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Supplementary Table 1 from Expression of Id-1 Is Regulated by MCAM/MUC18: A Missing Link in Melanoma Progression

Supplementary Figure 2 from Expression of Id-1 Is Regulated by MCAM/MUC18: A Missing Link in Melanoma Progression

Relative mRNA expression of NFAT1, IL-8, and MMP-3

Schematic presentation of the IL-8 and MMP-3 promoters for NFAT1 binding sites

Supplementary Methods and Figure Legend from Overexpression of Protease-Activated Receptor-1 Contributes to Melanoma Metastasis via Regulation of Connexin 43

Supplementary Figure 4 from Expression of Id-1 Is Regulated by MCAM/MUC18: A Missing Link in Melanoma Progression

Supplementary Figure 1 from Expression of Id-1 Is Regulated by MCAM/MUC18: A Missing Link in Melanoma Progression

Effect of NFAT1 silencing or overexpression on melanoma cell viability

Top potential genes downregulated after silencing NFAT1

Immunohistochemistry of tumor samples after silencing or overexpressing NFAT1 in melanoma cells

Supplementary Figure 3 from Expression of Id-1 Is Regulated by MCAM/MUC18: A Missing Link in Melanoma Progression

Nuclear factor of activated T cell (NFAT1, NFATC2) is a transcription factor that binds and positively regulates IL2 expression during T-cell activation. NFAT1 has important roles in both innate and adaptive immune responses, but its involvement in cancer is not completely understood. We previously demonstrated that NFAT1 contributes to melanoma growth and metastasis by regulating the autotaxin gene (Enpp2). Here, we report a strong correlation between NFAT1 expression and metastatic potential in melanoma cell lines and tumor specimens. To elucidate the mechanisms underlying NFAT1 overexpression during melanoma progression, we conducted a microarray on a highly metastatic melanoma cell line in which NFAT1 expression was stably silenced. We identified and validated two downstream targets of NFAT1, IL8, and MMP3. Accordingly, NFAT1 depletion in metastatic melanoma cell lines was associated with reduced IL8 and MMP3 expression, whereas NFAT1 overexpression in a weakly metastatic cell line induced expression of these targets. Restoration of NFAT1 expression recovered IL8 and MMP3 expression levels back to baseline, indicating that both are direct targets of NFAT1. Moreover, in vivo studies demonstrated that NFAT1 and MMP3 promoted melanoma tumor growth and lung metastasis. Collectively, our findings assign a new role for NFAT1 in melanoma progression, underscoring the multifaceted functions that immunomodulatory factors may acquire in an unpredictable tumor microenvironment. Cancer Res; 76(11); 3145–55. ©2016 AACR.

Supplementary Figure 1 from Overexpression of Protease-Activated Receptor-1 Contributes to Melanoma Metastasis via Regulation of Connexin 43

Supplementary Figure Legends

Supplementary Materials and Methods, Figure Legends 1-6, Table Legend from Expression of Id-1 Is Regulated by MCAM/MUC18: A Missing Link in Melanoma Progression

Over the past few years, numerous nanotechnology-based drug delivery systems have been developed in an effort to maximize therapeutic effectiveness of conventional drug delivery, while limiting undesirable side effects. Among these, carbon nanotubes (CNTs) are of special interest as potential drug delivery agents due to their numerous unique and advantageous physical and chemical properties. Here, we show in vivo favorable biodistribution and enhanced therapeutic efficacy of cisplatin (CDDP) encapsulated within ultra-short single-walled carbon nanotube capsules (CDDP@US-tubes) using three different human breast cancer xenograft models. In general, the CDDP@US-tubes demonstrated greater efficacy in suppressing tumor growth than free CDDP in both MCF-7 cell line xenograft and BCM-4272 patient-derived xenograft (PDX) models. The CDDP@US-tubes also demonstrated a prolonged circulation time compared to free CDDP which enhanced permeability and retention (EPR) effects resulting in significantly more CDDP accumulation in tumors, as determined by platinum (Pt) analysis via inductively-coupled plasma mass spectrometry (ICP-MS).Over the past decade, drug-loaded nanocarriers have been widely fabricated and studied to enhance tumor specific delivery. Among the diverse classes of nanomaterials, carbon nanotubes (CNTs), or more specifically ultra-short single-walled carbon nanocapsules (US-tubes), have been shown to be a popular, new platform for the delivery of various medical agents for both imaging and therapeutic purposes. Here, for the first time, we have shown that US-tubes can be utilized as a drug delivery platform in vivo to deliver the chemotherapeutic drug, cisplatin (CDDP) as CDDP@US-tubes. The studies have demonstrated the ability of the US-tube platform to promote the delivery of encapsulated CDDP by increasing the accumulation of drug in breast cancer resistance cells, which reveals how CDDP@US-tubes help overcome CDDP resistance.

