BRMS1 Expression Research Articles

Location, location, location: the BRMS1 protein and melanoma progression

The metastasis suppressor, BRMS1, has been demonstrated to cause dramatic regression of metastatic lesions without blocking orthotopic tumor growth. The role of BRMS1 is well-documented for several non-melanoma malignancies, such as breast cancer, ovarian cancer and non-small-cell lung cancer. However, its role in melanoma is just beginning to be understood, with a recent article by Slipicevic et al. highlighting the levels of expression of BRMS1 in benign nevi, primary and metastatic melanoma samples. Their findings emphasize that the intracellular location of BRMS1 protein (cytoplasmic or nuclear), appears to have a significant impact upon the metastatic capacity of melanoma cells. Interestingly, this selective localization translates into a statistically significant decrease in the relapse-free period in melanoma patients, further associated with a thicker Breslow's depth of primary melanomas. However, and more importantly, this study begins to define a clearer role for BRMS1 in melanoma that is strictly dependent upon its cellular location, with nuclear expression associated with invasive and metastatic capacity and cytoplasmic expression resulting in repressive effects upon progression and metastasis.Please see related article: http://www.biomedcentral.com/1471-2407/12/73

P1-09-06: Single Nucleotide Polymorphisms in the BRMS1 and SIPA1 Metastasis Suppressor Genes as Prognostic Markers in Breast Cancer Patients.

Abstract Introduction: Single nucleotide polymorphisms (SNPs) in the metastasis suppressors BRMS1 and SIPA1 may affect metastatic efficiency. BRMS1 affects apoptosis, colonization, cell adhesion, and invasive potential. Loss of BRMS1 expression has been correlated with younger age at diagnosis and reduced survival time in patients with progesterone-receptor (PR) negative, HER2−positive tumors. SIPA1 affects extracellular matrix gene expression and cell adhesion, and while SNPs have been associated with node positive, estrogen-receptor (ER)/PR negative tumors, evidence for a relationship with survival has been conflicting. Identifying SNPs that affect risk of recurrence and survival may improve the ascertainment of patients who require aggressive adjuvant therapy following a diagnosis of breast cancer. We evaluated associations between seven BRMS1 and SIPA1 SNPs and recurrence and survival in patients with primary breast cancer. Methods: We identified 1,015 incident breast cancer patients who received surgery at Roswell Park Cancer Institute (RPCI) and participated in the DataBank and BioRepository (DBBR) resource. Participants completed an epidemiologic questionnaire and provided a blood sample prior to surgery or other treatment. Clinical and pathologic data were linked to de-identified participant data in the DBBR database. SNPs in BRMS1 (rs11537993, rs3116068, and rs1052566) and SIPA1 (rs75894763, rs746429, rs3741378, and rs2306364) were genotyped by RPCI's Genomics facility using Sequenom® iPLEX Gold and Taqman® real-time PCR assays. Cox proportional hazards regression was used to estimate hazard ratios and 95% confidence intervals. Results: The median follow-up time was 33 months, and 49 deaths and 42 recurrences occurred. Tumors were more likely to be larger, node positive, ER/PR negative, and high grade among patients who experienced a recurrence or death. Recurrence was less likely in older patients and those with higher body mass index, although the latter association was nonsignificant (p=0.06). Patients with at least one variant allele of the BRMS1 rs3116068 genotype experienced shorter overall survival compared to patients with the homozygous common genotype (HR=2.05, 95% CI 1.15−3.63, rs3116068 AG+AA compared to GG). The remaining SNPs were not associated with overall survival, and none of the SNPs were associated with recurrence. Conclusions: In our data, the variant allele of rs3116068 was more common among women whose breast cancer was node negative and HER2−positive, compared to those with the common rs3116068 allele. The rs3116068 variant allele is also associated with poorer survival. While our findings do not support a role for common SNPs in the SIPA1 gene in breast cancer prognosis, BRMS1 rs3116068 may be a useful prognostic biomarker. Future goals are to examine additional SNPs in BRMS1 and other metastasis-related genes in a larger, racially diverse population. Funding: This research was supported by a gift from the Jayne and Phil Hubbell Family. The DBBR and Genomics Facility are RPCI Cancer Center Support Grant shared resources, supported by P30 CA016056-32. Ms. Roberts is a DOD Predoctoral Award recipient (BC10068) and Dr. Ambrosone is funded by the Breast Cancer Research Foundation. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-09-06.

