ADAM15 Is Functionally Associated with the Metastatic Progression of Human Bladder Cancer.

ADAM15 is a member of a family of catalytically active disintegrin membrane metalloproteinases that function as molecular signaling switches, shed membrane bound growth factors and/or cleave and inactivate cell adhesion molecules. Aberrant metalloproteinase function of ADAM15 may contribute to tumor progression through the release of growth factors or disruption of cell adhesion. In this study, we utilized human bladder cancer tissues and cell lines to evaluate the expression and function of ADAM15 in the progression of human bladder cancer. Examination of genome and transcriptome databases revealed that ADAM15 ranked in the top 5% of amplified genes and its mRNA was significantly overexpressed in invasive and metastatic bladder cancer compared to noninvasive disease. Immunostaining of a bladder tumor tissue array designed to evaluate disease progression revealed increased ADAM15 immunoreactivity associated with increasing cancer stage and exhibited significantly stronger staining in metastatic samples. About half of the invasive tumors and the majority of the metastatic cases exhibited high ADAM15 staining index, while all low grade and noninvasive cases exhibited negative or low staining. The knockdown of ADAM15 mRNA expression significantly inhibited bladder tumor cell migration and reduced the invasive capacity of bladder tumor cells through MatrigelTM and monolayers of vascular endothelium. The knockdown of ADAM15 in a human xenograft model of bladder cancer inhibited tumor growth by 45% compared to controls. Structural modeling of the catalytic domain led to the design of a novel ADAM15-specific sulfonamide inhibitor that demonstrated bioactivity and significantly reduced the viability of bladder cancer cells in vitro and in human bladder cancer xenografts. Taken together, the results revealed an undescribed role of ADAM15 in the invasion of human bladder cancer and suggested that the ADAM15 catalytic domain may represent a viable therapeutic target in patients with advanced disease

HER2 and EGFR Overexpression Support Metastatic Progression of Prostate Cancer to Bone.

Activation of the EGF receptors EGFR (ErbB1) and HER2 (ErbB2) drives the progression of multiple cancer types through complex mechanisms that are still not fully understood. In this study, we report that HER2 expression is elevated in bone metastases of prostate cancer independently of gene amplification. An examination of HER2 and NF-κB receptor (RANK) coexpression revealed increased levels of both proteins in aggressive prostate tumors and metastatic deposits. Inhibiting HER2 expression in bone tumor xenografts reduced proliferation and RANK expression while maintaining EGFR expression. In examining the role of EGFR in tumor-initiating cells (TIC), we found that EGFR expression was required for primary and secondary sphere formation of prostate cancer cells. EGFR expression was also observed in circulating tumor cells (CTC) during prostate cancer metastasis. Dual inhibition of HER2 and EGFR resulted in significant inhibition of tumor xenograft growth, further supporting the significance of these receptors in prostate cancer progression. Overall, our results indicate that EGFR promotes survival of prostate TIC and CTC that metastasize to bone, whereas HER2 supports the growth of prostate cancer cells once they are established at metastatic sites. Cancer Res; 77(1); 74-85. ©2016 AACR

Knockdown of ADAM15 inhibits bladder tumor cell motility.

<p><i>A)</i> ADAM15 immunoblot of UM-UC-9 bladder cancer cells expressing empty vector and scrambled ADAM15 sequences (ctl) and short hairpin RNA (shA15) and UM-UC-6 expressing scrambled ADAM15 sequences (ctl) and short hairpin RNA (shA15). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. <i>B)</i> Wound healing assay comparing extent of wound closure in shA15 UM-UC-9 and shA15 UM-UC-6 cells compared to controls (ctl). Bars represent mean ± SD (n = 4) of one of two independent experiments (UM-UC-9, <i>top</i>) and mean ± SEM (N = 3) (UM-UC-6, <i>bottom</i>). <i>C)</i> Proliferation assays of both knockdown (shA15) and control (ctl) UM-UC-9 and UM-UC-6 cells over a 72 hour time course. Results represent mean ± SD of one of three independent experiments.</p

<i>In vivo</i> inhibition of ADAM15 activity reduces tumor growth.

