Tumor and Host Determinants of Pulmonary Metastasis in Bladder Cancer
Despite the recent advances in the diagnosis of bladder cancer, recurrence after surgical intervention for muscle invasive disease is still problematic as nearly half of patients harbor occult distant metastases. Clinical data from human disease revealed that, invasive and metastatic bladder cancer cells can metastasize to lungs, and this in turn is associated with poor 5-year survival rate. Experimental rodent models of carcinogenesis and metastasis are available to study this phenomenon. Comparative gene expression profiling, proteomic and computational studies identified an intertwined network of metastasis promoters and suppressors that modulate the interactions between the components of the pulmonary milieu and cancer cells inflammatory mediators, ECM molecules, as well as peptide hormones. In this chapter we provide select exemplar of some of the molecular mechanisms underlying lung colonization by bladder cancer.
KeywordsBladder Cancer Bladder Cancer Cell Urothelial Cancer Human Bladder Cancer Bladder Cancer Cell Line
Urothelial cancer (UC) is the most common malignancy affecting the urinary system. When it affects the bladder, it leads to an estimated 70,530 new cases in the United States, with a male to female ratio of 3:1 and approximately 14,680 deaths, expected in 2010 . UC arises from the mucosal lining and is frequently multifocal. Numerous factors, including chromosomal markers, genetic polymorphisms, and genetic and epigenetic alterations may be involved in tumorigenesis, progression and metastasis. At initial presentation, 70% of patients with UC present with non-muscle invasive (formerly known as “superficial”), and 30% present with muscle invasive disease . Despite a good prognosis for patients with the former, recurrence is common and is associated with development of muscle invasive disease in up to 30%. Also, 50% of patients presenting with muscle invasive UC develop distant metastases leading to poor 5-year survival rate . Clinical data from human disease as well as experimental rodent models of carcinogenesis and metastasis reveals that when it occurs, metastasis of this tumor is commonly found in the regional lymph node metastasis and the lungs. Given that metastases are responsible for most of the deaths from this disease [3, 4], understanding of the process will aid in the development of new approaches for treatment.
14.2 Multistep Process of Metastasis
The high incidence of pulmonary metastases in cancer patients was initially believed to be a random process based on blood flow predominance as lungs receive significant cardiac output [5, 6]. This was supported by the observation that metastases often initiate in pulmonary arterioles and later traverse the basement membrane into the lung parenchyma [6, 7]. However, growing body of literature supports the patterns of metastases is also a consequence of the “seed and soil” theory put forth by Stephen Paget in 1889 . Hence, the development of lung metastasis is likely an active highly selective process instigated by tumor cells, and is strongly influenced by the interactions between tumor and host cells, and by both the immediate and extended tumor microenvironments [3, 7, 8, 9, 10, 11, 12, 13, 14].
14.2.1 The Lung Microenvironment as a Host for Metastatic Tumor Cells
The premetastatic niche may also be responsible for metastases to specific organs. The key tumor-secreted factors that determine metastatic sites and mediate premetastatic niche formation have yet to be identified, although the roles for cytokines/chemokines, MMPs and growth factors’ signaling in pulmonary endothelial cells and macrophages have been reported [25, 26]. These make the lung microenvironment more receptive to cancers cells. We have recently reported the roles of endothelin-1 in as an important mediator of lung inflammation and colonization of bladder cancer cells (discussed below) . Elevated fibronectin expression by fibroblasts and fibroblast-like cells resident at premetastatic sites seems to be an important factor in the development of the premetastatic niche . In addition, the secretion of versican by tumor cells and subsequently by inflammatory macrophages has been recently found to play a role in lung metastasis [10, 16, 27].
Overexpression of tumor-derived immunosuppressive factors such as TGFβ, VEGF, IL-6, MCP1/CCL2, MMPs and Cox-2/prostanoids in the inflammatory premetastatic niche dampens the tumor-suppressing activities of cells responsible of immune surveillance as natural killer cells and antigen-presenting cells and supports an inflammatory phenotype of tumor-associated macrophages, TAM; promote tumor progression by secreting growth factors and cytokines that stimulate tumor metastases [16, 17, 28]. TAMs can facilitate tumor formation and progression by activation of NF-κB and AP-1 [16, 17, 29], and are especially attracted to regions of hypoxia, where they secrete angiogenic inducers and proteases, and express high levels of the HIF-1 and -2 transcription factors [18, 30, 31, 32] that further augment macrophage recruitment and activation as well as tumor cell invasiveness. Furthermore, TAMs can release growth factors such as PDGF, EGF, cytokines/chemokines, and MMPs which enhance proliferation, survival, and invasion of tumor cells as well as secrete and induce secretion of ECM proteoglycans such as versican from tumor cells and fibroblasts in the lung milieu further augmenting invasiveness, inflammation and metastasis [10, 23, 33, 34, 35, 36, 37, 38].
