Functional evidence for a novel human breast carcinoma metastasis suppressor, BRMS1, encoded at chromosome 11q13

We previously showed that introduction of a normal, neomycin-tagged human chromosome 11 reduces the metastatic capacity of MDA-MB-435 (435) human breast carcinoma cells by 70-90% without affecting tumorigenicity, suggesting the presence of one or more metastasis suppressor genes encoded on human chromosome 11. To identify the gene(s) responsible, differential display comparing chromosome 11-containing (neo11/ 435) and parental, metastatic cells was done. We describe the isolation and functional characterization of a full-length cDNA for one of the novel genes, designated breast-cancer metastasis suppressor 1 (BRMS1), which maps to human chromosome 11q13.1-q13.2. Stably transfected MDA-MB-435 and MDA-MB-231 breast carcinoma cells still form progressively growing, locally invasive tumors when injected into mammary fat pads but are significantly less metastatic to lungs and regional lymph nodes. These data provide compelling functional evidence that breast-cancer metastasis suppressor 1 is a novel mediator of metastasis suppression in human breast carcinoma.     

Identification and characterization of the murine ortholog (brms1) of breast-cancer metastasis suppressor 1 (BRMS1).

We have cloned a novel metastasis-suppressor gene (BRMS1) by differential display, comparing metastatic human breast carcinoma cell line MDA-MB-435 to its metastasis-suppressed human chromosome 11 microcell hybrid. Screening of a murine cDNA library led to the identification of a 1.4 kb cDNA with a sequence revealing 85% homology to human BRMS1 within the open reading frame. The predicted protein sequence for the murine ortholog is 95% identical, suggesting that it is strongly conserved across these 2 species. The cloned cDNA was used to screen a murine strain SV129 BAC library to obtain brms1 genomic DNA. Three BAC clones [226(I4), 226(H4) and 239(N7)] were confirmed to encode the entire brms1 gene. Detailed analysis of BAC clone 226(I4) shows that the gene spans 8.5 kb and, like the human gene, is organized into 10 exons and 9 introns. While the exons share a high degree of homology, there are greater differences when comparing intron structures between the human and murine genes. The 5' upstream region shares about 64% homology with its human counterpart, retaining several of the many putative regulatory elements. Like the human genomic BRMS1, the murine ortholog of the iGnT gene is found upstream of brms1 and the murine ortholog of the RIN1 gene is found downstream of brms1. brms1 was then tested for suppression of metastasis of mouse mammary carcinoma cell line 66cl4 in syngeneic BALB/c mice. Transfection with brms1 did not inhibit 66cl4 primary tumor formation but significantly suppressed its metastatic capability. This suggests that the murine ortholog functions similarly to BRMS1.     

Functional evidence for a metastasis suppressor gene for rat prostate cancer within a 60-kilobase region on human chromosome 8p21-p12.

We recently demonstrated that the human chromosome 8p21-p12 region encodes a metastasis suppressor gene for rat prostate cancer. The presence of this region suppresses the metastatic ability of rat prostate cancer cells (N. Nihei et al., Genes Chromosomes Cancer, 17: 260-268, 1996). To define further the region harboring the metastasis suppressor gene, a truncated human chromosome 8 containing this region was transferred into highly metastatic AT6.3 rat prostate cancer cells by microcell-mediated chromosome transfer. The region of human chromosome 8 retained in each microcell hybrid was determined by a PCR analysis with sequence-tagged site markers, and this analysis placed the metastasis suppressor gene in the interval between D8S2249 and D8S2244 on human chromosome 8p21-p12. One of the metastasis-suppressed microcell hybrids was used for construction of representative yeast/bacterial artificial chromosome (YAC/BAC) library covering the candidate region using a transformation-associated recombination technology (N. Kouprina et al., Genomics, 53: 21-28, 1998). The final contig corresponding to the candidate region was assembled by four YAC/BAC clones. Each clone was transfected into the AT6.3 cells, and the resultant transfectants were tested for their metastatic ability in athymic nude mice. Introduction of a 60-kb YAC/BAC clone resulted in significant suppression of the metastatic ability without suppression of the tumorigenicity. In contrast, other YAC/BAC clones in the contig had neither metastasis nor tumor suppressor activity. This demonstrates that the 60-kb fragment from the human chromosome 8p21-p12 region contains the metastasis suppressor gene for the AT6.3 cells. Frequent loss of heterozygosity of this region is detected in human prostate cancer, which suggests that our target metastasis suppressor gene may also play an important role in the progression of human prostate cancer.     