<p><i>A)</i> ADAM15 immunoblot of UM-UC-6 bladder cancer cells expressing empty vector (Vec), control scrambled ADAM15 sequences (ctlA15UC6) and short hairpin RNA (shRNA) sequence (shA15UC6). GAPDH was utilized as loading control (<i>top</i>). Subcutaneous inoculation of shA15UC6 cells (n = 10) in SCID mice led to decreased tumor weight compared to ctlA15UC6 control cells (n = 10) (<i>bottom</i>). <i>B)</i> UM-UC-6 cells were subcutaneously implanted in SCID mice. Specific inhibition of the ADAM15 catalytic activity by daily treatment with adamastat 100 mg/kg (n = 5) during 3 weeks led to decrease in tumor growth compared to vehicle treated mice (n = 5). Bars represent mean tumor weight (mg) ± SD (<i>5A</i>,<i>B</i>).</p

Knockdown of ADAM15 decreases bladder cancer cell invasion and transmigration through vascular endothelium.

<p><i>A)</i> Extent of Matrigel^TM invasion of UM-UC-9 and UM-UC-6 bladder cancer cells expressing empty vector and scrambled ADAM15 sequences (ctl) and short hairpin RNA (shA15). The bars indicate mean invading cells ± SD (n = 3). The data are representative of 1 of 5 UM-UC-9 or 1 of 3 UM-UC-6 independent experiments. A representative 20X image of each condition is shown. <i>B)</i> Transendothelial migration assay revealed reduced transmigration through an endothelial HUV-EC-C monolayer of shA15UM-UC-9 and shA15UM-UC-6 cells when compared with control cells (ctl). Bars represent mean ± SEM (N = 3). A representative 20X image of each condition is shown.</p

ADAM15 staining index in bladder cancer microarrays.

<p>ADAM15 staining index in bladder cancer microarrays.</p

Novel ADAM15-specific sulfonamide inhibitor reduces viability of bladder cancer cells.

<p><i>A)</i> Surface model of adamastat binding to the ADAM15 catalytic. <i>B)</i> A mesh representation of adamastat binding in the S1’ pocket of the catalybtic domain of ADAM15. <i>C)</i> Fluorescence Resonance Energy Transfer assay indicating that inhibition of the recombinant ADAM15 catalytic domain (rA15cat) by adamastat is as effective as two other previously tested metalloproteinase inhibitors over the same concentration range. FI, Fluorescence Intensity. <i>D)</i> Viability of adamastat treated SV-HUC-1, UM-UC-9 and UM-UC-6 cells. Vehicle represents 0.1% DMSO control. Bars show mean cell viability % ± SD in 1 out of three independent experiments.</p

The metastatic progression of human bladder cancer is associated with elevated DNA copy number and increased ADAM15 expression.

<p>Analysis utilizing the Oncomine^TM (Compendia Bioscience) gene browser. <i>A)</i> Boxplots summarize mean copy number and standard deviation (SD) of a bladder cancer gene expression data set. ADAM15 copy number was elevated in invasive bladder cancer (N1+) compared to noninvasive disease (N0). <i>B)</i> ADAM15 mRNA is overexpressed in invasive bladder cancer compared to noninvasive bladder cancer. Bars represent ADAM15 mRNA levels in three different published mRNA expression studies. <i>C)</i> Microphotographs of ADAM15 immuno-staining of a bladder cancer progression tissue arrays. Three pathological stages are represented (normal tissue, noninvasive, and invasive bladder cancer). <i>D)</i> Boxplots represent the ADAM15 staining index in this TMA as mean ± SD. Invasive and metastatic bladder cancer specimens exhibited significantly increase staining index compared to noninvasive disease.</p