Our laboratory has identified two major molecules implicated in bladder cancer metastasis to the lungs, the metastasis suppressor RhoGDI2 and the pro-metastatic Ral A/B.
14.2.2 The Metastasis Suppressor RhoGDI2
Rho family GDP Dissociation Inhibitor 2 (RhoGDI2) protein has been identified as functional metastasis suppressor during studies of the differential invasive and metastatic properties of isogenic human bladder carcinoma cell lines, T24 (non-metastatic), and T24T (highly invasive and metastatic) using experimental metastasis models and comparative genomic studies . RhoGDI2 was also found to be a prognostic marker in patients after cystectomy that its reduced expression was associated with decreased patient survival. However, approximately 35% of patients with moderate or high levels of RhoGDI2 protein-developed metastatic disease suggesting that not only the expression level, but other mechanisms might regulate the metastasis suppressor effect of RhoGDI2 . Phosphorylation, binding to specific partners, truncations, proteolytic cleavage, or change in subcellular localization was considered given the existing data on the other members of the RhoGDI family (reviewed in ). Comparative gene expression profiling of human bladder cancer tissues and cell lines identified that the down-stream effectors downregulated by RhoGDI2 are molecules that have long been implicated in pulmonary vascular diseases: Endothelin-1 (ET-1)  and versican [14, 27, 43].
14.2.3 Endothelin Axis
The association between increased levels of versican and progression of cancer to disseminated disease suggests that versican is important in promoting cancer cell proliferation, motility, invasion and metastasis [10, 16, 23, 33, 34, 35, 36, 37, 38, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65]. More recent studies  identified cancer cell-secreted versican, as a macrophage activator that acts through TLR2/6 inducing TNF-alpha secretion by myeloid cells, generating an inflammatory microenvironment hospitable for metastatic growth. In vitro functional studies, revealed versican is a key mediator of cancer cell-macrophages cross-talk and significantly inhibited their adhesion to and invasion of ECM molecules as well as pulmonary microvascular endothelial cells [27, 66]. Interestingly, comparative gene expression profiling of invasive/metastatic bladder cancer cells with clinical bladder cancer specimens identified versican as the top upregulated gene, positively correlated with bladder cancer stage and poor prognosis, and in vitro with invasiveness of bladder cancer cell lines [27, 43]. It remains to be seen if genetic manipulation of versican expression in bladder cancer cells, either by stable overexpression or knockdown demonstrates a role of tumor versican in metastasis.
14.2.5 Ral GTPases
CD24, is a glycosyl phosphatidyl inositol–linked surface protein , that has been identified as a downstream target of Ral signaling by profiling the expression of RalA/B–depleted bladder carcinoma cells [67, 68]. Originally identified as a surface differentiation marker of peripheral B-lymphocytes , CD24 received recent attention as a marker of stem cell populations of a variety of cancers [74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87]. CD24 overexpression has been associated with lung metastasis  as it promoted cancer cell rolling and adhesion to P-selectin in murine lung vasculature and platelets [76, 78, 79, 80, 85, 89, 90, 91, 92, 93, 94, 95]. Consistently, CD24 has been recently reported to be a hypoxia-regulated gene in some cancer cell lines [96, 97] that was down-regulated by Cox-2 inhibitor, Celecoxib, suggesting role in inflammation associated with metastasis [98, 99, 100]. CD24 is highly expressed in urothelial as well as other cancers. Immunohistological localization of CD24 revealed high levels of expression correlated with the stromal/muscle invasion, stage, grade and shorter patient disease-free survival [67, 101]. Depletion of CD24 function in bladder cancer cell lines was found to be associated with decreased cell proliferation and anchorage-independent growth, changes in the actin cytoskeleton, and induction of apoptosis . In addition, monoclonal antibodies against CD24 that were efficacious in inhibiting in vitro and in vivo growth of colorectal and pancreatic cancer cells as well as sensitizing them to conventional adjuvant chemotherapeutics [98, 99, 100, 102, 103, 104], significantly reduced the number of lung metastases that developed after tail vein injection of the metastatic UMUC3 cell line in an experimental model of bladder cancer . However, the exact mechanism of CD24 involvement in lung metastasis is still elusive.
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