Nuclear factor of activated T cell (NFAT1, NFATC2) is a transcription factor that binds and positively regulates IL2 expression during T-cell activation. NFAT1 has important roles in both innate and adaptive immune responses, but its involvement in cancer is not completely understood. We previously demonstrated that NFAT1 contributes to melanoma growth and metastasis by regulating the autotaxin gene (Enpp2). Here, we report a strong correlation between NFAT1 expression and metastatic potential in melanoma cell lines and tumor specimens. To elucidate the mechanisms underlying NFAT1 overexpression during melanoma progression, we conducted a microarray on a highly metastatic melanoma cell line in which NFAT1 expression was stably silenced. We identified and validated two downstream targets of NFAT1, IL8, and MMP3. Accordingly, NFAT1 depletion in metastatic melanoma cell lines was associated with reduced IL8 and MMP3 expression, whereas NFAT1 overexpression in a weakly metastatic cell line induced expression of these targets. Restoration of NFAT1 expression recovered IL8 and MMP3 expression levels back to baseline, indicating that both are direct targets of NFAT1. Moreover, in vivo studies demonstrated that NFAT1 and MMP3 promoted melanoma tumor growth and lung metastasis. Collectively, our findings assign a new role for NFAT1 in melanoma progression, underscoring the multifaceted functions that immunomodulatory factors may acquire in an unpredictable tumor microenvironment. Cancer Res; 76(11); 3145–55. ©2016 AACR.

The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors and protease-activated receptors, both of which are GPCRs, are central to the metastatic melanoma phenotype and may serve as potential targets in novel therapies against melanoma and other cancers.

Progression of melanoma is dependent on cross-talk between tumor cells and the adjacent microenvironment. The thrombin receptor, protease-activated receptor-1 (PAR-1), plays a key role in exerting this function during melanoma progression. PAR-1 and its activating factors, which are expressed on tumor cells and the surrounding stroma, induce not only coagulation but also cell signaling, which promotes the metastatic phenotype. Several adhesion molecules, cytokines, growth factors, and proteases have recently been identified as downstream targets of PAR-1 and have been shown to modulate interactions between tumor cells and the microenvironment in the process of melanoma growth and metastasis. Inhibiting such interactions by targeting PAR-1 could potentially be a useful therapeutic modality for melanoma patients. Cancer Res; 71(21); 6561–6. ©2011 AACR.

We investigated the clinical and biological significance of p130cas, an important cell signaling molecule, in ovarian carcinoma.Expression of p130cas in ovarian tumors, as assessed by immunohistochemistry, was associated with tumor characteristics and patient survival. The effects of p130cas gene silencing with small interfering RNAs incorporated into neutral nanoliposomes (siRNA-DOPC), alone and in combination with docetaxel, on in vivo tumor growth and on tumor cell proliferation (proliferating cell nuclear antigen) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) were examined in mice bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) or taxane-resistant (HeyA8-MDR) ovarian tumors (n = 10 per group). To determine the specific mechanisms by which p130cas gene silencing abrogates tumor growth, we measured cell viability (MTT assay), apoptosis (fluorescence-activated cell sorting), autophagy (immunoblotting, fluorescence, and transmission electron microscopy), and cell signaling (immunoblotting) in vitro. All statistical tests were two-sided.Of 91 ovarian cancer specimens, 70 (76%) had high p130cas expression; and 21 (24%) had low p130cas expression. High p130cas expression was associated with advanced tumor stage (P.001) and higher residual disease (1 cm) following primary cytoreduction surgery (P = .007) and inversely associated with overall survival and progression-free survival (median overall survival: high p130cas expression vs low expression, 2.14 vs 9.1 years, difference = 6.96 years, 95% confidence interval = 1.69 to 9.48 years, P.001; median progression-free survival: high p130cas expression vs low expression, 1.04 vs 2.13 years, difference = 1.09 years, 95% confidence interval = 0.47 to 2.60 years, P = .01). In mice bearing orthotopically implanted HeyA8 or SKOV3ip1 ovarian tumors, treatment with p130cas siRNA-DOPC in combination with docetaxel chemotherapy resulted in the greatest reduction in tumor growth compared with control siRNA therapy (92%-95% reduction in tumor growth; P.001 for all). Compared with control siRNA therapy, p130cas siRNA-DOPC reduced SKOV3ip1 cell proliferation (31% reduction, P.001) and increased apoptosis (143% increase, P.001) in vivo. Increased tumor cell apoptosis may have persisted despite pan-caspase inhibition by the induction of autophagy and related signaling pathways.Increased p130cas expression is associated with poor clinical outcome in human ovarian carcinoma, and p130cas gene silencing decreases tumor growth through stimulation of apoptotic and autophagic cell death.

