Meta-Analysis of the Luminal and Basal Subtypes of Bladder Cancer and the Identification of Signature Immunohistochemical Markers for Clinical Use

AbstractBackgroundIt has been suggested that bladder cancer can be divided into two molecular subtypes referred to as luminal and basal with distinct clinical behaviors and sensitivities to chemotherapy. We aimed to validate these subtypes in several clinical cohorts and identify signature immunohistochemical markers that would permit simple and cost-effective classification of the disease in primary care centers.MethodsWe analyzed genomic expression profiles of bladder cancer in three cohorts of fresh frozen tumor samples: MD Anderson (n=132), Lund (n=308), and The Cancer Genome Atlas (TCGA) (n=408) to validate the expression signatures of luminal and basal subtypes and relate them to clinical follow-up data. We also used an MD Anderson cohort of archival bladder tumor samples (n=89) and a parallel tissue microarray to identify immunohistochemical markers that permitted the molecular classification of bladder cancer.FindingsBladder cancers could be assigned to two candidate intrinsic molecular subtypes referred to here as luminal and basal in all of the datasets analyzed. Luminal tumors were characterized by the expression signature similar to the intermediate/superficial layers of normal urothelium. They showed the upregulation of PPARγ target genes and the enrichment for FGFR3, ELF3, CDKN1A, and TSC1 mutations. In addition, luminal tumors were characterized by the overexpression of E-Cadherin, HER2/3, Rab-25, and Src. Basal tumors showed the expression signature similar to the basal layer of normal urothelium. They showed the upregulation of p63 target genes, the enrichment for TP53 and RB1 mutations, and overexpression of CD49, Cyclin B1, and EGFR. Survival analyses showed that the muscle-invasive basal bladder cancers were more aggressive when compared to luminal cancers. The immunohistochemical expressions of only two markers, luminal (GATA3) and basal (KRT5/6), were sufficient to identify the molecular subtypes of bladder cancer with over 90% accuracy.InterpretationThe molecular subtypes of bladder cancer have distinct clinical behaviors and sensitivities to chemotherapy, and a simple two-marker immunohistochemical classifier can be used for prognostic and therapeutic stratification.FundingU.S. National Cancer Institute and National Institute of Health

In-Frame cDNA Library Combined with Protein Complementation Assay Identifies ARL11-Binding Partners

<div><p>The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5′-untranslated regions (UTRs) that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5′-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an “in-frame cDNA library.” We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11), cellular retinoic acid binding protein 2 (CRABP2), and phosphoglycerate mutase 1 (PGAM1). Thus, the in-frame cDNA library without 5′-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.</p> </div

Identification of the CRABP2 and PGAM1 proteins as ARL11-binding partners using the in-frame cDNA expression library.

<p>(<b>A</b>) Western blot analysis of N-terminal YFP1-tagged fusion proteins expressed by the constructs with and without 5′-UTRs in HEK-293T cells. Expression of a construct containing only YFP1 was used as a control. An anti-GFP N-terminal antibody was used to visualize the expressed tagged proteins. (<b>B</b>) YFP fluorescence in HEK-293T cells after the co-transfection of YFP1-<i>CRABP2</i> with <i>ARL11</i>-YFP2 and YFP1-<i>PGAM1</i> with <i>ARL11</i>-YFP2. Nuclei were counterstained with DAPI. (<b>C</b>) Confirmation of the interaction between ARL11 and CRABP2 by western blotting and co-immunoprecipitation. HEK-293T cells were transfected with HA-tagged <i>ARL11</i> and FLAG-tagged <i>CRABP2</i> constructs without its 5′-UTR. Protein expression was verified by immunoblotting using anti-ARL11 and anti-CRABP2 antibodies in direct western blots (DWB). Immunoprecipitation with western blotting (IPWB) was performed by anti-HA antibody pull-down of ARL11 to detect CRABP2 binding (top panel). Results were confirmed using a complementary approach (HA-tagged CRABP2, anti-HA antibody immunoprecipitation, and anti-ARL11 immunoblotting (bottom panel). (<b>D</b>) Confirmation of ARL 11 and PGAM1 binding by IPWB. HEK-293T cells were transfected with HA-tagged <i>ARL11</i> and flag-tagged <i>PGAM1</i> constructs as indicated. Protein expression verified by immunoblotting with anti-PGAM1 (top panel) or anti-ARL11 (bottom panel) antibodies (DWBs). IPWBs were performed by anti-HA immunoprecipiation of ARL11 followed by immunoblotting with anti-PGAM1 (Top panel). Alternatively, immunoprecipitation was performed using HA-tagged PGAM1 followed by immunoblotting with anti-ARL11 (Bottom panel). (<b>E</b>) The in-frame cDNA library prevented interference caused by the <i>CRABP2</i> 5′-UTR that inhibits its binding to ARL11. HEK-293T cells were transfected with HA-<i>ARL11</i> and with YFP1-<i>CRABP2</i> or YFP1-5′-UTR-<i>CRABP2</i> as indicated. Protein expression was confirmed by immunoblotting with anti-CRABP2 antibody (top panel) or anti-YFP1 antibody (bottom panel). Alternatively, ARL11 was immunoprecipitated using the anti-HA antibody, and bound proteins were detected by immunoblotting with anti-CRABP2 (top panel) or anti-YFP1 (bottom panel) antibody. (<b>F</b>) The in-frame cDNA library prevents interference caused by the <i>PGAM1</i> 5′-UTR that prevents its binding to ARL11. HEK-293T cells were transfected with HA-<i>ARL11</i> and YFP1-<i>PGAM1</i>, or YFP1-5′-UTR-<i>PGAM1</i>. Protein expression was confirmed using anti-PGAM1 (top panel) or anti-YFP1 (bottom panel) antibodies (DWB). To identify ARL11-associated proteins (IPWB), ARL11 was immunoprecipitated using the anti-HA antibody and bound proteins were detected using either an anti-PGM1 (top panel) or anti-YFP (bottom panel) antibody.</p

