Mutant KRAS Downregulates the Receptor for Leukemia Inhibitory Factor (LIF) to Enhance a Signature of Glycolysis in Pancreatic Cancer and Lung Cancer.

Pancreatic cancer is characterized by aberrant activity of oncogenic KRAS, which is mutated in 90% of pancreatic adenocarcinomas. Because KRAS itself is a challenging therapeutic target, we focused on understanding key signaling pathways driven by KRAS as a way to reveal dependencies that are amenable to therapeutic intervention. Analyses in primary human pancreatic cancers and model systems revealed that the receptor for the cytokine leukemia inhibitory factor (LIF) is downregulated by mutant KRAS. Furthermore, downregulation of the LIF receptor (LIFR) is necessary for KRAS-mediated neoplastic transformation. We found LIFR exerts inhibitory effects on KRAS-mediated transformation by inhibiting expression of the glucose transporter GLUT1, a key mediator of the enhanced glycolysis found in KRAS-driven malignancies. Decreased LIFR expression leads to increased GLUT1 as well as increases in glycolysis and mitochondrial respiration. The repression of GLUT1 by LIFR is mediated by the transcription factor STAT3, indicating a tumor-suppressive role for STAT3 within cancer cells with mutated KRAS. Finally, reflecting a clinically important tumor-suppressive role of LIFR, decreased LIFR expression correlates with shorter survival in pancreatic cancer patients with mutated KRAS. Similar findings were found in non-small cell lung cancers driven by mutated KRAS, suggesting that silencing LIFR is a generalized mechanism of KRAS-mediated cellular transformation. These results indicate that the LIFR/STAT3 pathway may mediate either tumor-promoting or tumor-suppressive signaling pathways depending on the genetic background of tumor cells, and may play diverse roles within other cells in the tumor microenvironment. IMPLICATIONS: Mutant KRAS drives downregulation of the receptor for LIF, thereby allowing an increase in expression of the glucose transporter GLUT1 and increases in glycolysis and mitochondrial respiration.

Intracranial metastasis from prostate adenocarcinoma: a case report and literature review.

Intracranial metastasis from prostate adenocarcinoma is rare. A 70-year-old African American male with a history of prostate adenocarcinoma for the last 14 years, presented to our hospital complaining of generalized weakness for the past 2 weeks. He was found to have fever with left ptosis and mild eyelid edema. Brain MRI showed dural metastasis. Two months after the first presentation, he was readmitted with a suspected acute cerebral vascular accident (CVA). CT brain showed vasogenic edema in the right subcortical, likely from intracranial metastasis. His acute neurological symptoms improved with intravenous dexamethasone. This case highlights the possibility of intracranial metastasis from prostate adenocarcinoma. With the advent of novel therapies for prostate cancer, which prolong life expectancy, intracranial metastasis from prostate adenocarcinoma may become an increasingly frequent clinical scenario.

CDK5RAP3 is a co-factor for the oncogenic transcription factor STAT3.

The transcription factor STAT3 regulates genes governing critical cellular processes such as proliferation, survival, and self-renewal. While STAT3 transcriptional function is activated rapidly and transiently in response to physiologic signals, through a variety of mechanisms it can become constitutively activated in the pathogenesis of cancer. This leads to chronic expression of genes that underlie malignant cellular behavior. However, STAT3 is known to interact with other proteins, which may modulate its function. Understanding these interactions can provide insights into novel aspects of STAT3 function and may also suggest strategies to therapeutically target the large number of cancers driven by constitutively activated STAT3. To identify critical modulators of STAT3 transcriptional function, we performed an RNA-interference based screen in a cell-based system that allows quantitative measurement of STAT3 activity. From this approach, we identified CDK5 kinase regulatory-subunit associated protein 3 (CDK5RAP3) as an enhancer of STAT3-dependent gene expression. We found that STAT3 transcriptional function is modulated by CDK5RAP3 in cancer cells, and silencing CDK5RAP3 reduces STAT3-mediated tumorigenic phenotypes including clonogenesis and migration. Mechanistically, CDK5RAP3 binds to STAT3-regulated genomic loci, in a STAT3-dependent manner. In primary human breast cancers, the expression of CDK5RAP3 expression was associated with STAT3 gene expression signatures as well as the expression of individual STAT3 target genes. These findings reveal a novel aspect of STAT3 transcriptional function and potentially provide both a biomarker of enhanced STAT3-dependent gene expression as well as a unique mechanism to therapeutically target STAT3.