Ectopic expression of RhoBTB2 inhibits migration and invasion of human breast cancer cells

RhoBTB2, or Deleted in Breast Cancer 2 (DBC2), identified as a candidate tumor suppressor gene for breast cancer and other human malignancies, is an atypical member of a novel gene family encoding small GTPases. In this study, we found that ectopic expression of RhoBTB2 inhibits the migration and invasion of the human metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435 in a dose-dependent manner. Western blotting analysis revealed that ectopic expression of RhoBTB2 induces a significant increase in the breast cancer metastasis suppressor, BRMS1. siRNA suppression of BRMS1 expression markedly reversed the inhibitory effects of RhoBTB2 on the migration and invasion abilities of both cell lines. Ezrin is a member of the ezrin-radixin-moesin cytoskeleton-associated protein family and is a key signaling molecule that regulates cancer migration and invasion. Western blotting analysis demonstrated that ectopic expression of RhoBTB2 results in decreased phosphorylation of ezrin and Akt2 in both MDA-MB-231 and MDA-MB-435 cells. Therefore, we conclude that up-regulation of the breast cancer metastasis suppressor BRMS1 and down-regulation of the phosphorylation of the cancer metastasis-related gene, ezrin, contributed to RhoBTB2-induced inhibition of metastatic breast carcinoma cell migration and invasion. Our findings suggest that understanding RhoBTB2-mediated migration and suppression of invasion is critical to the development of new therapies designed to prevent and treat patients with breast cancer metastasis.

9248 Safety of romiplostim for treatment of chemotherapy-induced thrombocytopenia (CIT) in patients with advanced non-small cell lung cancer (NSCLC)

Circulating tumor cells (CTCs) are independent prognostic factors in the primary and metastatic breast cancer patients and play crucial role in hematogenous tumor dissemination. The aim of this study was to correlate the presence of CTCs in peripheral blood with the expression of proteins in tumor tissue that have a putative role in regulation of cell growth and metastatic potential. This prospective study included 203 primary breast cancer patients treated by definitive surgery. CTCs were detected by quantitative real-time PCR for the expression of epithelial (CK19) or epithelial-to-mesenchymal transition–inducing transcription factor genes (TWIST1, SNAIL1, SLUG, and ZEB1). Expression of APC, ADAM23, CXCL12, E-cadherin, RASSF1, SYK, TIMP3, BRMS1, and SOCS1 proteins in primary breast tumor tissue was evaluated by immunohistochemistry. CTCs with epithelial markers were found in 17 (9.2%) patients. Their occurrence was associated with inhibition of SOCS1 expression (odds ratio [OR] = 0.07; 95% confidence interval [CI], 0.03-0.13; P < .001). CTCs with positive epithelial-to-mesenchymal transition markers were detected in 30 (15.8%) patients; however, no association with analyzed protein expressions was found. Overall, CTCs were detected in 44 (22.9%) patients. Presence of any CTC marker was significantly associated with positive CXCL12 expression (OR = 3.08; 95% CI, 1.15-8.26; P = .025) and lack of SOCS1 expression (OR = 0.10; 95% CI, 0.04-0.25; P < .001) in patient’s tumor tissues. As both CXCL12 and SOCS1 proteins are involved in cytokine signaling, our results provide support for the hypothesis that aberrant signaling cross talk between cytokine and chemokine responses could have an important role in hematogenous dissemination of tumor cells in breast cancer.

