The thrombin receptor protease activated receptor-1 (PAR-1) is overexpressed in metastatic melanoma cell lines and tumor specimens. Previously, we demonstrated a significant reduction in tumor growth ...and experimental lung metastasis after PAR-1 silencing via systemic delivery of siRNA encapsulated into nanoliposomes. Gene expression profiling identified a 40-fold increase in expression of Maspin in PAR-1—silenced metastatic melanoma cell lines. Maspin promoter activity was significantly increased after PAR-1 silencing, suggesting that PAR1 negatively regulates Maspin at the transcriptional level. ChIP analyses revealed that PAR-1 decreases binding of Ets-1 and c-Jun transcription factors to the Maspin promoter, both known to activate Maspin transcription. PAR-1 silencing did not affect Ets-1 or c-Jun expression; rather it resulted in increased expression of the chromatin remodeling complex CBP/p300, as well as decreased activity of the CBP/p300 inhibitor p38, resulting in increased binding of Ets-1 and c-Jun to the Maspin promoter and higher Maspin expression. Functionally, Maspin expression reduced the invasive capability of melanoma cells after PAR-1 silencing, which was abrogated after rescuing with PAR-1. Furthermore, tumor growth and experimental lung metastasis was significantly decreased after expressing Maspin in a metastatic melanoma cell line. Moreover, silencing Maspin in PAR-1—silenced cells reverted the inhibition of tumor growth and experimental lung metastasis. Herein, we demonstrate a mechanism by which PAR-1 negatively regulates the expression of the Maspin tumor-suppressor gene in the acquisition of the metastatic melanoma phenotype, thus attributing an alternative function to PAR-1 other than coagulation.
Purpose Expression of various members of the ErbB family (epidermal growth factor receptor/ErbB-1, ErbB-2, ErbB-3 and ErbB-4) is associated with disease stage and survival in patients with urothelial ...carcinoma. We examined the correlation of ErbB family receptor expression with the progression of urothelial carcinoma and survival. Materials and Methods A urothelial carcinoma tissue array was constructed from 248 archival paraffin blocks and quality control studies were ascertained. The tissue microarray was stained for epidermal growth factor receptor, ErbB-2, ErbB-3 and ErbB-4, and analyzed using an automated reader. Patient data included grade, stage, growth pattern, recurrence and survival. Results Kaplan-Meier estimates of 5-year overall and recurrence-free survival were 58% and 27%, respectively. Patients with high grade, invasive or nonpapillary disease had a worse prognosis than patients with low grade, superficial or papillary disease (p <0.0001). High epidermal growth factor receptor or low ErbB-4 expression was associated with nonpapillary, high grade and invasive tumors as well as with significantly shorter recurrence-free and overall survival (p <0.002, 0.028 and 0.047, respectively). Levels of ErbB-2 and ErbB-3 expression were not associated with overall or recurrence-free survival. Conclusions The expression profiles of ErbB-4 and epidermal growth factor receptor are prognostic in urothelial carcinoma. They may help in selecting patients at high risk with bladder cancer for more aggressive therapy.
Epidermal growth factor receptor (EGFR) is an attractive target for the treatment of urothelial carcinoma, but a clinical response can be expected in only a small proportion of patients. The aim of ...this study was to define molecular markers of response to cetuximab therapy in a panel of urothelial carcinoma cell lines.
Eleven cell lines were investigated for antiproliferative response to cetuximab based on (3)Hthymidine incorporation. A variety of markers, including EGFR expression, phosphorylation, and gene amplification, as well as the expression of other growth factor receptors, their ligands, and markers of epithelial-to-mesenchymal transition were investigated. Cohen's kappa statistic was used to estimate the agreement between response and expression of these markers. E-cadherin was silenced by small interfering RNA in two sensitive cell lines, and the effect on the response to cetuximab was measured.
We were able to identify a panel of relevant markers pertaining especially to alternate growth factor receptor expression and epithelial-to-mesenchymal transition that predicted response to cetuximab. The data suggested that expression of intact HER-4 (kappa, 1.00; P = 0.008), E-cadherin (kappa, 0.81; P = 0.015), and beta-catenin (kappa, 0.81; P = 0.015) and loss of expression of platelet-derived growth factor receptor beta (kappa, 0.57; P = 0.167) were associated with response to cetuximab therapy. Silencing E-cadherin in two sensitive cell lines reduced responsiveness to cetuximab in both (P < 0.001).