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. Cancer Res; 71(10); 3494–504. ©2011 AACR.

The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP) and the metastatic phenotype are not very well defined. However, some of the genes involved in this process and their transcriptional regulation are beginning to be elucidated. For example, the switch from RGP to VGP and the metastatic phenotype is associated with loss of the AP-2α transcription factor. AP-2α regulates the expression of c-KIT, MMP-2, VEGF, and the adhesion molecule MCAM/MUC18. Recently, we reported that AP-2α also regulates two G-protein coupled receptors (GPCRs) PAR-1 and PAFR. In turn, the thrombin receptor, PAR-1, regulates the expression of the gap junction protein Connexin-43 and the tumor suppressor gene Maspin. Activation of PAR-1 also leads to overexpression and secretion of proangiogenic factors such as IL-8, uPA, VEGF, PDGF, as well certain integrins. PAR-1 also cooperates with PAFR to regulate the expression of the MCAM/MUC18 via phosphorylation of CREB. The ligands for these GPCRs, thrombin and PAF, are secreted by stromal cells, emphasizing the importance of the tumor microenvironment in melanoma metastasis. The metastatic phenotype of melanoma is also associated with overexpression and function of CREB/ATF-1. Loss of AP-2α and overexpression of CREB/ATF-1 results in the overexpression of MCAM/MUC18 which by itself contributes to melanoma metastasis by regulating the inhibitor of DNA binding-1 (Id-1). CREB/ATF-1 also regulates the angiogenic factor CYR-61. Our recent data indicate that CREB/ATF-1 regulates the expression of AP-2α, thus, supporting the notion that CREB is an important "master switch" in melanoma progression.

Melanoma remains as the deadliest form of skin cancer with limited and inefficient treatment options available for patients with metastatic disease. Within the last decade, the thrombin receptor, Protease Activated Receptor-1, has been described as an essential gene involved in the progression of human melanoma. PAR-1 is known to activate adhesive, invasive and angiogenic factors to promote melanoma metastasis. It is overexpressed not only in metastatic melanoma cell lines but is also highly expressed in metastatic lesions as compared to primary nevi and normal skin. Recently, PAR-1 has been described to regulate the gap junction protein Connexin 43 and the tumor suppressor gene Maspin to promote the metastatic melanoma phenotype. Herein, we review the role of PAR-1 in the progression of melanoma as well as utilizing PAR-1-regulated genes as potential therapeutic targets for melanoma treatment.

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.

The cellular and molecular pathways that regulate platelet activation, blood coagulation, and inflammation are emerging as critical players in cancer progression and metastasis. Here, we demonstrate a novel signaling mechanism whereby protease-activated receptor 1 (PAR1) mediates expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), a critical marker of melanoma metastasis, via activation of platelet-activating factor receptor (PAFR) and cAMP-responsive element-binding protein (CREB). We found that PAR1 silencing with small hairpin RNA inhibits MUC18 expression in metastatic melanoma cells by inhibiting CREB phosphorylation, activity, and binding to the MUC18 promoter. We further demonstrate that the PAF/PAFR pathway mediates MUC18 expression downstream of PAR1. Indeed, PAR1 silencing down-regulates PAFR expression and PAF production, PAFR silencing blocks MUC18 expression, and re-expression of PAFR in PAR1-silenced cells rescues MUC18 expression. We further demonstrate that the PAR1-PAFR-MUC18 pathway mediates melanoma cell adhesion to microvascular endothelial cells, transendothelial migration, and metastatic retention in the lungs. Rescuing PAFR expression in PAR1-silenced cells fully restores metastatic phenotype of melanoma, indicating that PAFR plays critical role in the molecular mechanism of PAR1 action. Our results link the two pro-inflammatory G-protein-coupled receptors, PAR1 and PAFR, with the metastatic dissemination of melanoma and suggest that PAR1, PAFR, and MUC18 are attractive therapeutic targets for preventing melanoma metastasis.