Study of Cathepsin B inhibition in VEGFR TKI treated human renal cell carcinoma xenografts.

Several therapeutic options are available for metastatic RCC, but responses are almost never complete, and resistance to therapy develops in the vast majority of patients. Consequently, novel treatments are needed to combat resistance to current therapies and to improve patient outcomes. We have applied integrated transcriptome and proteome analyses to identify cathepsin B (CTSB), a cysteine proteinase of the papain family, as one of the most highly upregulated gene products in established human RCC xenograft models of resistance to vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKI). We used established RCC models to test the significance of CTSB in the progression of renal cancer. Our evaluation of CTSB showed that stable CTSB knockdown suppressed RCC growth in vitro and in vivo. Stable over-overexpression of wild-type CTSB (CTSBwt/hi), but not of an CTSB active site mutant (CTSBN298A), rescued cell growth in CTSB knockdown cells and abolished the efficacy of VEGFR TKI treatment. Genome-wide transcriptome profiling of CTSB knockdown cells demonstrated significant effects on multiple metabolic and stem cell-related pathways, with ALDHA1A (ALDH1) as one of the most significantly downregulated genes. Importantly, survival analysis across 16 major TCGA cancers revealed that CTSB overexpression is associated with low rates of three and five year patient survival rates (P = 2.5e-08, HR = 1.4). These data strongly support a contribution of CTSB activity to RCC cell growth and tumorigenicity. They further highlight the promise of CTSB inhibition in development of novel combination therapies designed to improve efficacy of current TKI treatments of metastatic RCC.

The kinases IKBKE and TBK1 regulate MYC-dependent survival pathways through YB-1 in AML and are targets for therapy.

To identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.

The STAT3 Target Gene TNFRSF1A Modulates the NF-κB Pathway in Breast Cancer Cells.

The transcription factor STAT3 is activated inappropriately in 70% of breast cancers, most commonly in triple negative breast cancer (TNBC). Although the transcriptional function of STAT3 is essential for tumorigenesis, the key target genes regulated by STAT3 in driving tumor pathogenesis have remained unclear. To identify critical STAT3 target genes, we treated TNBC cell lines with two different compounds that block STAT3 transcriptional function, pyrimethamine and PMPTP. We then performed gene expression analysis to identify genes whose expression is strongly down-regulated by both STAT3 inhibitors. Foremost among the down-regulated genes was TNFRSF1A, which encodes a transmembrane receptor for TNFα. We showed that STAT3 binds directly to a regulatory region within the TNFRSF1A gene, and that TNFRSF1A levels are dependent on STAT3 function in both constitutive and cytokine-induced models of STAT3 activation. Furthermore, TNFRSF1A is a major mediator of both basal and TNFα-induced NF-κB activity in breast cancer cells. We extended these findings to primary human breast cancers, in which we found that high TNFRSF1A transcript levels correlated with STAT3 activation. In addition, and consistent with a causal role, increased TNFRSF1A expression was associated with an NF-κB gene expression in signature in breast cancers. Thus, TNFRSF1A is a STAT3 target gene that regulates the NF-κB pathway. These findings reveal a novel functional crosstalk between STAT3 and NF-κB signaling in breast cancer. Furthermore, elevated TNFRSF1A levels may predict a subset of breast tumors that are sensitive to STAT3 transcriptional inhibitors, and may be a biomarker for response to inhibition of this pathway.

Gene expression-based discovery of atovaquone as a STAT3 inhibitor and anticancer agent.