A panel of predictive markers for cetuximab response has been established in vitro and is currently being evaluated in a prospective clinical trial of neoadjuvant EGFR-targeted therapy. Most importantly, E-cadherin seems to play a central role in modulation of EGFR response in urothelial carcinoma.
Protease-activated receptor-1 (PAR-1) is a key player in melanoma metastasis with higher expression seen in metastatic melanoma cell lines and tissue specimens. cDNA microarray and Western blot ...analyses reveal that the gap junctional intracellular communication molecule connexin 43 (Cx-43), known to be involved in tumor cell diapedesis and attachment to endothelial cells, is significantly decreased after PAR-1 silencing in metastatic melanoma cell lines. Furthermore, Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells, suggesting that PAR-1 regulates Cx-43 at the transcriptional level. Chromatin immunoprecipitation studies showed a reduction in the binding of SP-1 and AP-1 transcription factors to the promoter of Cx-43. Both transcription factors have been shown previously to be required for maximal Cx-43 promoter activity. These results were corroborated by mutating the AP-1 and SP-1 binding sites resulting in decreased Cx-43 promoter activity in PAR-1-positive cells. Moreover, as Cx-43 has been shown to facilitate arrest of circulating tumor cells at the vascular endothelium, melanoma cell attachment to endothelial cells was significantly decreased in PAR-1-silenced cells, with this effect being abrogated after PAR-1 rescue. Herein, we report that up-regulation of PAR-1 expression, seen in melanoma progression, mediates high levels of Cx-43 expression. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Indeed, Cx-43 expression was restored following PAR-1 rescue in PAR-1-silenced cells. Taken together, our data support the tumor promoting function of Cx-43 in melanoma.
Expression of interleukin-8 (IL-8) by human melanoma cells correlates with their metastatic potential in vivo. Moreover, UVB irradiation of primary cutaneous melanoma induces IL-8 mRNA and protein ...production and increases both tumor growth and metastasis in nude mice. Although IL-8 has been shown to be an angiogenic factor, the biological consequences of increased IL-8 production by melanoma cells and the role of IL-8 in the metastatic process remains unclear. The purpose of this review is to determine the role of IL-8 in tumor growth and metastasis of human melanoma. Transfection of nonmetastatic and IL-8-negative melanoma cells with the IL-8 gene rendered them highly tumorigenic and increased their metastatic potential in nude mice. The IL-8-transfected cells displayed upregulation of MMP-2 expression and activity and increased invasiveness through Matrigel-coated filters. Activation of MMP-2 by IL-8 can enhance the invasion of host stroma by the tumor cells and increase angiogenesis and, hence, metastasis. In addition to UVB, IL-8 can also be upregulated by hypoxia conditions, suggesting that the environment plays a major role in regulating IL-8 expression and metastasis. The studies summarized in this review suggest that in melanoma, IL-8 may serve as the angiogenic factor distinguishing benign from malignant cells.
Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF) 1 contributes to melanoma progression and metastasis at least in part by promoting tumor ...cell survival and stimulating matrix metalloproteinase (MMP) 2 expression. However, little is known about the regulation of CREB and ATF-1 activities and their phosphorylation within the tumor microenvironment. We analyzed the effect of platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, for its ability to activate CREB and ATF-1 in eight cultured human melanoma cell lines, and we found that PAF receptor (PAFR) was expressed in all eight lines. In metastatic melanoma cell lines, PAF induced CREB and ATF-1 phosphorylation via a PAFR-mediated signal transduction mechanism that required pertussis toxin-insensitive Gαq protein and adenylate cyclase activity and was antagonized by a cAMP-dependent protein kinase A and p38 MAPK inhibitors. Addition of PAF to metastatic A375SM cells stimulated CRE-dependent transcription, as observed in a luciferase reporter assay, without increasing the CRE DNA binding capacity of CREB. Furthermore, PAF stimulated the gelatinase activity of MMP-2 by activating transcription and MMP-2 expression. MMP-2 activation correlated with the PAF-induced increase in the expression of an MMP-2 activator, membrane type 1 MMP. PAF-induced expression of pro-MMP-2 was causally related to PAF-induced CREB and ATF-1 phosphorylation; it was prevented by PAFR antagonist and inhibitors of p38 MAPK and protein kinase A and was abrogated upon quenching of CREB and ATF-1 activities by forced overexpression of a dominant-negative form of CREB. PAF-induced MMP-2 activation was also down-regulated by p38 MAPK and protein kinase A inhibitors. Finally, PAFR antagonist PCA4248 inhibited the development of A375SM lung metastasis in nude mice. This result indicated that PAF acts as a promoter of melanoma metastasis in vivo. We proposed that metastatic melanoma cells overexpressing CREB/ATF-1 are better equipped than nonmetastatic cells to respond to PAF within the tumor microenvironment.