BRMS1 contributes to the negative regulation of uPA gene expression through recruitment of HDAC1 to the NF-κB binding site of the uPA promoter

The BRMS1 metastasis suppressor was recently shown to negatively regulate NF-kappaB signaling and down regulate NF-kappaB-dependent uPA expression. Here we confirm that BRMS1 expression correlates with reduced NF-kappaB DNA binding activity in independently derived human melanoma C8161.9 cells stably expressing BRMS1. We show that knockdown of BRMS1 expression in these cells using small interfering RNA (siRNA) leads to the reactivation of NF-kappaB DNA binding activity and re-expression of uPA. Further, we confirm that BRMS1 expression does not alter IKKbeta kinase activity suggesting that BRMS1-dependent uPA regulation does not occur through inhibition of the classical upstream activators of NF-kappaB. BRMS1 has been implicated as a corepressor of HDAC1 and consistent with this, we show that BRMS1 promotes HDAC1 recruitment to the NF-kappaB binding site of the uPA promoter and is associated with reduced H3 acetylation. We also confirm that BRMS1 expression stimulates disassociation of p65 from the NF-kappaB binding site of the uPA promoter consistent with its reduced DNA binding activity. These data suggest that BRMS1 recruits HDAC1 to the NF-kappaB binding site of the uPA promoter, modulates histone acetylation of p65 on the uPA promoter, leading to reduced NF-kappaB binding activity on its consensus sequence, and reduced transactivation of uPA expression.

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.

High level of messenger RNA for BRMS1 in primary breast carcinomas is associated with poor prognosis

BRMS1 is regarded as a metastasis suppressor gene for its ability to reduce metastatic potential of human and murine breast cancer cells as well as human melanoma cells. However, BRMS1 association to human tumor progression is not clearly understood. In the present study we analyzed BRMS1 mRNA expression in tumor progression and its potential prognostic value for breast carcinoma. BRMS1 mRNA expression level was quantified by real-time PCR in 47 tumoral, in 14 peritumoral and in 15 metastatic microdissected cellular populations from 47 breast cancer patients with 10-year follow up. We found BRMS1 expression to be higher in carcinoma cells than in matching normal epithelial cell populations in 10 out of 14 cases (p = 0.0005), while lymph-nodal carcinoma cells showed lower BRMS1 expression in 9 out of 15 cases (p = 0.001). Using both in vivo (human mammary breast carcinomas) and in vitro systems (breast cancer cell lines) we were able to demonstrate that BRMS1 overexpression was not a bias effect induced by cell proliferation rate. BRMS1 expression levels did not correlate with standard breast cancer prognostic factors but BRMS1 higher expression was associated with patient shorter disease-free and overall survival. Our findings are apparently inconsistent with the concept of BRMS1 as a metastasis suppressor gene. One possible explanation is that epithelial cells increase their BRMS1 expression as a compensatory response to tumor formation or metastasis progression, which is elevated in proportion to tumor aggressiveness, whereas those cells of the primary tumor that cannot upregulate BRMS1 escape to form metastasis.

Suppression of human ovarian carcinoma metastasis by the metastasis-suppressor gene, BRMS1

