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Breast cancer progresses toward increasingly malignant behavior in tumorigenic and metastatic stages. In the series of events in the metastatic stage, tumor cells leave the primary tumor in breast and travel to distant sites where they establish secondary tumors, or metastases. In this report, we demonstrate that cell-cell communication via gap junctions is restored in the metastatic human breast carcinoma cell line MDA-MB-435 when it is transfected with breast metastasis suppressor 1 (BRMS1) cDNA. Furthermore, the expression profile of connexins (Cxs), the protein subunits of gap junctions, changes. Specifically, the expression of BRMS1 in MDA-MB-435 cells increases Cx43 expression and reduces Cx32 expression, resulting in a gap junction phenotype more similar to normal breast tissue. Taken together, these results suggest that gap junctional communication and the Cx expression profile may contribute to the metastatic potential of these breast cancer cells.

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.

Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).

Metastasis requires cytoskeletal remodeling for migration, adhesion, and extravasation of metastatic cells. Although protein kinase C (PKC) is involved in tumor promotion/progression and cytoskeletal remodeling, its role in metastasis has not been defined. PKCdelta levels are increased in highly metastatic 13762NF mammary tumor cells (MTLn3) compared with less metastatic, parental cell lines. To determine whether the increase in endogenous PKCdelta is functionally related to their increased metastatic potential, we prepared MTLn3 cells that express the inhibitory regulatory domain fragment of PKCdelta (RDdelta) under the control of a tetracycline-inducible promoter. RDdelta expression attenuated endogenous PKCdelta activity, as demonstrated by decreased phosphorylation of the PKCdelta substrate adducin in migrating cells. Thus, in MT cells, RDdelta appears to primarily influence cytoskeleton-dependent processes rather than cell cycle progression. To determine whether RDdelta expression influenced metastatic potential in vivo, MTLn3/RDdelta cells were either grown in the mammary fat pad or injected into the tail vein of syngeneic rats, and effects of doxycycline-induced RDdelta expression on pulmonary metastases were studied. Consistent with the in vitro data, induction of RDdelta significantly reduced the number of lung metastases without affecting growth of the primary tumor. These results suggest that interfering with endogenous PKCdelta activity by expressing the inhibitory RDdelta fragment inhibits cytoskeleton-regulated processes important for MTLn3 cell metastasis.

Based on the observation that chromosome 1q deletions are not infrequent in late-stage human breast carcinomas, we tested whether the recently discovered human melanoma metastasis suppressor gene, KiSS-1, which maps to chromosome 1q32-q41, could suppress metastasis of the human breast carcinoma cell line MDA-MB-435. Parental, vector-only transfectants and KiSS-1 transfectant clones were injected into the mammary fat pads of athymic nude mice and assessed for tumor growth and spontaneous metastasis to regional lymph nodes and lungs. Expression of KiSS-1 reduced metastatic potential by 95% compared to control cells but did not suppress tumorigenicity. Metastasis suppression correlated with a decreased clonogenicity in soft (0.3%) and hard (0.9%) agar. Although the overall rate of cell adhesion to extracellular matrix components was unaffected, KiSS-1 transfectants spread on immobilized type-IV collagen more rapidly than did control populations. Invasion and motility were unaffected by KiSS-1. Based on the predicted structure of the KiSS-1 protein, our results imply a mechanism whereby KiSS-1 regulates events downstream of cell-matrix adhesion, perhaps involving cytoskeletal reorganization. In addition to its already described role in melanoma, our results show that KiSS-1 also functions as a metastasis suppressor gene in at least some human breast cancers.

To determine the relevance of genetic information on chromosome 11 in the development of metastatic breast tumors, we introduced a normal human chromosome 11 into the highly metastatic MDA-MB-435 breast carcinoma cell line via the microcell-mediated chromosome transfer technique. Although the MDA-MB-435 recipient cell line and four randomly selected microcell hybrid clones remained tumorigenic in nude mice, the hybrids were95% suppressed for metastasis to lung and regional lymph nodes (p0.01). We also tested whether chromosome 6 harbors a metastasis-suppressor gene for breast cancer as observed previously for human melanoma. Grouped together, the four neo6 microcell hybrids had no statistically significant reduction in the incidence or number of lung or lymph node metastases compared to the weakly metastatic, subcloned parent cell line, MDA-MB-453.7. Expression of nm23-H1 (NME1), a known metastasis-suppressor gene in this breast cancer cell line, did not correlate with metastasis suppression in the microcell hybrids. These results further demonstrate that control of metastasis is molecularly distinct from tumorigenic potential. They also indicate that chromosome 11 encodes a metastasis-suppressor gene for human breast cancer.

