The outcome of bladder cancer treatment is largely dependent upon the pathobiology of individual bladder tumors 
. Previous microarray-based gene expression studies of various stage bladder cancers have recognized the distinct behaviors and genetic nature of superficial and muscle-invasive tumors, suggesting the possibility that the two are discrete pathobiological entities 
. However, no biomarkers have proven able to predict progression to muscle-invasive disease, and the physiological mechanisms that drive bladder tumor progression and invasive behavior are not well understood.
We have analyzed a large collection of expression microarray-derived data to develop a functional picture of bladder cancer progression to muscle-invasive disease. We achieved this by using only publicly available microarray data and open-access computational resources. This avoids the need to generate custom tools to perform specific analyses to analyze large amounts of multi-dimensional data. Moreover, this strategy avoided the need to invest the time and resources to acquire appropriate clinical samples in large enough numbers to allow a meaningful analysis, as well as the time and resources to perform a large number of microarrays analyses. This is especially important given that a large amount of data already exists and is available to the biomedical research community. By re-purposing public data to address our specific interests, we have not only avoided significant expenses but we also help to increase the value drawn from existing data. This has resulted in a novel systems-level model of TCC progression and muscle-invasive disease that integrates gene expression data with functional information and relationships from previous experimental observations.
In the present study, we identified specific expression differences that occur with bladder cancer progression and muscle-invasive tumors. Our results show that progression from papillary Ta to more advanced stage tumors is associated with a general increase in expression of a network of genes involved in Ras/MAPK and associated signaling pathways (). These pathways are closely associated with proliferation and are frequently disregulated in cancers, including bladder cancer. Interestingly, these pathways are downstream of the EGFR and ErbB2 family proteins whose expression and activity are related to bladder cancer progression, as well as ErbB3, FGFR3 and HRAS that are active in superficial tumors 
. The increased expression of genes in Ras/MAPK and PI3K pathways may be related to the disregulation of p53, Rb and other tumor suppressors in muscle-invasive bladder tumors 
. The increased expression of components of this large pathway network may act to promote and enhance the effects of EGFR and ErbB2 activity, but the dependence of muscle-invasive tumors on the activities of these pathways in concert has not been investigated. Conversely, the relatively low expression of Ras/MAPK and PI3K pathways in superficial tumors may compensate for the elevated activity of ErbB3, FGFR3 and RAS isoforms, possibly a factor that limits the progression to muscle-invasive disease. These and other issues will be further addressed through the course of developing a systems-level understanding of bladder cancer progression.
We also used a similar approach to identify gene expression changes specifically associated with muscle-invasive tumors (, , ). We found that these initially selected genes occur sporadically across the initial network rather than focused on a particular pathway within the network, but the expression of these genes is consistent and distinct in superficial and muscle-invasive tumor samples (). We then tested whether the expression differences of selected genes in our initial dataset were consistent with data from outside studies by using Oncomine to measure the median expression of each genes in 3 to 8 independent studies that include superficial and muscle-invasive bladder tumors, selecting a final set of 7 genes () 
. Taking into account the number of samples within each study, these results ultimately represent the gene expression data of hundreds of individual tumors, supporting the statistical validity of our observations.
The selected 7 genes can be separated into extracellular matrix proteins (COL3A1, COL5A1, COL11A1, FN1) and kinase signaling proteins (CDC25C, MAPK10, ErbB3). While the variation in expression data does not support the use of any of these genes as biomarkers individually (Data Not Shown), their expression may support a more specific means to identify muscle-invasive bladder tumors in the future. FN1 itself is a potential urine biomarker for bladder cancer detection 
. While well supported, their functional relationships and relevance to muscle-invasive bladder cancer were not immediately apparent. To aid in our interpretation, the data were analyzed using text-mining programs that identify instances where gene names occur in potentially interactive contexts within abstracts and full texts of published reports in PubMed 
. We found that these genes have been associated with cancer, bladder cancer, metastasis, angiogenesis, and invasion, suggesting that their involvement in cancer progression and/or muscle-invasive behaviors have been previously observed in various systems ().