Construction of mouse A9 clones containing a single human chromosome tagged with neomycin-resistance gene via microcell fusion

Normal human fibroblasts (MRC-5 or NTI-4) were transfected with pSV2-neo plasmid DNA. Fifty G418-resistant fibroblast clones were isolated and independently fused to mouse A9 cells. The cell hybrids were selected and isolated in the medium containing G418 plus ouabain. Since micronuclei were more efficiently induced in these hybrids compared to parental human fibroblasts by colcemid treatment, the transfer of neo-tagged human chromosomes in the hybrids to mouse A9 cells was performed via microcell fusion. Two hundred A9 microcell hybrids were isolated and karyotyped. Among them, thirteen microcell clones, each containing a single human chromosome 1, 2, 5, 6, 7, 8, 10, 11, 12, 15, 18, 19 or 20 were established. Isozyme analyses conformed the presence of each human chromosome in these A9 microcell clones. The results of Southern blot and chromosomal in situ hybridization analyses indicate that the human chromosomes in these clones were tagged with pSV2-neo plasmid DNA.     

Suppression of tumorigenicity of a Wilms' tumor cell line is associated with a decrease in synthesis of two proteins.

Wilms' tumor has been associated with deletions in two loci on chromosome 11, and the introduction of a translocated human chromosome [t(X;11)] into a Wilms' tumor cell line (G401.6TG.6) by microcell hybridization suppresses tumor formation in nude mice. The tumorigenic phenotype is restored in segregants of these microcell hybrids, in which the introduced chromosome is lost. We have used ultrahigh-resolution 'giant' two-dimensional gel electrophoresis of metabolically labeled cellular proteins and in vitro translation products of isolated mRNA to identify changes in cellular gene expression that occur in these cell lines. The changes in gene expression associated with these chromosomal manipulations per se are quite minimal. However, we have identified two proteins (p16 and p28) whose synthesis is consistently decreased in three non-tumorigenic (suppressed) microcell hybrid clones relative to parental and segregant tumorigenic lines. They are also decreased at the level of mRNA in at least two of the non-tumorigenic clones. The decrease of these proteins represents markers of the suppressed phenotype, and their down-regulation may conceivably mediate the suppression of tumorigenicity.     

Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer

Introduction  

The molecular mechanisms involved in breast cancer metastasis still remain unclear to date. In our previous study, differential expression of peroxiredoxin 6 was found between the highly metastatic MDA-MB-435HM cells and their parental counterparts, MDA-MB-435 cells. In this study, we investigated the effects of peroxiredoxin 6 on the proliferation and metastatic potential of human breast cancer cells and their potential mechanism.     

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.     

