Breast Carcinoma Metastasis Suppressor, BRMS1
Breast Carcinoma Metastasis Suppressor, BRMS1
By Rajeev S. Samant, PhD, Mohammed Jabed Seraj, MD, PhD and Danny R. Welch, PhD
Breast cancer is a leading cause of death among women worldwide. Mortality from breast carcinoma in the United States exceeded 40,000 in 1999.1 When breast carcinoma cells are confined to breast tissue, long-term survival rates are high. Cure rate and the quality of life drops significantly once tumor cells leave the primary site and colonize distant tissues (Stage IV, metastatic disease).2,3 Thus, effective treatment and/or prevention of metastatic disease is of great importance.
Control of the multi-step metastatic cascade involves an interplay between many genes. Metastasis-regulatory genes can be classified as metastasis-promoting and metastasis-suppressing. Metastasis-promoting genes drive the conversion of a non-metastatic tumor to a metastatic state. Metastasis-suppressing genes, although akin to tumor suppressors, are distinct in that they block the spread of the tumor cells without affecting primary tumor formation. A tumor suppressor, on the other hand, also blocks metastasis since tumorigenicity is a prerequisite to metastasis.4 Interestingly, while metastasis requires coordinated expression of many genes, it takes only one gene to inhibit metastasis at any step of the cascade.3,4 To date, only six metastasis-suppressing genes (NME1, KiSS1, KAI1, CAD1, MKK4, and TIMPs) have been shown to suppress metastasis using in vivo models.3
Two general approaches were used to identify these metastasis-suppressing genes. The first involved comparison of gene expression in non-metastatic or poorly metastatic cells with genetically related metastasis-proficient cells. The second took clues from clinical observations that identified non-random chromosomal changes that occur during tumor progression. A recent cataloging of differential gene/protein expression and chromosomal abnormalities occurring during progression of breast carcinoma revealed that some karyotypic alterations typically occur (at loci 1p,1q, 3p, 6q, 7q, 11p, and 11q ) in the later stages of breast cancer.3 Among the most common changes (40-65% of cases) in both familial and sporadic breast carcinomas are alterations of chromosome 11q, particularly surrounding the region near 11q13. This observation captured our attention, leading to studies combining both strategies outlined above.
Discovery of BRMS1
We hypothesized that chromosome 11q encodes metastasis suppressor genes. To test this hypothesis in a gross way, we (in collaboration with Dr. Bernard Weissman) introduced a neomycin-tagged normal human chromosome 11 (neo11) into a highly metastatic MDA-MB-435 (435) cell line by microcell-mediated chromosome transfer. (See Figure.) Testing the chromosome 11 hybrid cell lines in athymic mice for metastasis from an orthotopic (mammary fat pad) site showed that chromosome 11 significantly suppressed the metastatic ability of 435 cell line without affecting tumorigenicity.5
To identify the gene(s) responsible for this phenotypic change, the gene expression profile in metastasis competent 435 cells was compared to that of metastasis-suppressed (neo11/435) variants using differential display.6 Out of 64 differentially expressed mRNAs, six candidate transcripts with at least five-fold greater expression in neo11/435 were identified and differential expression confirmed by RNA blotting. Three of the six were novel transcripts with no significant homology in the combined molecular databases. Human tissue expression profile (breast tissue was not available on the commercially available blots) of the three candidates showed high level in kidney tissue, with unique transcript sizes of 1, 1.2, and 1.5 kb. A full-length clone corresponding to the 1.5 kb transcript was obtained by screening a human kidney cDNA library. This novel cDNA was termed the Breast Metastasis Suppressor 1 (BRMS1).7
BRMS1 cDNA encompassed an open reading frame of 741 bp with a predicted polypeptide of 246 amino acids (Mr ~28,500). Sequence homology searches using various search engines revealed potential nuclear localization sequence, coiled-coil domain, but BRMS1 did not appear to be a part of any known major protein families. BRMS1 cDNA was used to screen bacterial artificial chromosome libraries at Genome Systems Inc. (St. Louis, MO) to obtain a genomic clone of BRMS1. The genomic sequence analysis revealed that BRMS1 is organized into 10 exons spanning ~10 kb (unpublished data, 2000). Fluorescence in situ hybridization analysis mapped BRMS1 gene at human chromosome 11q13.1-q13.2, a region frequently implicated in the later stages of breast carcinoma.3,8