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. [Cancer Res 2009;69(16):6730–7]

The thrombin receptor [protease-activated receptor-1 (PAR-1)] is overexpressed in highly metastatic melanoma cell lines and in patients with metastatic lesions. Activation of PAR-1 leads to cell signaling and up-regulation of genes involved in adhesion, invasion, and angiogenesis. Herein, we stably silence PAR-1 through the use of lentiviral short hairpin RNA and found significant decreases in both tumor growth (P < 0.01) and metastasis (P < 0.001) of highly metastatic melanoma cell lines in vivo. The use of viruses for therapy is not ideal as it can induce toxic immune responses and possible gene alterations following viral integration. Therefore, we also used systemic delivery of PAR-1 small interfering RNA (siRNA) incorporated into neutral liposomes [1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)] to decrease melanoma growth and metastasis in vivo. Significant decreases in tumor growth, weight, and metastatic lung colonies (P < 0.001 for all) were found in mice treated with PAR-1 siRNA-DOPC. The in vivo effects of PAR-1 on invasion and angiogenesis were analyzed via immunohistochemistry. Concomitant decreases in vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-2 expression levels, as well as decreased blood vessel density (CD31), were found in tumor samples from PAR-1 siRNA-treated mice, suggesting that PAR-1 is a regulator of melanoma cell growth and metastasis by affecting angiogenic and invasive factors. We propose that siRNA incorporated into DOPC nanoparticles could be delivered systemically and used as a new modality for melanoma treatment. [Cancer Res 2008;68(21):9078–86]

The incidence of melanoma has been steadily increasing over the last 3 decades. Currently, there are several approved treatments for metastatic melanoma, including chemotherapy and biologic therapy as both single treatments and in combination, but none is associated with a significant increase in survival. The chemotherapeutic agent dacarbazine is the standard treatment for metastatic melanoma, with a response rate of 15-20%, although most responses are not sustained. One of the main problems with melanoma treatment is chemotherapeutic resistance. The mechanisms of resistance of melanoma cells to chemotherapy have yet to be elucidated. Following treatment with dacarbazine, melanoma cells activate the extracellular signal-regulated kinase pathway, which results in over-expression and secretion of interleukin (IL)-8 and vascular endothelial growth factor. Melanoma cells utilize this mechanism to escape from the cytotoxic effect of the drug. We have previously reported on the development of fully human neutralizing antibodies against IL-8 (anti-IL-8-monoclonal-antibody [ABX-IL8]). In preclinical studies, ABX-IL8 inhibited tumor growth, angiogenesis, and metastasis of human melanoma in vivo. We propose that combination treatment with dacarbazine and IL-8 will potentiate the cytotoxic effect of the drug. Furthermore, formation of metastasis is a multistep process that includes melanoma cell adhesion to endothelial cells. Melanoma cell adhesion molecule (MUC18) mediates these processes in melanoma and is therefore a good target for eliminating metastasis. We have developed a fully human antibody against MUC18 that has shown promising results in preclinical studies. Since resistance is one of the major obstacles in the treatment of melanoma, we propose that utilization of antibodies against IL-8 or MUC18 alone, or as part of a 'cocktail' in combination with dacarbazine, may be a new treatment modality for metastatic melanoma that overcomes resistance of the disease to chemotherapy and significantly improves survival of patients.

Expression and overexpression of the epidermal growth factor receptor (EGFR) have been described in several solid tumors including bladder, breast, colorectal, NSCLC, prostate, and ovarian cancers. In addition to gene amplification, point mutations within the kinase domain also occur. Previous reports indicate that the patient's response to gefitinib depends on either the presence of mutations within the kinase domain of EGFR or the expression of the most frequent alteration, the truncated EGFR variant III (EGFRvIII). Therefore, it is important to determine if these EGFR alterations are present in urothelial carcinoma. The kinase domain of EGFR (exons 18-21) from 11 bladder cancer cell lines as well as from 75 patient tumors was analyzed by automated sequencing. No mutations were detected in all samples tested. Furthermore, analysis of EGFRvIII by immunohistochemistry revealed that almost half of all the patient samples expressed this truncation in a urothelial carcinoma tissue microarray. However, there have been previous reports of inconsistencies in detecting EGFRvIII by immunohistochemistry owing to the specificity of the antibodies and the methodologies utilized. Therefore, these results were validated by reverse transcription PCR, real-time PCR and western blot analysis. In these assays, none of the samples tested positive for EGFRvIII. Taken together, these results indicate that mutations within the tyrosine kinase domain of EGFR and expression of EGFRvIII are rare events in bladder cancer and therefore do not contribute to the malignant phenotype of this tumor. These results have clinical implications in selecting tyrosine kinase inhibitors for the therapy of urothelial carcinoma.

