, muscle invasion, and drug resistance, and could serve as clinical biomarkers or therapeutic targets in bladder cancer.