BRMS1 Suppresses Metastasis
To assess the effect of BRMS1 on breast carcinoma metastasis, BRMS1 (under the control of a constitutive promoter) was transfected into two independently derived metastatic human breast carcinoma cell lines, MDA-MB-435 and MDA-MB-231.9 Clones expressing low, medium, or high levels of BRMS1 were isolated and tested for tumorigenicity and metastasis in athymic mice. MDA-MB-435 shows metastasis to lungs and regional lymph nodes following primary tumor formation at the mammary fat pad. This assay most closely mimics the situation in patients (i.e., all steps of the metastatic cascade must be completed). MDA-MB-231, on the other hand, forms a primary tumor, but does not metastasize from this site. However, it does form lung metastasis when injected intravenously into the lateral tail vein. In both cell lines, BRMS1 expression suppressed metastasis significantly. As expected, BRMS1 did not influence the growth of primary tumor.
How Does BRMS1 Suppress Metastasis?
The mechanism by which BRMS1 suppresses meta-statis is currently being investigated with respect to various steps in the metastatic cascade. In vitro assays examining individual steps predict a complex role for this molecule. It apparently blocks metastasis downstream of local invasion since invasive cords are present in histologic sections of primary tumor. Likewise matrix metalloproteinase (MMP-2 and MMP-9) expression (RNA blots) and activity (zymography) are practically unaltered. Blockage at steps subsequent to intravasation also is supported by data with 231 cells injected intravenously (unpublished data, 2000). There is a modest (30-60%) reduction in mobility as measured by in vitro wound healing assays (unpublished data, 2000).
Another potentially novel mechanism of action is restoration of homotypic gap-junction mediating intercellular communication (GJIC). Gap junctions are channels that allow passage of small molecules (< 1 kDa). It is observed that GJIC is diminished or absent in many neoplastic cell lines and primary tumors.10 Moreover, GJIC loss tends to correlate inversely with progression in neoplastic mammary tissue.11 When doubly fluorescence-labeled (10 mM 1-1’-dioctadecyl-3,3,3’3’-tetra-methylindocarboncyanine [diI] and 10 mM calcein AM) cells (435, 231, or their respective BRMS1 transfectants) were dropped onto a confluent monolayer of unlabeled acceptor cells, parental cells (435 and 231) did not exhibit capacity to transfer calcein. (Note: diI does not transfer. It is included to unequivocally identify the parachuted cells.) However, BRMS1-transfected cell lines showed restored capacity to establish GJIC (~90 min), indicated by calcein transfer to unlabeled acceptor cells.
Conclusion
Although recently discovered, BRMS1 offers promise in clinical medicine. This novel gene suppresses metastasis of human cancer in an animal models. That it is encoded at a locus frequently altered in late-stage breast cancer, further supports a role in human disease. With a novel mechanism of action, BRMS1 may represent a new target for attacking breast cancer metastasis. (Dr. Samant is a Postdoctoral Fellow and Dr. Welch is an Associate Professor at the Jake Gittlen Cancer Research Institute, Pennsylvania State University College of Medicine in Hershey. Dr. Seraj is a Senior Research Associate in the Department of Urology at the University of Virginia in Charlottesville.)
Acknowledgement
The GJIC data were collected in collaboration with Henry J. Donahue, PhD, and Marnie M. Saunders, PhD, Department of Orthopedics and Rehabilitation at The Pennsylvania State University College of Medicine.
This research was supported by grant DAMD-96-1-6152 from the United States Army Medical Research and Materiel Command, with additional support from NIH grants CA-62168 and CA-87728, the National Foundation for Cancer Research, and the Jake Gittlen Memorial Golf Tournament.
References
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