Metastasis suppressor genes: basic biology and potential clinical use

Metastatic disease remains a significant contributor to morbidity and mortality in patients with breast cancer. An improved molecular and biochemical understanding of the metastatic process is expected to fuel the development of new therapeutic approaches. The suppression of tumor metastasis, despite tumor cell expression of oncogenes and metastasis-promoting events, has become a diverse and fruitful field of investigation. Although many genetic events promote metastasis, several genes show relatively reduced expression levels in metastatic tumor cells in mouse model systems and in aggressive human tumors. Re-expression of a metastasis-suppressor gene in a metastatic tumor cell line results in a significant reduction in metastatic behavior in vivo with no effect on tumorigenicity. The known metastasis-suppressor gene products nm23, KAI1, mitogen-activated protein kinase kinase 4, breast cancer metastasis suppressor-1, KiSS1, RHOGDI2, CRSP3, and vitamin D3-upregulated protein/thioredoxin interacting protein exhibit unexpected biochemical functions that have shed new light on signaling events that are important in metastasis. Most metastasis suppressors function at the translationally important stage of outgrowth of micrometastatic tumor cells at a distant site. We hypothesize that elevation of metastasis suppressor gene expression in micrometastatic tumor cells in the adjuvant high-risk population of patients with breast cancer will halt metastatic colonization and have a clinical benefit. DNA methylation inhibitors have shown limited promise in increasing metastasis-suppressor gene expression, and ligands of the nuclear hormone receptor family are currently under investigation in vitro and in vivo. Clinical testing of agents that increase metastasis-suppressor gene expression is expected to require tailored trial designs.     

Increased actin cable organization after single chromosome introduction: Association with suppression of in vitro cell growth rather than tumorigenic suppression

We previously showed that introduction of a single human chromosome 1, 6, or 9 derived from normal fibroblasts into HHUA endometrial carcinoma cells resulted in suppression of tumorigenicity. The tumorigenic suppression was accompanied by remarkable morphological changes in the microcell hybrids containing an extra copy of chromosome 1. The study presented here was undertaken to search for target cytoskeletal components affected by chromosome 1 transfer into endometrial carcinoma cells. We found that the microcell hybrids containing an extra copy of chromosome 1 were characterized by intracellular actin bundle formation and an excessive accumulation of actin and vinculin. The latter was a result of increased stabilization of the proteins. Additionally, chromosome 3 introduction into RCC23 human renal carcinoma cells resulted in prolongation of cell division and in senescence of a significant proportion of the microcell hybrids. In these microcell hybrids, the intracellular actin network was also reorganized, but the amounts of actin and vinculin protein were not increased. These findings suggest that the increased actin organization, which appeared not to cause tumorigenic suppression in the microcell hybrids, is associated with complementation of tumor suppressor genes and senescence by multiple mechanisms. © 1994 Wiley-Liss, Inc.     

Downregulation of osteopontin contributes to metastasis suppression by breast cancer metastasis suppressor 1

Breast cancer metastasis suppressor 1 (BRMS1) inhibits the ability of multiple human and murine cancer cell lines to metastasize to lymph nodes, bones and lungs. Comparison of mRNA expression in metastatic MDA-MB-435 human carcinoma cells (435) and metastasis-suppressed BRMS1 transfectants (435/BRMS1) showed a marked (>90%) reduction of osteopontin (OPN) mRNA and protein expression in BRMS1-overexpressing cells. OPN expression is associated with disease progression in patients, with higher levels of OPN produced by cancer cells associated with poorer patient survival. Furthermore, OPN has been suggested to promote survival of cancer cells in response to stress, although the mechanisms by which this may occur remain poorly understood. This study tested the hypothesis that re-expression of OPN in metastasis-suppressed 435/BRMS1 cells would reverse metastasis suppression and confer protection from stress-induced apoptosis. A stable pooled population of OPN overexpressing 435/BRMS1 cells was created (435/BRMS1/OPN). OPN re-expression did not affect in vitro cell growth rates; however, increased anchorage independent growth/survival and protection from hypoxia-induced apoptosis was observed (p < 0.05). In vivo, OPN re-expression in BRMS1 transfected cells did not affect in vivo primary tumor growth but did increase the incidence of spontaneous metastasis to lymph nodes and lungs in mice. These novel findings suggest that OPN downregulation by BRMS1 may be responsible, at least in part, for BRMS1-mediated metastasis suppression by sensitizing cancer cells to stress induced apoptosis. These studies clarify one mechanism by which BRMS1 can suppress metastasis.     

