Brandon J. Metge

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

BackgroundOsteopontin (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.ResultsThe 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.ConclusionOur 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.

Osteopontin Knockdown Suppresses Tumorigenicity of Human Metastatic Breast Carcinoma, MDA-MB-435

Elevated expression of osteopontin (OPN), a secreted phosphoglycoprotein, is frequently associated with many transformed cell lines. Various studies suggest that OPN may contribute to tumor progression as well as metastasis in multiple tumor types. High levels of OPN have been reported in patients with metastatic cancers, including breast. We found that the expression of OPN corroborates with the aggressive phenotype of the breast cancer cells i.e. the expression of OPN is acquired as the breast cancer cells become more aggressive. To assess the role(s) of OPN in breast carcinoma, expression of endogenous OPN was knocked down in metastatic MDA-MB-435 human breast carcinoma cells using RNA interference. We targeted multiple regions of the OPN transcript for RNA interference, along with ‘scrambled’ and ‘non-targeting siRNA pool’ controls to distinguish between target-specific and potential off-target effects including interferon-response gene (PeIF2-α) induction. The OPN knockdown by shRNA suppressed tumor take in immunocompromised mice. The ‘silenced’ cells also showed significantly lower invasion and migration in modified Boyden chamber assays and reduced ability to grow in soft agar. Thus, in addition to the widely reported roles of OPN in late stages of tumor progression, these results provide functional evidence that OPN contributes to breast tumor growth as well.

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.

Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer

IntroductionMammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntingtons disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below.MethodsExpression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L).ResultsWe found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding α2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes.ConclusionCollectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.

Epigenetic silencing contributes to the loss of BRMS1 expression in breast cancer

Breast Cancer Metastasis Suppressor 1 (BRMS1) suppresses metastasis of human breast cancer, ovarian cancer and melanoma in athymic mice. Studies have also shown that BRMS1 is significantly downregulated in some breast tumors, especially in metastatic disease. However, the mechanisms which regulate BRMS1 expression are currently unknown. Upon examination of the BRMS1 promoter region by methylation specific PCR (MSP) analysis, we discovered a CpG island (−3477 to −2214), which was found to be hypermethylated across breast cancer cell lines. A panel of 20 patient samples analyzed showed that 45% of the primary tumors and 60% of the matched lymph node metastases, displayed hypermethylation of BRMS1 promoter. Furthermore, we found a direct correlation between the methylation status of the BRMS1 promoter in the DNA isolated from tissues, with the loss of BRMS1 expression assessed by immunohistochemistry. There are several studies investigating the mechanism by which BRMS1 suppresses metastasis; however thus far there is no study that reports the cause(s) of loss of BRMS1 expression in aggressive breast cancer. Here we report for the first time that BRMS1 is a novel target of epigenetic silencing; and aberrant methylation in the BRMS1 promoter may serve as a cause of loss of its expression.

Effect of Niclosamide on Basal-like Breast Cancers

Basal-like breast cancers (BLBC) are poorly differentiated and display aggressive clinical behavior. These tumors become resistant to cytotoxic agents, and tumor relapse has been attributed to the presence of cancer stem cells (CSC). One of the pathways involved in CSC regulation is the Wnt/β-catenin signaling pathway. LRP6, a Wnt ligand receptor, is one of the critical elements of this pathway and could potentially be an excellent therapeutic target. Niclosamide has been shown to inhibit the Wnt/β-catenin signaling pathway by causing degradation of LRP6. TRA-8, a monoclonal antibody specific to TRAIL death receptor 5, is cytotoxic to BLBC cell lines and their CSC-enriched populations. The goal of this study was to examine whether niclosamide is cytotoxic to BLBCs, specifically the CSC population, and if in combination with TRA-8 could produce increased cytotoxicity. Aldehyde dehydrogenase (ALDH) is a known marker of CSCs. By testing BLBC cells for ALDH expression by flow cytometry, we were able to isolate a nonadherent population of cells that have high ALDH expression. Niclosamide showed cytotoxicity against these nonadherent ALDH-expressing cells in addition to adherent cells from four BLBC cell lines: 2LMP, SUM159, HCC1187, and HCC1143. Niclosamide treatment produced reduced levels of LRP6 and β-catenin, which is a downstream Wnt/β-catenin signaling protein. The combination of TRA-8 and niclosamide produced additive cytotoxicity and a reduction in Wnt/β-catenin activity. Niclosamide in combination with TRA-8 suppressed growth of 2LMP orthotopic tumor xenografts. These results suggest that niclosamide or congeners of this agent may be useful for the treatment of BLBC. Mol Cancer Ther; 13(4); 800–11. ©2014 AACR.

