Douglas R. Hurst

Breast cancer metastasis suppressor 1 up-regulates miR-146, which suppresses breast cancer metastasis.

Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that differentially regulates expression of multiple genes, leading to suppression of metastasis without blocking orthotopic tumor growth in multiple human and murine cancer cells of diverse origins. We hypothesized that miR-146 may be involved in the ability of BRMS1 to supress metastasis because miR-146 expression is altered by BRMS1 and because BRMS1 and miR-146 are both associated with decreased signaling through the nuclear factor-kappaB pathway. BRMS1 significantly up-regulates miR-146a by 6- to 60-fold in metastatic MDA-MB-231 and MDA-MB-435 cells, respectively, and miR-146b by 40-fold in MDA-MB-435 as measured by real-time quantitative reverse transcription-PCR. Transduction of miR-146a or miR-146b into MDA-MB-231 down-regulated expression of epidermal growth factor receptor, inhibited invasion and migration in vitro, and suppressed experimental lung metastasis by 69% and 84%, respectively (mean +/- SE: empty vector = 39 +/- 6, miR-146a = 12 +/- 1, miR-146b = 6 +/- 1). These results further support the recent notion that modulating the levels of miR-146a or miR-146b could have a therapeutic potential to suppress breast cancer metastasis.

Metastamir: the field of metastasis-regulatory microRNA is spreading.

Despite advancements in knowledge from more than a century of metastasis research, the genetic programs and molecular mechanisms required for cancer metastasis are still incompletely understood. Genes that specifically regulate the process of metastasis are useful tools to elucidate molecular mechanisms and may become markers and/or targets for antimetastatic therapy. Recently, several noncoding regulatory RNA genes, microRNA (miRNA), were identified, which play roles in various steps of metastasis, some without obvious roles in tumorigenesis. Understanding how these metastasis-associated miRNA, which we term metastamir, are involved in metastasis will help identify possible biomarkers or targets for the most lethal attribute of cancer: metastasis.

Matrix Metalloproteinase Inhibitors as Prospective Agents for the Prevention and Treatment of Cardiovascular and Neoplastic Diseases

Acting on a broad spectrum of extracellular, intracellular, and membrane-associated substrates, the matrix metalloproteinases (MMPs) are critical to the biological processes of organisms; when aberrantly expressed, many pathological conditions may be born or exacerbated. The prospect of MMP inhibition for therapeutic benefit in cancer, cardiovascular disease, and stroke is reviewed here. MMP inhibitor (MMPI) development constitutes an important branch of research in both academic and industrial settings and advances our knowledge on the structure-function relationship of MMPs. Targeting MMPs in disease treatment is complicated by the fact that MMPs are indispensable for normal development and physiology and by their multi-functionality, possible functional redundancy or contradiction, and context-dependent expression and activity. This complexity was revealed by previous efforts to inhibit MMP activity in the treatment of cancer patients that yielded unsatisfactory results. This review focuses on MMPI development since the late 90s, in terms of natural products and their derivatives, and synthetic compounds of low molecular mass incorporating specific zinc-binding groups (ZBGs). A few polyphenols and flavonoids that exhibit MMPI activities may have chemopreventive and neuro- and cardiovascular-protective effects. A new generation of potent and selective MMPIs with novel ZBGs and inhibition mechanisms have been designed, synthesized, and tested. Although only one collagenase inhibitor (Periostat, doxycycline hyclate) has been approved by the Food and Drug Administration as a drug for the treatment of periodontal disease, new hope is emerging in the form of natural and synthetic MMPIs for the prevention and treatment of stroke, cardiovascular disease, cancer, and other medical conditions.

Metastasis Suppressor Genes: At the Interface Between the Environment and Tumor Cell Growth

The molecular mechanisms and genetic programs required for cancer metastasis are sometimes overlapping, but components are clearly distinct from those promoting growth of a primary tumor. Every sequential, rate-limiting step in the sequence of events leading to metastasis requires coordinated expression of multiple genes, necessary signaling events, and favorable environmental conditions or the ability to escape negative selection pressures. Metastasis suppressors are molecules that inhibit the process of metastasis without preventing growth of the primary tumor. The cellular processes regulated by metastasis suppressors are diverse and function at every step in the metastatic cascade. As we gain knowledge into the molecular mechanisms of metastasis suppressors and cofactors with which they interact, we learn more about the process, including appreciation that some are potential targets for therapy of metastasis, the most lethal aspect of cancer. Until now, metastasis suppressors have been described largely by their function. With greater appreciation of their biochemical mechanisms of action, the importance of context is increasingly recognized especially since tumor cells exist in myriad microenvironments. In this chapter, we assemble the evidence that selected molecules are indeed suppressors of metastasis, collate the data defining the biochemical mechanisms of action, and glean insights regarding how metastasis suppressors regulate tumor cell communication to-from microenvironments.