Multiple chromosomes carrying tumor suppressor activity for a uterine endometrial carcinoma cell line identified by microcell-mediated chromosome transfer

Putative tumor suppressor genes can be mapped to specific chromosomes by the introduction of individual chromosomes derived from normal cells via microcell fusion. We have examined whether a highly malignant human uterine endometrial carcinoma cell line, HHUA, can be suppressed by only one normal chromosome or by multiple chromosomes. A library of mouse A9 clones containing different human chromosomes tagged with the pSV2-neo plasmid DNA were constructed. Transfer by microcell fusion of either chromosome 1, 6, 9, 11, or 19 into the HHUA tumor cell line was performed, and the abilities of the microcell hybrids to form tumors in nude mice were examined. The introduction of a chromosome 19 had no effect on the tumorigenicity of the cells, whereas microcell-hybrid clones with an introduced chromosome 1, 6 or 9 were completely suppressed for tumorigenicity. A decrease in tumor-take incidence in some but not all clones was observed following the introduction of a chromosome 11. The nontumorigenic microcell hybrids with an introduced chromosome 1 differed from the nontumorigenic microcell hybrids with an introduced chromosome 6, 9, or 11. A large percentage of hybrids with chromosome 1 senesced and/or showed alterations in cellular morphology and transformed growth properties in vitro. No growth or morphology alterations were observed following transfer of the other chromosomes. These results may indicate that more than one chromosome carries a tumor suppressor gene(s) for this human uterine endometrial carcinoma cell line and support the hypothesis that multiple tumor suppressor genes control the tumorigenic phenotype in the multistep process of neoplastic development.     

Suppression of malignant growth of human breast cancer cells by ectopic expression of integrin-linked kinase

Allelic loss at the short arm of chromosome 11 is one of the most common and potent events in the progression and metastasis of breast cancer. Here, we present evidence that the Integrin-Linked Kinase (ILK) gene maps to the commonly deleted chromosome 11p15.5 and suppresses malignant growth of human breast cancer cells both in vitro and in vivo. ILK is expressed in normal breast tissue but is downregulated in metastatic breast cancer cell lines and in advanced breast cancers. Transfection of wild-type ILK into the MDA-MB-435 mammary carcinoma cells potently suppressed their growth and invasiveness in vitro and reduced the cells' ability to induce tumors and metastasize in athymic nude mice. Conversely, expression of the ankyrin repeat or catalytic domain mutants of ILK failed to suppress the growth of these cells. Growth suppression by ILK is not due to apoptosis but is mediated by its ability to block cell-cycle progression in the G1 phase and by modulating the levels of integrins. These findings directly demonstrate that ILK deficiency facilitates neoplastic growth and invasion and suggest a novel role for the ILK gene in the suppression of tumor metastasis.     

Localization of metastasis suppressor gene(s) for prostatic cancer to the short arm of human chromosome 11.

Previous studies using somatic cell hybridization of highly metastatic and nonmetastatic rat prostatic cancer cells demonstrated that the resultant hybrids were nonmetastatic if all of the parental chromosomes were retained. Somatic hybrid segregants which underwent nonrandom chromosomal losses reexpressed high metastatic ability. These results demonstrated that there are gene(s) the expression of which can suppress metastatic ability of prostatic cancer cells. To identify the location of homologous gene(s) in the human, specific human chromosomes were introduced into highly metastatic rat prostatic cancer cells using the microcell-mediated chromosome transfer. Introduction of human chromosome 11 into highly metastatic rat prostate cancer cells results in suppression of metastatic ability without suppression of the in vivo growth rate or tumorigenicity of the hybrid cells. Spontaneous deletion of portions of human chromosome 11 in some of the clones delineated the minimal portion of human chromosome 11 capable of suppressing prostatic cancer metastases as the region between 11p11.2-13 but not including the Wilms' tumor-1 locus.     