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated inappropriately in a wide range of hematological and solid cancers, but clinically available therapies targeting STAT3 are lacking. Using a computational strategy to identify compounds opposing the gene expression signature of STAT3, we discovered atovaquone (Mepron), an antimicrobial approved by the US Food and Drug Administration, to be a potent STAT3 inhibitor. We show that, at drug concentrations routinely achieved clinically in human plasma, atovaquone inhibits STAT3 phosphorylation, the expression of STAT3 target genes, and the viability of STAT3-dependent hematological cancer cells. These effects were also observed with atovaquone treatment of primary blasts isolated from patients with acute myelogenous leukemia or acute lymphocytic leukemia. Atovaquone is not a kinase inhibitor but instead rapidly and specifically downregulates cell-surface expression of glycoprotein 130, which is required for STAT3 activation in multiple contexts. The administration of oral atovaquone to mice inhibited tumor growth and prolonged survival in a murine model of multiple myeloma. Finally, in patients with acute myelogenous leukemia treated with hematopoietic stem cell transplantation, extended use of atovaquone for Pneumocystis prophylaxis was associated with improved relapse-free survival. These findings establish atovaquone as a novel, clinically accessible STAT3 inhibitor with evidence of anticancer efficacy in both animal models and humans.

Cooperation of imipramine blue and tyrosine kinase blockade demonstrates activity against chronic myeloid leukemia.

The use of tyrosine kinase inhibitors (TKI), including nilotinib, has revolutionized the treatment of chronic myeloid leukemia (CML). However current unmet clinical needs include combating activation of additional survival signaling pathways in persistent leukemia stem cells after long-term TKI therapy. A ubiquitous signaling alteration in cancer, including CML, is activation of reactive oxygen species (ROS) signaling, which may potentiate stem cell activity and mediate resistance to both conventional chemotherapy and targeted inhibitors. We have developed a novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, imipramine blue (IB) that targets ROS generation. ROS levels are known to be elevated in CML with respect to normal hematopoietic stem/progenitor cells and not corrected by TKI. We demonstrate that IB has additive benefit with nilotinib in inhibiting proliferation, viability, and clonogenic function of TKI-insensitive quiescent CD34+ CML chronic phase (CP) cells while normal CD34+ cells retained their clonogenic capacity in response to this combination therapy in vitro. Mechanistically, the pro-apoptotic activity of IB likely resides in part through its dual ability to block NF-κB and re-activate the tumor suppressor protein phosphatase 2A (PP2A). Combining BCR-ABL1 kinase inhibition with NADPH oxidase blockade may be beneficial in eradication of CML and worthy of further investigation.

Deregulation of SOCS5 suppresses dendritic cell function in chronic lymphocytic leukemia.

One cause of morbidity and mortality in chronic lymphocytic leukemia (CLL) is infection, which results from defects in a number of components of the immune system. In particular, dendritic cells (DCs) are functionally defective in patients with CLL. To understand the molecular mechanism for this abnormality, we focused on signal transduction pathways that regulate the function of monocyte-derived dendritic cells (Mo-DCs). Monocytes from CLL patients exhibit high IL-4Rα expression due to the enhanced activation of STAT3. However, IL-4R signaling is decoupled from activation of its downstream mediator STAT6 by enhanced levels of the negative regulator SOCS5. This impairs differentiation of functionally mature DCs leading to decreased expression of HLA-DR and costimulatory molecules, and reduced secretion of pro-inflammatory cytokines in LPS-activated DCs. Moreover, Mo-DCs from CLL patients display a decreased ability to induce pro-inflammatory T-cell responses. IL-10-treatment of monocytes from healthy donors mimics the alteration in signaling observed in CLL patients, through enhanced STAT3-dependent expression of SOCS5. The higher level of SOCS5 inhibits STAT6 activation and leads to defective DC differentiation. These findings indicate that SOCS5 mediates the impaired function of DCs in CLL patients, and has the potential to be a new therapeutic target for reversing cancer-associated immune suppression.

Development of Selective Covalent Janus Kinase 3 Inhibitors.