Purpose: It has previously been reported that the patient response to gefitinib depends on the presence of mutations within the kinase domain of epidermal growth factor receptor (EGFR) or the expression of its truncated form, EGFR variant III (EGFRvIII). The focus of this study was to determine if these alterations are present within the tyrosine kinase and ligand-binding domain of EGFR in urothelial carcinoma. Experimental Design: The kinase domain found within exons 18 to 21 of the EGFR from 11 bladder cancer cell lines and 75 patient tumors were subjected to automated sequencing. EGFRvIII expression was determined by immunohistochemistry using a urothelial carcinoma tissue microarray, and its expression was subsequently verified by reverse transcription PCR, real-time PCR, and Western blot analysis, using an EGFRvIII-transfected glioblastoma cell line and glioblastoma tumors as positive controls. Results: Our analysis failed to detect mutations within the tyrosine kinase domain of EGFR in the 11 cell lines and 75 patients tested. The initial analysis of EGFRvIII expression by immunohistochemistry revealed that at least 50% of the patient tumors expressed EGFRvIII in a urothelial carcinoma tissue microarray. Conflicting reports exist, however, regarding the extent of EGFRvIII expression in tissues owing to the specificity of the antibodies and the methodologies used. Therefore, we sought to validate this observation by reverse transcription PCR, real-time PCR, and Western blot analysis. In these assays, none of the samples were positive for EGFRvIII except for control transfectants and glioblastomas. Conclusions: When our results are taken together, we conclude that alterations within the tyrosine kinase domain and expression of EGFRvIII are rare events in bladder cancer. The present study has clinical implications in selecting tyrosine kinase inhibitors for the therapy of urothelial carcinoma.

Nature Communications 4: Article number: 1403 (2013); Published: 29 January 2013; Updated: 29 July 2013. The original version of this Article contained a typographical error in the spelling of the author Mian M. K. Shahzad, which was incorrectly given as Mian M. K. Shazhad. This has now been corrected in both the PDF and HTML versions of the Article.

Background The loss of AP-2α and increased activity of cAMP-responsive element binding (CREB) protein are two hallmarks of malignant progression of cutaneous melanoma. However, the molecular mechanism responsible for the loss of AP-2α during melanoma progression remains unknown. Methodology/Principal Findings Herein, we demonstrate that both inhibition of PKA-dependent CREB phosphorylation, as well as silencing of CREB expression by shRNA, restored AP-2α protein expression in two metastatic melanoma cell lines. Moreover, rescue of CREB expression in CREB-silenced cell lines downregulates expression of AP-2α. Loss of AP-2α expression in metastatic melanoma occurs via a dual mechanism involving binding of CREB to the AP-2α promoter and CREB-induced overexpression of another oncogenic transcription factor, E2F-1. Upregulation of AP-2α expression following CREB silencing increases endogenous p21Waf1 and decreases MCAM/MUC18, both known to be downstream target genes of AP-2α involved in melanoma progression. Conclusions/Significance Since AP-2α regulates several genes associated with the metastatic potential of melanoma including c-KIT, VEGF, PAR-1, MCAM/MUC18, and p21Waf1, our data identified CREB as a major regulator of the malignant melanoma phenotype.

Metastatic progression of melanoma is associated with overexpression and activity of cAMP-response element-binding protein (CREB). However, the mechanism by which CREB contributes to tumor progression and metastasis remains unclear. Here, we demonstrate that stably silencing CREB expression in two human metastatic melanoma cell lines, A375SM and C8161-c9, suppresses tumor growth and experimental metastasis. Analysis of cDNA microarrays revealed that CREB silencing leads to increased expression of cysteine-rich protein 61 (CCN1/CYR61) known to mediate adhesion, chemostasis, survival, and angiogenesis. Promoter analysis and chromatin immunoprecipitation assays demonstrated that CREB acts as a negative regulator of CCN1/CYR61 transcription by directly binding to its promoter. Re-expression of CREB in CREB-silenced cells rescued the low CCN1/CYR61 expression phenotype. CCN1/CYR61 overexpression resulted in reduced tumor growth and metastasis and inhibited the activity of matrix metalloproteinase-2. Furthermore, its overexpression decreased melanoma cell motility and invasion through Matrigel, which was abrogated by silencing CCN1/CYR61 in low metastatic melanoma cells. Moreover, a significant decrease in angiogenesis as well as an increase in apoptosis was seen in tumors overexpressing CCN1/CYR61. Our results demonstrate that CREB promotes melanoma growth and metastasis by down-regulating CCN1/CYR61 expression, which acts as a suppressor of melanoma cell motility, invasion and angiogenesis.