Osteopontin can act as an effector for a germline mutation of BRCA1 in malignant transformation of breast cancer‐related cells

Breast cancer‐associated 1 (BRCA1) plays an important role in breast cancer initiation and progression through its functions in the cell cycle and DNA repair processes; however, its role in metastatic development in human breast cancer is still poorly understood. We have previously shown that osteopontin (OPN) expression was suppressed by wild‐type BRCA1 (Wt.BRCA1) and that a natural mutant allele of BRCA1 (Mut.BRCA1) diminished the effect of Wt.BRCA1 on OPN in vitro. In this study, we show that while Wt.BRCA1 suppresses OPN‐induced metastasis in a rat syngeneic system, Mut.BRCA1 enhances the development of metastasis through OPN, suggesting that OPN and BRCA1 work closely to regulate metastatic development in the rat. To test whether these findings are relevant to human breast cancer, we have investigated the relationship between BRCA1, OPN, and metastatic properties in human breast cancer‐related cells. Using western blot analysis, we show that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN protein expression; and in parallel that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN‐mediated in vitro properties associated with the metastatic state in both MCF‐7 and MDA MB435s cells. Overall, these results suggest that Mut.BRCA1 can elicit some of the changes involved in metastatic progression in human breast cancer via the overexpression of OPN. (Cancer Sci 2010)     

Expression of the nm23-2/NDP kinase alpha gene in rat mammary and oral carcinoma cells of varying metastatic potential.

Reduced expression of the putative metastasis-suppressor gene, nm23-1, has previously been correlated with high tumour metastatic potential. The involvement of the related and proximally-located nm23-2 gene in the suppression of tumour metastasis, however, has not yet been tested. In this study, we compared nm23-2 RNA levels in cell lines derived from three independent rat mammary carcinomas. Northern blot analysis revealed no correlation between nm23-2 RNA levels and metastatic potential in parent or clonal cell lines derived from chemically-induced (MAT 13762, DMBA-8) or spontaneous (BC1) rat mammary carcinomas. Cloning and sequencing of an nm23-2 cDNA from metastatic BC1 cells demonstrated that the predicted coding sequence of nm23-2 RNA in these cells was not inactivated by mutation. Further analysis showed that the nm23-2 gene was not down-regulated in H-ras-transfected metastatic clones or other metastatic cell lines derived from a spontaneous rat paraoral squamous cell carcinoma, B10. The data do not suggest a correlation between nm23-2 gene expression and metastasis-suppression in these tumours.     

Mapping a novel cellular-senescence gene to human chromosome 2q37 by irradiation microcell-mediated chromosome transfer

To identify the subchromosomal region that carries the cellular-senescence–restoring program of the human cervical carcinoma cell line SiHa, we constructed by irradiation microcell-mediated chromosome transfer a library of mouse A9 cells containing various fragments of human chromosome 2 tagged with pSV2neo in 2p11-p12. Eighty-seven clones were isolated and screened for the presence of human sequences by inter-Alu and inter-L1 polymerase chain reaction (PCR), and six clones exhibiting PCR-laddering patterns that differed from those of the A9 cells containing an intact chromosome 2 were examined further. Chromosome analysis and fluorescence in situ hybridization (FISH) using human-specific repetitive sequences revealed that four of these clones contained single subchromosomal transferable fragments (STFs). Southern blot hybridization of 14 cosmid markers revealed that the STFs in A9 cells were derived from human chromosome 2. These STFs were transferred into SiHa cells by microcell fusion, and one of the STFs restored the cellular-senescence program. The concordance of the cellular-senescence–restoring program with the presence or absence of specific DNA fragments of chromosome 2 indicated that the putative cellular-senescence gene was located in 2q32-qter. For more detailed mapping, we constructed mouse A9 cells containing STFs derived from human chromosome 2 tagged with pSTneo at different regions in 2q31-qter. PCR-laddering and FISH analyses were used to identify six clones that contained different STFs. These STFs were transferred into SiHa cells, and one of the three clones that restored cellular senescence contained a small fragment of human chromosome 2. This STF was shown by PCR analysis using 14 human chromosome 2–specific primer pairs to be smaller than 12.2 cM and was mapped to the 2q37 region by FISH anaysis with inter-Alu PCR. β-Galactosidase activity, which is a biomarker of senescent cells, and telomerase activity similar to that found in parental SiHa cells were detected in SiHa microcell hybrids, suggesting that the putative cellular-senescence gene was not involved in a telomerase pathway but rather in an alternate pathway of cellular senescence. Mol. Carcinog. 22:34–45, 1998. © 1998 Wiley-Liss, Inc.     