BRMS1 Suppresses Breast Cancer Experimental Metastasis to Multiple Organs by Inhibiting Several Steps of the Metastatic Process

Breast cancer metastasis suppressor 1 (BRMS1) inhibits formation of macroscopic lung metastases in breast, ovary, and melanoma xenograft models. Because it is unclear which step(s) of the metastatic cascade are affected by BRMS1, the major aim of this study was to determine when and how BRMS1 acts to suppress metastasis. We also examined whether BRMS1 expression globally blocks metastasis or selectively inhibits metastatic outgrowths in specific tissues. Metastatic human breast carcinoma cell lines MDA-MB-231 and -435 expressing enhanced green fluorescent protein (GFP; 231 GFP and 435 GFP) and cell lines transduced with the BRMS1 gene (231 GFP-BRMS1 and 435 GFP-BRMS1) were injected into the left cardiac ventricle to achieve the widest possible cellular distribution, by minimizing first-pass clearance in the lungs. Compared with parental cells, BRMS1-expressing clones formed significantly fewer metastases in all organs tested. When cells were injected directly into the vasculature, fewer of the BRMS1-expressing cells reached lungs or bone compared with parental cells, suggesting that restoration of BRMS1 expression increased cell death during transit. Susceptibility to anoikis was verified in vitro by demonstrating decreased survival on poly-hydroxyethyl methacrylate-coated dishes. Most of the BRMS1-expressing cells reaching secondary sites failed to proliferate, suggesting that BRMS1 also inhibits colonization. Coupled with previous reports showing modest effects of BRMS1 on adhesion and invasion, our results indicate that BRMS1 inhibits metastases in multiple organs by blocking several steps in the metastatic cascade.

Breast cancer metastasis suppressor 1 coordinately regulates metastasis-associated microRNA expression

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis of multiple tumor types without blocking tumorigenesis. BRMS1 forms complexes with SIN3, histone deacetylases and selected transcription factors that modify metastasis‐associated gene expression (e.g., EGFR, OPN, PI4P5K1A, PLAU). microRNA (miRNA) are a recently discovered class of regulatory, noncoding RNA, some of which are involved in neoplastic progression. Based on these data, we hypothesized that BRMS1 may also exert some of its antimetastatic effects by regulating miRNA expression. MicroRNA arrays were done comparing small RNAs that were purified from metastatic MDA‐MB‐231 and MDA‐MB‐435 and their nonmetastatic BRMS1‐transfected counterparts. miRNA expression changed by BRMS1 were validated using SYBR Green RT‐PCR. BRMS1 decreased metastasis‐promoting (miR‐10b, ‐373 and ‐520c) miRNA, with corresponding reduction of their downstream targets (e.g., RhoC which is downstream of miR‐10b). Concurrently, BRMS1 increased expression of metastasis suppressing miRNA (miR‐146a, ‐146b and ‐335). Collectively, these data show that BRMS1 coordinately regulates expression of multiple metastasis‐associated miRNA and suggests that recruitment of BRMS1‐containing SIN3:HDAC complexes to, as yet undefined, miRNA promoters might be involved in the regulation of cancer metastasis.

Heparanase-mediated loss of nuclear syndecan-1 enhances histone acetyltransferase (HAT) activity to promote expression of genes that drive an aggressive tumor phenotype.

Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins known to drive tumor progression (e.g. VEGF, MMP-9, hepatocyte growth factor (HGF), and RANKL). However, the mechanism whereby this enzyme regulates gene expression remains unknown. We previously reported that elevation of heparanase levels in myeloma cells causes a dramatic reduction in the amount of syndecan-1 in the nucleus. Because syndecan-1 has heparan sulfate chains and because exogenous heparan sulfate has been shown to inhibit the activity of histone acetyltransferase (HAT) enzymes in vitro, we hypothesized that the reduction in nuclear syndecan-1 in cells expressing high levels of heparanase would result in increased HAT activity leading to stimulation of protein transcription. We found that myeloma cells or tumors expressing high levels of heparanase and low levels of nuclear syndecan-1 had significantly higher levels of HAT activity when compared with cells or tumors expressing low levels of heparanase. High levels of HAT activity in heparanase-high cells were blocked by SST0001, an inhibitor of heparanase. Restoration of high syndecan-1 levels in heparanase-high cells diminished nuclear HAT activity, establishing syndecan-1 as a potent inhibitor of HAT. Exposure of heparanase-high cells to anacardic acid, an inhibitor of HAT activity, significantly suppressed their expression of VEGF and MMP-9, two genes known to be up-regulated following elevation of heparanase. These results reveal a novel mechanistic pathway driven by heparanase expression, which leads to decreased nuclear syndecan-1, increased HAT activity, and up-regulation of transcription of multiple genes that drive an aggressive tumor phenotype.

Breast Cancer Metastasis Suppressor-1 Differentially Modulates Growth Factor Signaling

That metastatic tumor cells grow in selective non-native environments suggests an ability to differentially respond to local microenvironments. BRMS1, like other metastasis suppressors, halts ectopic growth (metastasis) without blocking orthotopic tumor formation. BRMS1-expressing tumor cells reach secondary sites but do not colonize distant tissues, compelling the hypothesis that BRMS1 selectively restricts the ability of tumor cells to respond to exogenous regulators in different tissues. Here we report that BRMS1 expression in metastatic human breast cancer cells leads to a selective reduction in epidermal growth factor receptor expression and downstream (AKT) signaling. Signaling through another receptor tyrosine kinase, hepatocyte growth factor receptor (c-Met), remains unaltered despite reduced levels of the signaling intermediate phosphatidylinositol (4,5)-bisphosphate. Interestingly, reduced downstream calcium signaling is observed following treatment with platelet-derived growth factor, consistent with decreased phosphatidylinositol (4,5)-bisphosphate. However, platelet-derived growth factor receptor expression is unaltered. Thus, BRMS1 differentially attenuates cellular responses to mitogenic signals, not only dependent upon the specific signal received, but at varying steps within the same signaling cascade. Specific modulation of signaling responses received from the microenvironment may ultimately dictate which environments are permissive/restrictive for tumor cell growth and provide insights into the biology underlying metastasis.

Metastasis suppressors and the tumor microenvironment.

The most lethal and debilitating attribute of cancer cells is their ability to metastasize. Throughout the process of metastasis, tumor cells interact with other tumor cells, host cells and a variety of molecules. Tumor cells are also faced with a number of insults, such as hemodynamic sheer pressure and immune selection. This brief review explores how metastasis suppressor proteins regulate interactions between tumor cells and the microenvironments in which tumor cells find themselves.

Alterations of BRMS1-ARID4A Interaction Modify Gene Expression but Still Suppress Metastasis in Human Breast Cancer Cells

The BRMS1 metastasis suppressor interacts with the protein AT-rich interactive domain 4A (ARID4A, RBBP1) as part of SIN3·histone deacetylase chromatin remodeling complexes. These transcriptional co-repressors regulate diverse cell phenotypes depending upon complex composition. To define BRMS1 complexes and their roles in metastasis suppression, we generated BRMS1 mutants (BRMS1mut) and mapped ARID4A interactions. BRMS1L174D disrupted direct interaction with ARID4A in yeast two-hybrid genetic screens but retained an indirect association with ARID4A in MDA-MB-231 and -435 human breast cancer cell lines by co-immunoprecipitation. Deletion of the first coiled-coil domain (BRMS1ΔCC1) did not disrupt direct interaction in yeast two-hybrid screens but did prevent association by co-immunoprecipitation. These results suggest altered complex composition with BRMS1mut. Although basal transcription repression was impaired and the pro-metastatic protein osteopontin was differentially down-regulated by BRMS1L174D and BRMS1ΔCC1, both down-regulated the epidermal growth factor receptor and suppressed metastasis in MDA-MB-231 and -435 breast cancer xenograft models. We conclude that BRMS1mut, which modifies the composition of a SIN3·histone deacetylase chromatin remodeling complex, leads to altered gene expression profiles. Because metastasis requires the coordinate expression of multiple genes, down-regulation of at least one important gene, such as the epidermal growth factor receptor, had the ability to suppress metastasis. Understanding which interactions are necessary for particular biochemical/cellular functions may prove important for future strategies targeting metastasis.