The reported functions and interactions of the selected genes suggest a novel model of muscle-invasive disease in bladder cancer (). COL3A1, COL5A1 and COL11A1 are fibrillar collagens, which act as “tracks” for metastatic invasion of breast tumors into secondary organs 
. Formation of these fibrils is initiated by interactions between COL5A1 and COL11A1 proteins 
. Tumor-secreted proteases diffuse along collagen fibrils and modify existing matrix to allow tumor cells a path of least-resistance along fibrils 
. Collagens and FN1 are among many ligands for integrins, a class of surface receptor proteins that are functional heterodimers of 8 α and 18 β subunits that each bind specific ligands 
. COL3A1 and COL5A1 are functionally related in connective tissue disorders 
, and expression of different collagens are associated with various cancers. FN1 “decorates” collagen fibrils and affects integrin binding specificity 
. Integrin signaling stimulates RAS/MAPK signaling and modifies the activity of receptor tyrosine kinases, including EGFR/ErbB proteins, which are also upstream of Ras/MAPK 
. Interestingly, FN1 has been reported to preferentially induce the activity of EGFR, ErbB2 and ErbB4 but not ErbB3 activity 
. Our identification of decreased ErbB3 expression in muscle-invasive tumors is well supported by previous studies 
. Formation of ErbB3/ErbB2 is kinetically preferred among ErbB family receptors, and ErbB3 is the only ErbB receptor that directly associates with and activates the PI3K signaling pathway 
. In all, this evidence suggests a “class-switch” from ErbB3 to EGFR/ErbB2 mediated ErbB signaling, mediated in part by collagen and fibronectin-stimulated integrin signaling, is a key mechanism promoting muscle-invasion of bladder tumors. This may affect trafficking of active receptor proteins and impact the intensity and duration of downstream signaling 
, as well as alter the pathways that stimulate PI3K signaling.
Model of muscle-invasive bladder cancer based on expression data and published observations.
Importantly, the effects of these gene expression differences in muscle-invasive tumors are likely augmented by increased expression of genes in mitogenic pathways that we find associated with general bladder cancer progression in the absence of activation mutations in RAS and PI3K pathways that are prevalent in superficial tumors. The ErbB and collagen-activated integrin proteins are upstream activators of Ras/MAPK signaling, which is associated with proliferative and tumorigenic activity and which our above expression data shows is generally increased in muscle-invasive bladder cancer (). Changes in how Ras/MAPK and PI3K signaling are regulated by upstream receptors may fundamentally alter the nature of the responses to those signaling pathways. The MAPK family protein MAPK10/JNK3 is generally involved in stress signaling and acts counter to Erk MAPK and PI3K signaling 
. The decreased expression of antagonistic signaling proteins such as MAPK10 would further promote Ras/Erk MAPK signaling activity. Ultimately these activities modulate the expression and activities of proteins that affect proliferative, pro-metastatic and invasive behavior, including the CDC25C phosphatase which stimulates cyclin/CDK activity and mitosis. Metastasis and progression are further promoted by the disruption of tumor suppressors in muscle-invasive bladder cancer, including p53, Rb, myc and others that would act to limit proliferative and metastatic behavior 
. As well as providing a basis for future systems-level studies of TCC, this model will inform efforts to identify and develop the therapeutic targets and predictive biomarkers that will guide the clinical treatment of muscle-invasive bladder cancer.
In summary, we have analyzed public expression microarray data of bladder tumors across stages, identifying the increased expression of the proliferative and pro-survival Ras/MAPK and PI3K pathways as a potential regulatory hallmark of tumor progression, while related genes, including fibrillar collagens are associated specifically with muscle-invasive bladder cancer. Translation of this information into clinical treatment may potentially improve the identification, treatment and outcome of bladder cancer in patients as well as in the treatment of other cancers.