The interaction of tumor cells and the host stroma (microenvironment) is essential for tumor progression and metastasis. The melanoma tumor microen- vironment has emerged within the last decade as a significant player in melanoma progression from the radial growth phase to the vertical growth phase by providing the necessary elements for growth, invasion and survival. Two receptors involved in this transition that are not only activated by factors from the tumor microenvironment but also in turn secrete factors into the microenvironment are the Protease Activated Receptor 1 (PAR-1) and the Platelet Activating Factor Receptor (PAFR). Throm- bin, which is abundant in the microenvironment milieu, activates PAR-1 causing cell signaling via G-proteins resulting in upregulation and secretion of gene products in- volved in adhesion (integrins), invasion (MMP-2) and angiogenesis (IL-8, VEGF, PDGF, bFGF). PAF, which is secreted by platelets, macrophages, neutrophils, en- dothelial cells and keratinocytes within the tumor microenvironment, will activate PAFR and signal through p38 MAPK to phosphorylate the CREB/ATF-1 transcrip- tion factors. Phosphorylation of CREB/ATF-1 results in overexpression and secretion of MMP-2 and MT1-MMP. Since only metastatic melanoma cells express activated CREB/ATF-1, we propose that they are better equipped to respond to PAF than their non-metastatic counterparts. These two G-protein coupled receptors that play major roles in melanoma progression highlight the crucial interactions between the tumor microenvironment and melanoma cells in the acquisition of the metastatic phenotype.

Interleukin-8 (IL-8) is a proangiogenic cytokine that is overexpressed in many human cancers. We investigated the clinical and biologic significance of IL-8 in ovarian carcinoma using human samples and orthotopic mouse models.Tumor expression of IL-8 was assessed by immunohistochemistry among ovarian cancer patients (n = 102) with available clinical and survival data. We examined the effect of IL-8 gene silencing with small interfering RNAs incorporated into neutral liposomes (siRNA-DOPCs), alone and in combination with docetaxel, on in vivo tumor growth, angiogenesis (microvessel density), and tumor cell proliferation in mice (n = 10 per treatment group) bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) and taxane-resistant (SKOV3ip2.TR) ovarian tumors. All statistical tests were two-sided.Of the 102 cancer specimens, 43 (42%) had high IL-8 expression and 59 (58%) had low or no IL-8 expression; high IL-8 expression was associated with advanced tumor stage (P = .019), high tumor grade (P = .031), and worse survival (median survival for patients with high vs low IL-8 expression: 1.62 vs 3.79 years; P.001). Compared with empty liposomes, IL-8 siRNA-DOPC reduced the mean tumor weight by 32% (95% confidence interval [CI] = 14% to 50%; P = .03) and 52% (95% CI = 27% to 78%; P = .03) in the HeyA8 and SKOV3ip1 mouse models, respectively. In all three mouse models, treatment with IL-8 siRNA-DOPC plus the taxane docetaxel reduced tumor growth the most compared with empty liposomes (77% to 98% reduction in tumor growth; P.01 for all). In the HeyA8 and SKOV3ip1 models, tumors from mice treated with IL-8 siRNA-DOPC alone had lower microvessel density than tumors from mice treated with empty liposomes (HeyA8: 34% lower, 95% CI = 32% to 36% lower [P = .002]; SKOV3ip1: 39% lower, 95% CI = 34% to 44% lower [P = .007]). Compared with empty liposomes, IL-8 siRNA-DOPC plus docetaxel reduced tumor cell proliferation by 35% (95% CI = 25% to 44%; P.001) and 38% (95% CI = 28% to 48%; P.001) in the HeyA8 and SKOV3ip1 models, respectively.Increased IL-8 expression is associated with poor clinical outcome in human ovarian carcinoma, and IL-8 gene silencing decreases tumor growth through antiangiogenic mechanisms.

Melanoma growth, angiogenesis and metastatic progression are strongly promoted by the inflammatory tumor microenvironment due to high levels of cytokine and chemokine secretion by the recruited inflammatory and stromal cells. In addition, platelets and molecular components of procoagulant pathways have been recently emerging as critical players of tumor growth and metastasis. In particular, thrombin, through the activity of its receptor protease-activated receptor-1 (PAR-1), regulates tumor cell adhesion to platelets and endothelial cells, stimulates tumor angiogenesis, and promotes tumor growth and metastasis. Notably, in many tumor types including melanoma, PAR-1 expression directly correlates with their metastatic phenotype and is directly responsible for the expression of interleukin-8, matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor, platelet-derived growth factor, and integrins. Another proinflammatory receptor–ligand pair, platelet-activating factor (PAF) and its receptor (PAFR), have been shown to act as important modulators of tumor cell adhesion to endothelial cells, angiogenesis, tumor growth and metastasis. PAF is a bioactive lipid produced by a variety of cells from membrane glycerophospholipids in the same reaction that releases arachidonic acid, and can be secreted by platelets, inflammatory cells, keratinocytes and endothelial cells. We have demonstrated that in metastatic melanoma cells, PAF stimulates the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) and activating transcription factor 1 (ATF-1), which results in overexpression of MMP-2 and membrane type 1-MMP (membrane type 1-MMP). Since only metastatic melanoma cells overexpress CREB/ATF-1, we propose that metastatic melanoma cells are better equipped than their non-metastatic counterparts to respond to PAF within the tumor microenvironment. The evidence supporting the hypothesis that the two G-protein coupled receptors, PAR-1 and PAFR, contribute to the acquisition of the metastatic phenotype of melanoma is presented and discussed.

