Profiling Bladder Cancer Organ Site-Specific Metastasis Identifies LAMC2 as a Novel Biomarker of Hematogenous Dissemination

Abstract

Little is known about which genes mediate metastasis in bladder cancer, which accounts for much of the mortality of this disease. We used human bladder cancer cell lines to develop models of two clinically common metastatic sites, lung and liver, and evaluated their gene expression with respect to human tumor tissues. Parental cells were injected into either the murine spleen to generate liver metastases or tail vein to generate lung metastases with sequential progeny derived by re-injection and comparisons made of their organ-specific nature by crossed-site injections. Both genomic and transcriptomic analyses of organselected cell lines found salient differences and shared core metastatic profiles, which were then screened against gene expression data from human tumors. The expression levels of laminin V gamma 2 (LAMC2) contained in the core metastatic signature were increased as a function of human tumor stage, and its genomic location was in an area of gain as measured by comparative genomic hybridization. Using immunohistochemistry in a human bladder cancer tissue microarray, LAMC2 expression levels were associated with tumor grade, but inversely with nodal status. In contrast, in node-negative patients, LAMC2 expression was associated with visceral metastatic recurrence. In summary, LAMC2 is a novel biomarker of bladder cancer metastasis that reflects the propensity of cells to metastasize via either lymphatic or hematogenous routes. Metastasis represents a critical event in the natural history of disease for a cancer patient and is associated with significant reduction in patient survival.1 However, although a significant body of research has shed light on this process, less is known about the nature of organspecific metastasis.2,3 In vivo metastasis assays using cancer cell lines passaged through mouse models have been used to discover the role of specific genes relevant to metastatic behavior. 4 For example, in breast cancer, recent reports have addressed the question of the molecular regulation of organ site-specific metastasis. Using the spontaneously metastatic MDA-MB-231 human breast cancer cell line, one group reported the in vivo selection of sublines of differing metastatic potential to bone, lung, and adrenal gland, 5,6 and then evaluated in both animal models and metastatic tumor tissues from patients the gene expression profiles associated with site-specific metastasis. 7Bladder cancer is the fifth most commonly diagnosed cancer in the United States, 8 and the molecular lesions characterizing bladder carcinogenesis and progression are beginning to be better elucidated. 9 However, little is known about the pathways that regulate general metastatic propensity or organ site-specific tropism to lung or liver, which are two common sites of dissemination in this and other tumor types. Our prior work has focused on the discovery and investigation of genes that regulate lung metastasis, such as the metastasis suppressor RhoGDI2, by comparing metastatic T24T cells to their isogenic nonmetastatic relativ...

LAMC2 enhances the metastatic potential of lung adenocarcinoma

“…The cutoff of 30% was used for LAMC2 positivity as in previous reports with the same antibody. 11,14,50 Vimentin was considered to be positive if ≥ 1% of cancer cells were stained in the cytoplasm as reported previously. 51,52 Detailed methods are described in the Supplementary section.…”

“…8 Besides, LAMC2 monomer expression has been frequently found in the invasive front of epithelial tumors such as ADCs of the stomach, colon, pancreas, and lung, SCCs of the esophagus, head and neck, skin and lung, and transitional cell carcinoma of the bladder. 5,[9][10][11][12][13][14] Epithelialmesenchymal transition (EMT) has a critical role in the early stages of dissemination of malignant epithelial tumors leading to metastatic spread. 15 There is growing evidence that LAMC2 expression increases in the carcinoma cells undergoing EMT, [16][17][18] and that level of LAMC2 expression is regulated directly by the EMT master regulator ZEB1 and activated β-catenin in invasive colorectal carcinoma cells.…”

Extravasation during bladder cancer metastasis requires cortactin-mediated invadopodia formation

“…Bladder cancer cells (2x10 6 ) were suspended in 0.1 ml serum-free RPMI-1640 medium and injected into the tail vein of 6-to 8-week-old nude mice (BALB/cAJcI-nu/nu; Clea Japan, Inc., Tokyo, Japan). After 3 weeks, lungs were harvested and fixed with formalin.…”

“…In patients with aggressive bladder cancer, the most common site of hematogenous metastasis is the lungs (6). The circulating bladder cancer cells exit the lung microvessels by transendothelial invasion, to enter the lung tissue.…”

RREB1 Transcription Factor Splice Variants in Urologic Cancer

“…We performed IHC on a 142-sample TMA 35 from patients with bladder cancer who underwent cystectomy. The intensity of RREB1 staining was scored as negative, low/focal, moderate, and high ( Figure 6A).…”