The Janus kinases (JAKs) and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical immune cell signaling circuit, which is of fundamental importance in innate immunity, inflammation, and hematopoiesis, and dysregulation is frequently observed in immune disease and cancer. The high degree of structural conservation of the JAK ATP binding pockets has posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs. Here we report the discovery and optimization of 2,4-substituted pyrimidines as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Investigation of structure-activity relationship (SAR) utilizing biochemical and transformed Ba/F3 cellular assays resulted in identification of potent and selective inhibitors such as compounds 9 and 45. A 2.9 Å cocrystal structure of JAK3 in complex with 9 confirms the covalent interaction. Compound 9 exhibited decent pharmacokinetic properties and is suitable for use in vivo. These inhibitors provide a set of useful tools to pharmacologically interrogate JAK3-dependent biology.

Inhibiting STAT5 by the BET bromodomain inhibitor JQ1 disrupts human dendritic cell maturation.

Maturation of dendritic cells (DCs) is required to induce T cell immunity, whereas immature DCs can induce immune tolerance. Although the transcription factor STAT5 is suggested to participate in DC maturation, its role in this process remains unclear. In this study, we investigated the effect of STAT5 inhibition on LPS-induced maturation of human monocyte-derived DCs (Mo-DCs). We inhibited STAT5 by treating Mo-DCs with JQ1, a selective inhibitor of BET epigenetic readers, which can suppress STAT5 function. We found that JQ1 inhibits LPS-induced STAT5 phosphorylation and nuclear accumulation, thereby attenuating its transcriptional activity in Mo-DCs. The diminished STAT5 activity results in impaired maturation of Mo-DCs, as indicated by defective upregulation of costimulatory molecules and CD83, as well as reduced secretion of IL-12p70. Expression of constitutively activated STAT5 in JQ1-treated Mo-DCs overcomes the effects of JQ1 and enhances the expression of CD86, CD83, and IL-12. The activation of STAT5 in Mo-DCs is mediated by GM-CSF produced following LPS stimulation. Activated STAT5 then leads to increased expression of both GM-CSF and GM-CSFR, triggering an autocrine loop that further enhances STAT5 signaling and enabling Mo-DCs to acquire a more mature phenotype. JQ1 decreases the ability of Mo-DCs to induce allogeneic CD4(+) and CD8(+) T cell proliferation and production of proinflammatory cytokines. Furthermore, JQ1 leads to a reduced generation of inflammatory CD8(+) T cells and decreased Th1 differentiation. Thus, JQ1 impairs LPS-induced Mo-DC maturation by inhibiting STAT5 activity, thereby generating cells that can only weakly stimulate an adaptive-immune response. Therefore, JQ1 could have beneficial effects in treating T cell-mediated inflammatory diseases.

Targeting STAT5 in hematologic malignancies through inhibition of the bromodomain and extra-terminal (BET) bromodomain protein BRD2.

The transcription factor signal STAT5 is constitutively activated in a wide range of leukemias and lymphomas, and drives the expression of genes necessary for proliferation, survival, and self-renewal. Thus, targeting STAT5 is an appealing therapeutic strategy for hematologic malignancies. Given the importance of bromodomain-containing proteins in transcriptional regulation, we considered the hypothesis that a pharmacologic bromodomain inhibitor could inhibit STAT5-dependent gene expression. We found that the small-molecule bromodomain and extra-terminal (BET) bromodomain inhibitor JQ1 decreases STAT5-dependent (but not STAT3-dependent) transcription of both heterologous reporter genes and endogenous STAT5 target genes. JQ1 reduces STAT5 function in leukemia and lymphoma cells with constitutive STAT5 activation, or inducibly activated by cytokine stimulation. Among the BET bromodomain subfamily of proteins, it seems that BRD2 is the critical mediator for STAT5 activity. In experimental models of acute T-cell lymphoblastic leukemias, where activated STAT5 contributes to leukemia cell survival, Brd2 knockdown or JQ1 treatment shows strong synergy with tyrosine kinase inhibitors (TKI) in inducing apoptosis in leukemia cells. In contrast, mononuclear cells isolated form umbilical cord blood, which is enriched in normal hematopoietic precursor cells, were unaffected by these combinations. These findings indicate a unique functional association between BRD2 and STAT5, and suggest that combinations of JQ1 and TKIs may be an important rational strategy for treating leukemias and lymphomas driven by constitutive STAT5 activation.