The Editors-in-Chief of World Journal of Urology have decided to retract the article by G. J. Villares, M. Zigler, K. Blehm, C. Bogdan, D. McConkey, D. Colin and M. BarEli, Targeting EGFR in bladder cancer (World J Urol (2007) 25:573–579, doi:10.1007/s00345-007-0202-7). Substantial portions of this article were found to have been published previously by K. N. Blehm, P. E. Spiess, J. E. Bondaruk, M. E. Dujka, G. J. Villares, Yi-jue Zhao, O. Bogler, K. D. Aldape, H. B. Grossman, L. Adam, D. J. McConkey, B. A. Czerniak, C. P. Dinney and M. BarEli, Mutations within the kinase domain and truncations of the epidermal growth factor receptor are rare events in bladder cancer: implications for therapy (Clin Cancer Res (2006) 12:4671–4677, doi:10.1158/1078-0432.CCR-060407).

Clinical studies have demonstrated that chronic stress can influence cancer progression. However, the underlying mechanisms are not fully understood. To determine the molecular drivers of downstream signaling networks activated in response to chronic stress, we performed a phosphoproteomic analysis and determined that the non-receptor tyrosine kinase, Src, was the key regulator of these networks. Since Src plays an important role in cancer biology, we examined the biological and clinical significance of Src in stress-mediated tumor growth. Norepinephrine (NE) rapidly activated SrcY419 in β-adrenergic receptor (ADRB) positive ovarian cancer cell lines, but not in ADRB-null cells. Confocal microscopy showed that Src was rapidly recruited to the cellular membrane after NE exposure in ADRB positive ovarian cancer cells. Furthermore, treatment with different ADRB agonists and blockers determined that ADRB2 is required for SrcY419 phosphorylation. Treatment with a cAMP agonist or PKA agonist/antagonists demonstrated that cAMP/PKA signaling is required for NE-induced Src activation. The unexpected Src activation via cAMP/PKA was found to be mediated by direct phosphorylation of SrcS17 following NE treatment. In Src-/- cells transiently expressing WT Src, NE caused SrcY419 phosphorylation, which was not observed when cells were transfected with a Src S17A construct. In order to investigate how S17 phosphorylation leads to Src activation, we performed molecular dynamic simulations and observed that upon SrcS17 phosphorylation, Src undergoes significant structural changes that expose its Y419 residue. To understand the functional consequences of stress-induced Src activation, we performed migration and invasion assays. Exposure to NE resulted in an increase in ovarian cancer cell migration and invasion that was completely abrogated by Src-targeted siRNA (P < 0.01). In various orthotopic mouse models of ovarian carcinoma, chronic restraint stress significantly increased tumor weights (P < 0.05). Increased tumor growth was completely blocked by Src silencing with Src siRNA-DOPC or by the non-specific beta-blocker, propranolol (P < 0.05). To test the clinical significance of our biological findings, we examined 91 epithelial ovarian cancer samples. Elevated pSrcY419 was associated with worse patient survival (P < 0.001), high tumoral NE levels (P < 0.001) and high scores on the Center for Epidemiologic Studies Depression Scale (P = 0.008). To examine the potential clinical impact of our findings, we investigated whether beta-blocker usage by patients affected cancer-related mortality. This analysis revealed that beta-blockers reduced mortality by 17% across all major cancer types and 14.6% among patients with ovarian and other gynecologic cancers. This work is the first to demonstrate that an ADRB-PKA-Src axis mediates the effect of chronic stress on tumor growth and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 937. doi:10.1158/1538-7445.AM2011-937