Metastasis-suppressor genes, by definition, suppress metastasis without affecting tumorigenicity and, hence, present attractive targets as prognostic or therapeutic markers. BRMS1 (breast cancer metastasis suppressor) has recently been identified as a metastasis-suppressor gene for human breast cancer and melanoma. Expression of BRMS1 messenger RNA (mRNA) in multitissue including normal prostate, ovarian, testis, and colon has been detected by northern blot analysis. We hypothesize that the role of BRMS1 in tumor progression may not be limited to breast cancer and melanoma. We previously found that BRMS1 mRNA levels in primary ovarian epithelial carcinomas were significantly lower than that in normal ovarian and benign tumors (P < 0.05), and statistical analysis of BRMS1 mRNA levels revealed that BRMS1 mRNA levels were significantly higher in early tumor stages (I, II) compared with advanced tumor stages (III, IV) in which lymph node or distant metastases were present (P < 0.01). Our data showed that reduced BRMS1 mRNA seems to influence ovarian carcinoma metastatic ability. Therefore, we transfected BRMS1 plasmid into highly malignant ovarian carcinoma cell line, HO-8910PM, and examined cell biologic behaviors including proliferation, adhesion, invasion, and metastasis in vitro and in vivo. BRMS1 expression did not alter the proliferation of HO-8910PM cells in vitro and primary tumor formation in vivo. But, BRMS1 expression significantly suppressed the cell adhesion to extracellular matrix components and in vitro cell invasion in BRMS1-transfected HO-8910PM cells compared to parental HO-8910PM and vector-only transfectants (HO-8910PM-vect). Furthermore, motility of BRMS1 transfectants was inhibited. lung colony formation of intravenously injected BRMS1 transfectants in nude mice was significantly reduced. Also, BRMS1 transfectants form significantly less metastatic to organs of peritoneal cavity in orthotopically implanted ovarian tumor nude models. We further discovered that BRMS1 expression did downregulate expression of an actin-bundling protein associated with cell motility -fascin, which perhaps is the mechanism underlying BRMS1 suppression of metastasis. These data suggested that in addition to its already described role in breast cancer and melanoma, BRMS1 functions as a metastasis-suppressor gene in ovarian carcinoma by modifying several metastatic-associated phenotypes, offering a new target for therapeutic intervention.

Breast Cancer Metastasis Suppressor 1 Inhibits Gene Expression by Targeting Nuclear Factor-κB Activity

Breast cancer metastasis suppressor 1 (BRMS1) functions as a metastasis suppressor gene in breast cancer and melanoma cell lines, but the mechanism of BRMS1 suppression remains unclear. We determined that BRMS1 expression was inversely correlated with that of urokinase-type plasminogen activator (uPA), a prometastatic gene that is regulated at least in part by nuclear factor-kappaB (NF-kappaB). To further investigate the role of NF-kappaB in BRMS1-regulated gene expression, we examined NF-kappaB binding activity and found an inverse correlation between BRMS1 expression and NF-kappaB binding activity in MDA-MB-231 breast cancer and C8161.9 melanoma cells stably expressing BRMS1. In contrast, BRMS1 expression had no effect on activation of the activator protein-1 transcription factor. Further, we showed that suppression of both constitutive and tumor necrosis factor-alpha-induced NF-kappaB activation by BRMS1 may be due to inhibition of IkappaBalpha phosphorylation and degradation. To examine the relationship between BRMS1 and uPA expression in primary breast tumors, we screened a breast cancer dot blot array of normalized cDNA from 50 breast tumors and corresponding normal breast tissues. There was a significant reduction in BRMS1 mRNA expression in breast tumors compared with matched normal breast tissues (paired t test, P < 0.0001) and a general inverse correlation with uPA gene expression (P < 0.01). These results suggest that at least one of the underlying mechanisms of BRMS1-dependent suppression of tumor metastasis includes inhibition of NF-kappaB activity and subsequent suppression of uPA expression in breast cancer and melanoma cells.

Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases

Brain metastases are an increasingly common complication in breast cancer patients. The Metastasis Suppressor Genes (MSG) Nm23, KISS1, KAI1, BRMS1, and Mkk4 have been associated with the metastatic potential of breast cancer in vitro and in vivo. The mRNA expression of Nm23, KISS1, KAI1, BRMS1, and Mkk4 in fresh frozen tissue samples of brain metastases from ductal invasive breast cancer specimens was examined in relation to primary tumors. In a first step, mRNA expression screening was carried out using a semi-quantitative RT-PCR approach, in a second step quantitative real-time RT-PCR was performed on selected specimens. By immunohistochemical staining, gene products were visualized on the protein level. Semi-quantitative RT-PCR revealed reduced mRNA expression of Nm23, KISS1, KAI1, BRMS, and Mkk4 in brain metastases. Results for KISS1, KAI1, BRMS, and Mkk4 were confirmed by real-time RT-PCR. In detail, mRNA expression reduction in breast cancer brain metastases was tenfold. Expression of MSG could be confirmed by immunohistochemical staining on protein level. Our investigations revealed significantly reduced mRNA expression of metastases suppressor genes KISS1, KAI1, BRMS1, and Mkk4 in breast cancer brain metastasis. Particularly, in the case of KISS1 and Mkk4, an important role for future treatment of patients with breast cancer brain metastatic lesions can be assumed.

Transcriptional expression of genes involved in cell invasion and migration by normal and tumoral trophoblast cells.

Once initiated, invasion of trophoblast cells must be tightly regulated, particularly in early pregnancy. The mechanisms necessary for the invasion and migration of trophoblast cells are thought to be related to those involved in the invasive and metastatic properties of cancer cells. Quantitative PCR was used to measure, in trophoblast cells, the transcriptional expression profiles of four genes, INSL4, BRMS1, KiSS-1 and KiSS-1R, reported to be implicated in tumor invasion and metastasis. Laser capture microdissection and purification of trophoblast cells demonstrate that, as already known for INSL4, BRMS1, KiSS-1 and KiSS-1R are expressed by the trophoblast subset of placental tissues. Expression profiles of these genes studied in early placentas (7-9 weeks, n=55) and term placentas (n=11) showed that expression levels of BRMS1 are higher in term than in early placentas, while expression levels of KiSS-1R are higher in early than in term placentas. Low levels of expression of BRMS1 were observed in normal pregnancies, in molar pregnancies and in choriocarcinoma cell lines BeWo, JAR and JEG3 while, in striking contrast, the expression levels of INSL4, KiSS-1 and Kiss-1R were increased in both early placentas and molar pregnancies and were reduced in choriocarcinoma cells. These transcriptional expression profiles are in favor of a predominant role of INSL4, KiSS-1 and KiSS-1R in the control of the invasive and migratory properties of trophoblast cells.

BRMS-1 expression in human breast carcinoma

BRMS-1 expression in human breast carcinoma

Analysis of mechanisms underlying BRMS1 suppression of metastasis.

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

The relationship of BRMS1 and RhoGDI2 gene expression to metastatic potential in lineage related human bladder cancer cell lines.

We have recently characterized a human bladder cancer cell line T24 and a more aggressive lineage related variant of it, T24T. To gain further insights, we have studied their metastatic ability in an in vivo model system. Results show that T24 forms significantly fewer [4/12 (1/11) mice had metastases with 1-2 lesions/mouse] metastasis in SCID/bg mice than T24T [14/14 (6/6) mice had metastases with a mean of 24-28 lesions/mouse]. To begin exploring the mechanisms underlying this difference, we evaluated the mRNA and protein expression levels of metastasis-suppressor genes, known to be important in the progression of other cancers, in our model of bladder cancer progression. A higher mRNA expression of BRMS1, a metastasis suppressor in breast cancer, was observed in T24 cells. In addition, RhoGDI2 mRNA expression was only observed in T24 when compared to T24T, suggesting that Rho activation might play a significant role in the metastatic cascade. However, a basal level mRNA expression of KISS1, described as metastasis suppressor in melanoma and breast, was observed in both the lines and had slightly higher expression in T24T. No difference of Nm23-H1, KAI1, MKK4/SEK1 and E-Cadherin protein levels were noted between these two lines. In summary, it appears that the T24/T24T paired cell lines constitute a useful model for the study of human bladder cancer metastasis that will allow both the discovery and mechanistic evaluation of genes potentially involved in this process.