Loss of tumor suppressor Merlin in advanced breast cancer is due to post-translational regulation.

Background: The role of Merlin in breast cancer is unknown. Results: Merlin protein is degraded in advanced breast cancer due to osteopontin-initiated signaling. Conclusion: Merlin is regulated at the post-translational level in breast tumors. Significance: We have defined a functional role for Merlin in limiting breast tumor growth and elucidated the utility of Merlin as an important biomarker in breast cancer. Unlike malignancies of the nervous system, there have been no mutations identified in Merlin in breast cancer. As such, the role of the tumor suppressor, Merlin, has not been investigated in breast cancer. We assessed Merlin expression in breast cancer tissues by immunohistochemistry and by real-time PCR. The expression of Merlin protein (assessed immunohistochemically) was significantly decreased in breast cancer tissues (although the transcript levels were comparable) simultaneous with increased expression of the tumor-promoting protein, osteopontin (OPN). We further demonstrate that the loss of Merlin in breast cancer is brought about, in part, due to OPN-initiated Akt-mediated phosphorylation of Merlin leading to its proteasomal degradation. Restoring expression of Merlin resulted in reduced malignant attributes of breast cancer, characterized by reduced invasion, migration, motility, and impeded tumor (xenograft) growth in immunocompromised mice. The possibility of developing a model using the relationship between OPN and Merlin was tested with a logistic regression model applied to immunohistochemistry data. This identified consistent loss of immunohistochemical expression of Merlin in breast tumor tissues. Thus, we demonstrate for the first time a role for Merlin in impeding breast malignancy, identify a novel mechanism for the loss of Merlin protein in breast cancer, and have developed a discriminatory model using Merlin and OPN expression in breast tumor tissues.

microRNA-29 negatively regulates EMT regulator N-myc interactor in breast cancer

BackgroundN-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis.MethodWeb based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3β phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses.ResultsInvasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/β-catenin signaling resulting from inactivation of GSK3β.ConclusionAberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.

Spheroid-forming subpopulation of breast cancer cells demonstrates vasculogenic mimicry via hsa-miR-299–5p regulated de novo expression of osteopontin

The growth of cancer cells as multicellular spheroids has frequently been reported to mimic the in vivo tumour architecture and physiology and has been utilized to study antitumour drugs. In order to determine the distinctive characteristics of the spheroid‐derived cells compared to the corresponding monolayer‐derived cells, we enriched multicellular spheroid‐forming subpopulations of cells from three human breast cancer cell lines (MCF7, MCF10AT and MCF10DCIS.com). These spheroid‐derived cells were injected into female athymic nude mice to assess their tumorigenic potential and were profiled for their characteristic miRNA signature. We discovered that the spheroid‐derived cells expressed increased levels of osteopontin (OPN), an oncogenic protein that has been clinically correlated with increased tumour burden and adverse prognosis in patients with breast cancer metastasis. Our studies further show that increased OPN levels are brought about in part, by decreased levels of hsa‐mir‐299–5p in the spheroid‐forming population from all three cell lines. Moreover, the spheroid‐forming cells can organize into vascular structures in response to nutritional limitation; these structures recapitulate a vascular phenotype by the expression of endothelial markers CD31, Angiopoeitin‐1 and Endoglin. In this study, we have validated that hsa‐mir‐299–5p targets OPN; de novo expression of OPN in turn plays a critical role in enhancing proliferation, tumorigenicity and the ability to display vasculogenic mimicry of the spheroid‐forming cells.

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.