Breast cancer metastasis suppressor 1 (BRMS1) inhibits osteopontin transcription by abrogating NF-κB activation

BACKGROUND: Osteopontin (OPN), a secreted phosphoglycoprotein, has been strongly associated with tumor progression and aggressive cancers. MDA-MB-435 cells secrete very high levels of OPN. However metastasis-suppressed MDA-MB-435 cells, which were transfected with breast cancer metastasis suppressor 1 (BRMS1), expressed significantly less OPN. BRMS1 is a member of mSin3-HDAC transcription co-repressor complex and has been shown to suppress the metastasis of breast cancer and melanoma cells in animal models. Hence we hypothesized that BRMS1 regulates OPN expression. RESULTS: The search for a BRMS1-regulated site on the OPN promoter, using luciferase reporter assays of the promoter deletions, identified a novel NF-κB site (OPN/NF-κB). Electrophoretic mobility shift assays and chromatin immunoprecipitations (ChIP) confirmed this site to be an NF-κB-binding site. We also show a role of HDAC3 in suppression of OPN via OPN/NF-κB. CONCLUSION: Our results show that BRMS1 regulates OPN transcription by abrogating NF-κB activation. Thus, we identify OPN, a tumor-metastasis activator, as a crucial downstream target of BRMS1. Suppression of OPN may be one of the possible underlying mechanisms of BRMS1-dependent suppression of tumor metastasis

The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis

<p>Abstract</p> <p>Background</p> <p>The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples.</p> <p>Methods</p> <p>We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes.</p> <p>Results</p> <p>We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including <it>SPRR1A/B</it>, <it>KRT16/17</it>, <it>CD24</it>, <it>LOR</it>, <it>GATA3</it>, <it>MUC15</it>, and <it>TMPRSS4</it>, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as <it>MAGE</it>, <it>GPR19</it>, <it>BCL2A1</it>, <it>MMP14</it>, <it>SOX5</it>, <it>BUB1</it>, <it>RGS20</it>, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (<it>SPP-1</it>, <it>MITF</it>, <it>CITED-1</it>, <it>GDF-15</it>, <it>c-Met</it>, <it>HOX </it>loci) and suppressor genes (<it>PITX-1</it>, <it>CST-6</it>, <it>PDGFRL</it>, <it>DSC-3</it>, <it>POU2F3</it>, <it>CLCA2</it>, <it>ST7L</it>), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype.</p> <p>Conclusion</p> <p>The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.</p

Breast cancer metastasis suppressor 1 (BRMS1) inhibits osteopontin transcription by abrogating NF-κB activation

Abstract Background Osteopontin (OPN), a secreted phosphoglycoprotein, has been strongly associated with tumor progression and aggressive cancers. MDA-MB-435 cells secrete very high levels of OPN. However metastasis-suppressed MDA-MB-435 cells, which were transfected with breast cancer metastasis suppressor 1 (BRMS1), expressed significantly less OPN. BRMS1 is a member of mSin3-HDAC transcription co-repressor complex and has been shown to suppress the metastasis of breast cancer and melanoma cells in animal models. Hence we hypothesized that BRMS1 regulates OPN expression. Results The search for a BRMS1-regulated site on the OPN promoter, using luciferase reporter assays of the promoter deletions, identified a novel NF-κB site (OPN/NF-κB). Electrophoretic mobility shift assays and chromatin immunoprecipitations (ChIP) confirmed this site to be an NF-κB-binding site. We also show a role of HDAC3 in suppression of OPN via OPN/NF-κB. Conclusion Our results show that BRMS1 regulates OPN transcription by abrogating NF-κB activation. Thus, we identify OPN, a tumor-metastasis activator, as a crucial downstream target of BRMS1. Suppression of OPN may be one of the possible underlying mechanisms of BRMS1-dependent suppression of tumor metastasis.</p

The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis.

BackgroundThe process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples.MethodsWe have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes.ResultsWe find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including SPRR1A/B, KRT16/17, CD24, LOR, GATA3, MUC15, and TMPRSS4, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as MAGE, GPR19, BCL2A1, MMP14, SOX5, BUB1, RGS20, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (SPP-1, MITF, CITED-1, GDF-15, c-Met, HOX loci) and suppressor genes (PITX-1, CST-6, PDGFRL, DSC-3, POU2F3, CLCA2, ST7L), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype.ConclusionThe gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches

The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis-0

Measured utilizing Breslow's depth of invasion. All procured lymph node metastases were macroscopically involved, often completely replacing the entire lymph node parenchyma. Distant metastatic (subcutaneous and solid organ) melanoma often exhibited varying degrees of pigmentation, however, surrounding stroma was avoided in procurement of melanoma samples. B: A distinct change in the gene expression patterns is apparent within the comparative groups of thin/I.M. to thick PCM samples. Gene over-expression (upper graph) is evident at the I.M. thickness sample set, with an average Breslow's tumor thickness of 2.1 mm and 19 mm for thick melanomas. Contrary, there is a decrease in gene expression (lower graph) of the same set of genes, with a comparative difference in gene down-regulation evident at the same interphase of I.M. to thick PCM. Proceeding from left to right: normal skin, BCC, SCC, MIS, I.M., thick primary, metastatic melanoma (subcutaneous, lymph node and distant) and melanoma cell lines derived from patients with stage IV melanoma.<p><b>Copyright information:</b></p><p>Taken from "The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis"</p><p>http://www.biomedcentral.com/1755-8794/1/13</p><p>BMC Medical Genomics 2008;1():13-13.</p><p>Published online 28 Apr 2008</p><p>PMCID:PMC2408576.</p><p></p

The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis-2

Band (-), faintly visible band (+/-), visible band (+), strongly visible band (++), N = Not Done. β-actin served as the internal comparative control. The grey values of PCR products of each gene are analyzed by the AlphaEase 3 software and standardized according to β-actin in every sample. B: a, b, Daughter melanoma cell lines secrete SPP1 (osteopontin). The melanoma cell lysates and conditioned cell-free media was resolved by SDS-PAGE and transferred to a PVDF membrane. The blot was probed with anti-SPP1 antibody. , Antibodies for DSC-3 (c), CLCA2 (d), PDGFRL (e) and α-tubulin (f) as an internal control. Lanes 1–3 are PCM and lanes 4–6 are MM cell lines.<p><b>Copyright information:</b></p><p>Taken from "The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis"</p><p>http://www.biomedcentral.com/1755-8794/1/13</p><p>BMC Medical Genomics 2008;1():13-13.</p><p>Published online 28 Apr 2008</p><p>PMCID:PMC2408576.</p><p></p

The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis-1

oncogenes (GDF15, SPP-1) in PCM (n = 7) and MM (n = 13) samples. Relative quantitation of target gene expression for each sample was determined using the equation 2, where GAPDH was used as the internal reference and normal skin as the calibrator. Values were Log base 10 transformed (y-axis) so that all values below zero represent a down-regulation in gene expression and values above zero represent an up-regulation in gene expression, compared to normal skin. B: Correlative microarray analysis of gene expression levels in primary and metastatic melanoma samples compared with normal skin. The statistical differences of gene expression between primary (PCM) and metastatic melanoma (MM) samples were analyzed by Wilcoxon's signed rank test; two-tailed significance level was set at α = 0.05. Compared to PCM samples (n = 7), the expression levels of 4 putative tumor suppressor genes (CST6, p < 0.0001; DSC3, p < 0.0001; PITX1, p = 0.0043, POU2F3, p < 0.0001) were significantly decreased in MM samples (n = 40), while the expression of putative oncogenes (GDF15, p = 0.0027; SPP1, p < 0.0001) were significantly increased in MM samples.<p><b>Copyright information:</b></p><p>Taken from "The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis"</p><p>http://www.biomedcentral.com/1755-8794/1/13</p><p>BMC Medical Genomics 2008;1():13-13.</p><p>Published online 28 Apr 2008</p><p>PMCID:PMC2408576.</p><p></p

The Hedgehog Pathway Transcription Factor GLI1 Promotes Malignant Behavior of Cancer Cells by Up-regulating Osteopontin*

The role of Hedgehog (Hh) signaling as a developmental pathway is well established. Several recent studies have implicated a role for this pathway in multiple cancers. In this study we report that expression of GLI1 and osteopontin (OPN) increase progressively with the progression of melanoma from primary cutaneous cancer to metastatic melanoma in clinically derived specimens. We have further determined that OPN is a direct transcriptional target of GLI1. We have observed that OPN expression is stimulated in the presence of Hh ligands and inhibited in the presence of the Smoothened (SMO) inhibitor, cyclopamine. Transcriptional silencing of GLI1 negatively impacts OPN expression and compromises the ability of cancer cells to proliferate, migrate, and invade in vitro and interferes with their ability to grow as xenografts and spontaneously metastasize in nude mice. These altered attributes could be rescued by re-expressing OPN in the GLI1-silenced cells, suggesting that OPN is a critical downstream effector of active GLI1 signaling. Our observations lead us to conclude that the GLI1-mediated up-regulation of OPN promotes malignant behavior of cancer cells