Suppression of tumorigenicity of A549 lung adenocarcinoma cells by human chromosomes 3 and 11 introduced via microcell-mediated chromosome transfer

To map tumor suppressor genes for lung adenocarcinomas, we introduced normal human chromosomes 3, 7, and 11 into the A549 tumor cell line by microcell-mediated chromosome transfer to test which chromosomes had the ability to suppress tumorigenicity. These human chromosomes, which contain the neomycin gene as a selectable marker, were transferred into A549 lung adenocarcinoma cells at frequencies of 0.3–1.8 × 10^?6. Two microcell hybrid clones with an introduced chromosome 3, two with an introduced chromosome 7, and six with an introduced chromosome 11 were isolated and examined for their growth properties and tumorigenicity in nude mice. Whereas parental A549 cells formed tumors with an average latency of 68 d, both microcell hybrids with an introduced chromosome 3 failed to form tumors for over 360 d. Similar tumorigenicity results were obtained when the clones were implanted into denuded tracheas, a more orthotopic transplantation site. The two clones with an introduced chromosome 7 were still tumorigenic; they formed tumors within 100–123 d after injection and grew progressively, although the tumors grew slightly slower than the parental cells did. Among the six clones with an introduced chromosome 11, one clone was still highly tumorigenic but did not contain an extra copy of an intact introduced chromosome 11. Three clones with a single intact copy of introduced chromosome 11 formed tumors with latency periods significantly longer than those of the parental cells. Two clones had two copies of the introduced chromosome 11, and both failed to form tumors within 1 yr of injection. These results indicate that chromosomes 3 and 11 can suppress the tumorigenicity of A549 lung adenocarcinoma cells.     

Breast cancer metastatic potential: correlation with increased heterotypic gap junctional intercellular communication between breast cancer cells and osteoblastic cells

The breast cancer metastasis-suppressor gene BRMS1 is downregulated in metastatic breast cancer cells. Previous reports have shown restoration of gap junctional intercellular communication (GJIC) in the metastatic human breast carcinoma cell line MDA-MB-435 (435) transfected with BRMS1 cDNA. Metastasis, to a large extent in most breast cancers, occurs to bone. However, the reason for this preferential metastasis is not known. We explored cell-to-cell communication between 435 carcinoma cells and a human osteoblastic cell line, hFOB1.19, to determine whether carcinoma cells can form gap junctions with bone cells and to explore the role of these heterotypic gap junctions and the BRMS1 gene in breast cancer metastasis to bone. 435 cells displayed greater cell-to-cell communication with hFOB 1.19 cells than with themselves. Transfection of BRMS1 into 435 cells increased homotypic gap junctional communication but did not significantly affect heterotypic communication with hFOBs. However, heterotypic communication of BRMS1 transfectants with hFOB cells was reduced relative to homotypic communication. In contrast, parental 435 cells displayed greater heterotypic communication with hFOBs relative to homotypic communication. Our results suggest that there are differences in the relative homotypic and heterotypic GJIC of metastasis-capable and -suppressed cell lines.