STAT3 induction of miR-146b forms a feedback loop to inhibit the NF-κB to IL-6 signaling axis and STAT3-driven cancer phenotypes.

Interleukin-6 (IL-6)-mediated activation of signal transducer and activator of transcription 3 (STAT3) is a mechanism by which chronic inflammation can contribute to cancer and is a common oncogenic event. We discovered a pathway, the loss of which is associated with persistent STAT3 activation in human cancer. We found that the gene encoding the tumor suppressor microRNA miR-146b is a direct STAT3 target gene, and its expression was increased in normal breast epithelial cells but decreased in tumor cells. Methylation of the miR-146b promoter, which inhibited STAT3-mediated induction of expression, was increased in primary breast cancers. Moreover, we found that miR-146b inhibited nuclear factor κB (NF-κB)-dependent production of IL-6, subsequent STAT3 activation, and IL-6/STAT3-driven migration and invasion in breast cancer cells, thereby establishing a negative feedback loop. In addition, higher expression of miR-146b was positively correlated with patient survival in breast cancer subtypes with increased IL6 expression and STAT3 phosphorylation. Our results identify an epigenetic mechanism of crosstalk between STAT3 and NF-κB relevant to constitutive STAT3 activation in malignancy and the role of inflammation in oncogenesis.

Up-regulation of miR-182 expression after epigenetic modulation of human melanoma cells.

PURPOSE: We sought to investigate the epigenetic regulation of microRNAs (miRNAs) in melanoma. METHODS: We treated two highly metastatic human melanoma cell lines, C8161.9 and WM266-4, with the demethylating agents DAC (5-aza-2'-deoxycytidine) and trichostatin A. Locked nucleic acid-based miRNA expression profiling was utilized to examine the differential expression of miRNAs before and after treatment. RESULTS: We found that miR-182, a miRNA with oncogenic properties, was significantly up-regulated in human melanoma cells after epigenetic modulation. Genome sequence analysis revealed the presence of a prominent CpG island 8-10 kb upstream of mature miR-182. Methylation analysis showed that this genomic region was exclusively methylated in melanoma cells but not in human melanocytes, skin, or peripheral blood mononuclear cells. DISCUSSION: These results indicate that an epigenetic mechanism is likely involved in modulating the expression level of miR-182 in melanoma, and increased expression of oncogenic-like miR-182 could be a concern for melanoma patients after epigenetic therapy.

Elevated osteopontin levels in metastatic melanoma correlate with epigenetic silencing of breast cancer metastasis suppressor 1.

OBJECTIVE: Breast cancer metastasis suppressor 1 (BRMS1) has been shown to functionally reduce the metastatic potential of melanoma. We also previously reported that BRMS1 negatively regulates the expression of the oncoprotein osteopontin (OPN). This study was carried out to assess the clinical relevance of BRMS1 and OPN in melanoma. METHODS: Epigenetic regulation of BRMS1 was assessed by treating clinically derived melanoma cell lines with the demethylating agent 5-aza-2'-deoxycytidine (DAC) and the histone deacetylase inhibitor trichostatin A (TSA), followed by sodium bisulfite modification and methylation-specific PCR. Assessments of BRMS1 and OPN levels were performed using immunoblotting, quantitative real-time RT-PCR or reporter assays. RNA silencing was employed to abrogate the expression of OPN in melanoma-derived cell lines. The in vivo relevance of our findings was determined with experiments using athymic nude mice. RESULTS: The reduced expression of BRMS1 in surgically excised melanoma specimens correlated with increased OPN expression during the progression from primary to metastatic melanoma. Treatment with DAC and TSA elevated BRMS1 levels, but caused an inconsistent change in OPN gene expression. Abrogating the expression of OPN in BRMS1-deficient metastatic melanoma-derived cell lines retarded the growth of melanoma tumor xenografts in athymic nude mice. CONCLUSION: While treatment with DAC and TSA may not be a universally applicable treatment alternative in melanoma, silencing the expression of OPN in metastatic melanomas that have lost expression of BRMS1 is a potential option for therapeutic intervention.