The acquisition of the metastatic phenotype of melanoma is associated with increase of the melanoma cell adhesion molecule MCAM/MUC18. We have previously shown that MUC18 plays a major role in promoting cellular invasion by regulating MMP-2 expression and activity. Here, we aim to establish the molecular mechanism by which MUC18 regulates melanoma cell invasion to promote tumor growth and metastasis. Utilizing lentivirus-incorporated small hairpin RNA, we stably silenced MUC18 expression in the metastatic melanoma cell lines A375SM and C8161 and conducted cDNA gene expression microarray analyses. We identified the Inhibitor of DNA Binding-1 (Id-1) to be down regulated by 5.7 fold as a result of MUC18 silencing. Id-1 expression has been shown to correlate with the progression of melanoma and to promote cell invasion by regulating MT1-MMP. We have found that Id-1 expression decreased significantly after MUC18 silencing in both A375SM and C8161, thereby corroborating our microarray results. Additionally, we found that MUC18 regulates Id-1 expression at the transcriptional level through regulation of ATF-3. ATF-3 was upregulated by 6 fold in our cDNA microarray analysis. ChIP analysis showed increase binding of ATF-3 to the Id-1 promoter after MUC18 silencing. Moreover, mutation of the ATF-3 binding site on the Id-1 promoter increased Id-1 promoter activity in MUC18 silenced cells. To further confirm the specificity of regulation of Id-1 and ATF-3 by MUC18, we rescued MUC18 and found that their expression was indeed inverted. Moreover, overexpression of Id-1 in MUC18 silenced cells demonstrated rescue of the invasive phenotype as well as increased activity of MMP-2 and the transcription of MT1-MMP. Collectively, our results suggest that MUC18 contributes to the malignant phenotype of melanoma via regulation of Id-1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2369.

Background: p130Cas is a central node for many cancer signaling pathways. We have previously established the clinical significance of p130cas overexpression in ovarian carcinoma and the therapeutic efficacy of silencing p130cas with siRNA in an orthotopic mouse model of ovarian carcinoma. The focus of the current work is to establish the mechanism by which p130cas gene silencing decreases tumor growth. Methods: To determine the specific mechanisms by which p130cas gene silencing abrogates tumor growth, in vitro measures of cell viability (MTT), apoptosis (TUNEL by FACS), autophagy (acridine organ staining by FACS and immunoblotting), and cell signaling (immunoblotting) were performed. Results: p130Cas treatment with siRNA yielded a 21 (SKOV3TRip2, p Conclusions: Silencing p130Cas with siRNA decreases tumor cell viability by induction of both apoptotic and autophagic pathways establishing p130cas as a novel therapeutic target in ovarian carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1044.

The Activator protein-2 (AP-2) family of transcription factors consists of five different proteins which are involved in the regulation of cell proliferation, differentiation, apoptosis and carcinogenesis. Among these proteins, AP-2α has been suggested to function as a tumor suppressor in many cancers including melanoma. We have previously shown that in melanoma cell lines, acquisition of the metastatic phenotype is associated with loss of AP-2α expression and gain in expression of cAMP-responsive element (CRE)-binding protein (CREB). This inverse correlation is associated with increase of melanoma invasion and metastasis resulting in overexpression of the melanoma cell adhesion molecule MCAM/MUC18 and protease-activated receptor 1 (PAR-1). However, the molecular mechanism involved in AP-2α regulation during melanoma progression remains unknown. Analyses of AP-2α promoter for transcription factor binding sites showed two distinct CRE-like sites as well as four E2F1 sites. Herein, we propose that the inhibition of AP-2α during the transition from the Radial Growth Phase (RGP) to the Vertical Growth Phase (VGP) occurs at the transcriptional level and is regulated through binding of CREB and E2F1. Hence, AP-2α protein expression was found to be increased after utilizing small hairpin RNA (shRNA) targeting CREB in two human metastatic melanoma cell lines, A375SM and C8161. Interestingly, E2F1 expression level was found to be reduced by 50% after CREB silencing in both cell lines. We also found a 20-fold increase in the luciferase activity driven by the AP-2α promoter after silencing CREB in both cell lines. In addition, point mutations at CRE-like binding sites as well as E2F1 increased the AP-2α promoter activity by more then 5-fold. To further establish the involvement of E2F1 in AP-2α regulation, we transiently overexpressed E2F1 in a low-metastatic melanoma cell line, SB-2. The AP-2α protein level as well as the promoter activity was significantly inhibited as compared to control. Finally, using Chromatin Immunoprecipitation (ChIP) assays in both CREB silenced cell lines, we were able to confirm that CREB and E2F1 bind to the AP-2α promoter thereby inhibiting its expression. Taken together, our studies demonstrate that CREB regulates AP-2α expression, thus, contributing to the metastatic phenotype of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2353.