The acquisition of the metastatic melanoma phenotype is associated with increased expression of the melanoma cell adhesion molecule MCAM/MUC18 (CD146). However, the mechanism by which MUC18 ...contributes to melanoma metastasis remains unclear. Herein, we stably silenced MUC18 expression in two metastatic melanoma cell lines, A375SM and C8161, and conducted cDNA microarray analysis. We identified and validated that the transcriptional regulator, inhibitor of DNA binding-1 (Id-1), previously shown to function as an oncogene in several malignancies, including melanoma, was downregulated by 5.6-fold following MUC18 silencing. Additionally, we found that MUC18 regulated Id-1 expression at the transcriptional level via ATF-3, which itself was upregulated by 6.9-fold in our cDNA microarray analysis. ChIP analysis showed increased binding of ATF-3 to the Id-1 promoter after MUC18 silencing. To complement these studies, we rescued the expression of MUC18, which reversed the expression patterns of Id-1 and ATF-3. Moreover, we showed that MUC18 promotes melanoma invasion through Id-1, as overexpression of Id-1 in MUC18-silenced cells resulted in increased MMP-2 expression and activity. To our knowledge, this is the first demonstration that MUC18 is involved in cell signaling regulating the expression of Id-1 and ATF-3, thus contributing to melanoma metastasis.
In a previous study, we found that the small-molecule epidermal growth factor receptor (EGFR) inhibitor gefitinib (ZD1839, Iressa) blocked cell proliferation at biologically relevant concentrations ...in approximately one third (6 of 17) of human bladder cancer cell lines examined. Here, we studied the effects of gefitinib on apoptosis in a representative subset of the same panel of cells. The drug had modest effects on DNA fragmentation as a single agent at concentrations that produced strong growth inhibition (< or =1 micromol/L) and also failed to promote apoptosis induced by conventional chemotherapeutic agents (gemcitabine and paclitaxel). However, gefitinib did interact with recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce high levels of apoptosis in gefitinib-responsive but not gefitinib-unresponsive lines. The molecular mechanisms involved down-regulation of active AKT and X-linked inhibitor of apoptosis protein (XIAP) expression and were mimicked by chemical inhibitors of the phosphatidylinositol 3-kinase/AKT pathway but not of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway. Furthermore, direct small interfering RNA-mediated knockdown of AKT resulted in down-regulation of XIAP and TRAIL sensitization, and knockdown of XIAP itself was sufficient to reverse TRAIL resistance. Together, our results show that EGFR pathway activation limits TRAIL-induced apoptosis via an AKT- and XIAP-dependent mechanism in EGFR-dependent human bladder cancer cells, providing the conceptual framework for a further evaluation of the combination in relevant preclinical in vivo models.
Melanoma begins with benign nevi and progresses to radial growth phase (RGP) and to vertical growth phase (VGP), metastatic phenotype. The molecular changes associated with these transitions are not ...yet well defined. However, transcriptional regulation of some genes that are critical in melanoma progression is beginning to be elucidated. The first part of this review will focus on our recent studies demonstrating that progression of human melanoma is associated with loss of expression of the transcription factor AP-2. In metastatic melanoma cells, this loss resulted in overexpression of MCAM/MUC18 and MMP-2, and lack of expression of c-KIT. In further investigations, we inactivated AP-2 in SB-2 primary cutaneous melanoma cells by using a dominant-negative AP-2, the AP-2B gene. Expression of AP-2B in SB-2 cells augmented their tumorigenicity in nude mice and upregulated MMP-2 expression and activity. We have also recently demonstrated that loss of AP-2 expression in metastatic melanoma cells resulted in overproduction of the thrombin receptor, PAR-1. Other studies have shown that AP-2 regulates additional genes involved in melanoma development and progression, including E-cadherin, p21/WAF-1, HER2, Bcl-2, FAS/APO-1, IGF-R-1, and VEGF. We propose that loss of AP-2 is crucial in the development of malignant melanoma. Additionally, the transition of melanoma cells from RGP to VGP is associated with overexpression of two transcription factors, CREB and ATF-1, both of which may act as survival factors for human melanoma cells. The second part of the review will briefly discuss the role of other transcription factors, including ATF-2, SNAIL, MITF, and NFkappaB in the progression of human melanoma and will summarize recent knowledge on how changes in the expression of these transcription factors contribute to acquisition of the metastatic phenotype in human melanoma.
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