Mitf-Mdel, a novel melanocyte/melanoma-specific isoform of microphthalmia-associated transcription factor-M, as a candidate biomarker for melanoma.

BACKGROUND: Melanoma incidence is on the rise and advanced melanoma carries an extremely poor prognosis. Treatment options, including chemotherapy and immunotherapy, are limited and offer low response rates and transient efficacy. Thus, identification of new melanocyte/melanoma antigens that serve as potential novel candidate biomarkers in melanoma is an important area for investigation. METHODS: Full length MITF-M and its splice variant cDNA were cloned from human melanoma cell line 624 mel by reverse transcription polymerase chain reaction (RT-PCR). Expression was investigated using regular and quantitative RT-PCR in three normal melanocytes (NHEM), 31 melanoma cell lines, 21 frozen melanoma tissue samples, 18 blood samples (peripheral blood mononuclear cell; PBMC) from healthy donors and 12 non-melanoma cancer cell lines, including three breast, five glioma, one sarcoma, two kidney and one ovarian cancer cell lines. RESULTS: A novel splice variant of MITF-M, which we named MITF-Mdel, was identified. The predicted MITF-Mdel protein contains two in frame deletions, 56- and 6- amino acid deletions in exon 2 (from V32 to E87) and exon 6 (from A187 to T192), respectively. MITF-Mdel was widely expressed in melanocytes, melanoma cell lines and tissues, but almost undetectable in non-melanoma cell lines or PBMC from healthy donors. Both isoforms were expressed significantly higher in melanoma tissues than in cell lines. Two of 31 melanoma cell lines expressed only one isoform or the other. CONCLUSION: MITF-Mdel, a novel melanocyte/melanoma-specific isoform of MITF-M, may serve as a potential candidate biomarker for diagnostic and follow-up purposes in melanoma.

Functional characterization of the progestagen-associated endometrial protein gene in human melanoma.

Utilizing gene microarray profiling of melanoma samples, we have recently identified a novel gene overexpressed in both thick primary and metastatic melanomas. This gene, progestagen-associated endometrial protein (PAEP), has never before been implicated in the oncogenic processes of melanoma, with its true function in oncogenesis and tumour progression relatively unknown. Overexpression of the PAEP gene in freshly procured thick primary and metastatic melanoma samples (58%) and daughter cell lines (77%) is confirmed by quantitative RT-PCR, immunohistochemistry, Western blotting and mass spectrometric analysis. We suggest that PAEP gene overexpression is involved with melanoma tumour progression as well as an aggressive phenotype. Transfection of melanoma cells with PAEP small interfering RNA (siRNA) reveals a significant decrease in soft agar colony formation and a marked inhibition of both cell migration and cell invasion. Furthermore, we establish stable melanoma transfectants via PAEP lentiviral small hairpin RNA (shRNA), examine their growth characteristics in a murine xenograft model and reveal that tumour growth is significantly inhibited in two separate melanoma cell lines. Our data strongly implicate the PAEP gene as a tumour growth promoter with oncogenic properties and a potential therapeutic target for patients with advanced melanoma.

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.

Epigenetics in human melanoma.

BACKGROUND: Recent technological advances have allowed us to examine the human genome in greater detail than ever before. This has opened the door to an improved understanding of the gene expression patterns involved with cancer. METHODS: A review of the literature was performed to determine the role of epigenetic modifications in human melanoma. We focused the search on histone deacetylation, methylation of gene promoter regions, demethylation of CpG islands, and the role of microRNA. We examined the relationship between human melanoma epigenetics and their importance in tumorigenesis, tumor progression, and inhibition of metastasis. The development and clinical application of select pharmacologic agents are also discussed. RESULTS: We identified several articles that have extensively studied the role of epigenetics in melanoma, further elucidating the complex processes involved in gene regulation and expression. Several new agents directly affect epigenetic mechanisms in melanoma, with divergent affects on the metastatic potential of melanoma. CONCLUSIONS: Epigenetic mechanisms have emerged as having a central role in gene regulation of human melanoma, including the identification of several putative tumor suppressor genes and oncogenes. Further research will focus on the development of novel therapeutics that will likely target and alter such epigenetic changes.