Predicted expression of CRABP2 and PGAM1 proteins by the constructs with and without 5′-UTRs.

<p>(<b>A</b>) Comparison of <i>CRABP2</i> expression constructs with and without the <i>CRABP2</i> 5′-UTR. (<b>B</b>) Comparison of <i>PGAM1</i> expression constructs with and without the <i>PGAM1</i> 5′-UTR.</p

Analysis of the human 5′-UTR database, overview of the approach, and construction of the in-frame cDNA expression library.

<p>(<b>A</b>) Analysis of the human 5′-UTR database (<a href="http://utrdb.ba.itb.cnr.it/" target="_blank">http://utrdb.ba.itb.cnr.it/</a>) to predict their effects on expressed sequences following translation with a YFP1 tag peptide as fusion proteins during the construction of a prey cDNA library. (<b>B</b>) Overview of the screening procedure. (<b>C</b>) For the construction of the in-frame cDNA expression library, mRNA was isolated from normal human urothelial cells and was used as a template for first-strand cDNA synthesis using polyT primer. Double-stranded cDNAs without 5′-UTRs were synthesized using primers 1 and 2 (representing approximately 40% of the Kozak sequences that are present in vertebrate genomes) complemented with primer mixes 3 and 4 (representing the remaining 60% of the Kozak sequence combinations in vertebrates). In primer mixes 3 and 4, the combination of sequences referred to as “D” is an equal mixture of A, G and T, “H” is an equal mixture of A, C and T, “K” is an equal mixture of G and T, and “W” is an equal mixture of A and T. There are 19,683 and 157,464 possible sequence combinations in primer mixes 3 and 4, respectively. (<b>D</b>) Sequence analysis of the in-frame cDNA library was performed on 198 representative plasmids isolated from random colonies of the library.</p

Whole-Organ Genomic Characterization of Mucosal Field Effects Initiating Bladder Carcinogenesis

Summary: We used whole-organ mapping to study the locoregional molecular changes in a human bladder containing multifocal cancer. Widespread DNA methylation changes were identified in the entire mucosa, representing the initial field effect. The field effect was associated with subclonal low-allele frequency mutations and a small number of DNA copy alterations. A founder mutation in the RNA splicing gene, ACIN1, was identified in normal mucosa and expanded clonally with an additional 21 mutations in progression to carcinoma. The patterns of mutations and copy number changes in carcinoma in situ and foci of carcinoma were almost identical, confirming their clonal origins. The pathways affected by the DNA copy alterations and mutations, including the Kras pathway, were preceded by the field changes in DNA methylation, suggesting that they reinforced mechanisms that had already been initiated by methylation. The results demonstrate that DNA methylation can serve as the initiator of bladder carcinogenesis. : Majewski et al. report that methylation changes suppressing innate immunity and dysregulating Ras-related pathways initiate bladder carcinogenesis. Keywords: Whole-organ map, bladder cancer, DNA methylation, DNA copy alterations, founder mutation, field effect, clonal origins, clonal expansion, urothelial carcinoma, gene signatur

Dysregulation of EMT Drives the Progression to Clinically Aggressive Sarcomatoid Bladder Cancer

Summary: Sarcomatoid urothelial bladder cancer (SARC) displays a high propensity for distant metastasis and is associated with short survival. We report a comprehensive genomic analysis of 28 cases of SARC and 84 cases of conventional urothelial carcinoma (UC), with the TCGA cohort of 408 muscle-invasive bladder cancers serving as the reference. SARCs show a distinct mutational landscape, with enrichment of TP53, RB1, and PIK3CA mutations. They are related to the basal molecular subtype of conventional UCs and could be divided into epithelial-basal and more clinically aggressive mesenchymal subsets on the basis of TP63 and its target gene expression levels. Other analyses reveal that SARCs are driven by downregulation of homotypic adherence genes and dysregulation of the EMT network, and nearly half exhibit a heavily infiltrated immune phenotype. Our observations have important implications for prognostication and the development of more effective therapies for this highly lethal variant of bladder cancer. : Guo et al. report that sarcomatoid carcinoma of the bladder evolves by the progression of the basal subtype of conventional urothelial carcinoma with the enrichment of mutagenesis signature 1 and mutations of TP53, RB1, and PIK3CA. It is driven by the dysregulation of the EMT network and shows increased immune infiltrate with overexpression of PD-L1. Keywords: bladder cancer, sarcomatoid carcinoma, urothelial carcinoma, epithelial-mesenchymal transformation, genomic expression, chromatin remodeling, microRNA expression, molecular classification, basal subtype, immune phenotyp