The expression of metalloproteinases, such as type IV collagenase/gelatinase, enables tumor cells to degrade type IV collagen present in the basement membrane and correlates with metastatic potential of several tumor types. We found that increased levels of rat serum type IV collagenolytic activity are associated with increased 13762NF mammary adenocarcinoma metastases in lungs and lymph nodes of syngeneic rats. To investigate serum metalloproteinases responsible for type IV collagenolysis, we performed zymography and Western blot analysis of rat sera. A M(r) 92,000 progelatinase (progelatinase B, M(r) 92,000 type IV procollagenase, MMP-9) was detected on zymograms of rat sera within 16 days after intramammary fat pad inoculation of highly metastatic MTLn3 cells. The activated serum M(r) 92,000 progelatinase degraded sodium dodecyl sulfate-denatured type I and IV collagens but was not active on casein, albumin, hemoglobin, and immunoglobulin. Sera from rats with fat pad tumors and lung metastases formed from highly metastatic clones possessed greater than 7 times higher levels of serum M(r) 92,000 progelatinase than control sera or sera from rats bearing similar size fat-pad tumors of low or nonmetastatic clones. The results were confirmed by Western blot analysis of rat sera using rabbit anti-human M(r) 92,000 progelatinase antibodies. Similar results were obtained from the analysis of rat plasma samples. The serum and plasma M(r) 92,000 progelatinase levels correlated with the extent of metastases in the lung and lymph nodes. The results indicate that high levels of serum and plasma M(r) 92,000 progelatinase are associated with the presence of disseminated metastatic adenocarcinoma cells and suggest that this enzyme may facilitate the invasion of blood-borne tumor cells through vascular basement membranes.

Using rat 13762NF mammary tumor cell clones of varying spontaneous metastatic potentials and biochemical properties and known phenotypic stabilities we studied the relationship between cell colony growth in a clonogenic assay and the biological and biochemical properties of cells derived from different cell colonies. The spontaneous metastatic potential of in vivo or in vitro grown 13762NF tumor cells was not related to their in vitro cloning efficiencies; cells of both low and high metastatic potential formed colonies of various sizes and shapes during 14 days of growth in 0.3% or 0.6% semisolid agarose. A highly metastatic cell clone of relatively low growth potential in agarose was examined further. Individual tumor cell colonies derived from this cell clone were removed from agarose and their properties determined. Cells from small (less than 100-microns-diameter) or large (greater than 500-microns-diameter) agarose colonies had similar self-renewal capacities in agarose and formed variously sized cell colonies when replated in agarose medium. Metastatic potential, drug sensitivity parameters, and expression of a high Mr mucin-like glycoprotein antigen and type IV collagenolytic activity known to be associated with spontaneous metastasis of 13762NF tumor cells were dissimilar in cells from different colonies, and these characteristics were independent of original tumor cell colony size in agarose. In contrast, the expression of cell surface proteins of Mr less than 300,000 were similar among cells derived from different agarose colonies. The data indicate that heterogeneity exists in the ability of 13762NF adenocarcinoma cells of different biochemical and metastatic potentials and drug sensitivities to grow in semisolid agarose. In addition, the cells that grow in agarose to form detectable colonies (greater than 50 cells) are not necessarily those with a high potential of metastasizing spontaneously to distant sites.

Local recurrence occurs in 4-47% of breast cancer patients and is often associated with development of metastatic foci and resistant cell populations. Thus, recurrent breast cancer indicates a poor prognosis for the patient. Local tumor-derived 13762NF rat mammary adenocarcinoma cell clone MTF7(T20) was injected into the inguinal mammary fat pad and allowed to grow before surgical excision. Individual locally growing (primary) tumors were removed and established in short-term tissue culture. Corresponding local recurrences were excised after regrowth and established in short-term tissue culture. All sublines were tested for in vitro sensitivities to 5-fluoro-2'-deoxyuridine, Adriamycin, and ionizing X-irradiation. Using a clonogenic colony formation assay, responses of individual sublines ranged from 85 to 1500 ng/ml for Adriamycin and 65 to 10,000 nM for FdUrd. Some recurrences were significantly more resistant while others were more sensitive than the corresponding primary tumor lines. All recurrences had smaller 90% lethal dose values than the corresponding parent or primary tumor in response to Adriamycin; whereas, to 5-fluoro-2'-deoxyuridine, 90% lethal dose values revealed that most lines were quite resistant. Statistically significant differences in radiation survival were observed only for lines LR1a and LR5 (more sensitive). There was no apparent correlation between sensitivities to chemotherapy agents or X-irradiation and experimental metastatic potential in LR sublines. These dose-response data indicate that locally recurrent tumors are frequently, but not always, different from the original primary tumor in response to chemotherapy agents and ionizing X-irradiation. Although an exact mechanism is unknown, it is likely that "selective" pressures which eliminate large numbers of cells, in this case surgery, change tumor composition so that recurrent tumors may no longer be equivalent to the tumor mass that was originally excised. This suggests that treatment strategies should be planned accordingly.

A series of rat 13762NF mammary adenocarcinoma cell clones and subclones of various lung metastatic potentials were examined for their abilities to degrade rat lung subendothelial matrix and purified basement membrane type IV collagen. Metastatic potentials were simultaneously determined based on the ability to form "spontaneous" lung metastases after s.c. injection or "experimental" lung metastases after i.v. injection of cells. Microvessel endothelial cells isolated from rat lung were grown in vitro, and the subendothelial matrix containing type IV collagen was metabolically labeled with [3H]proline. When mammary adenocarcinoma cells were placed on the isolated subendothelial matrix, fragmentation and solubilization of [3H]proline-labeled components were observed; highly metastatic 13762NF cells solubilized the matrix at higher rates than did poorly metastatic cells. The 13762NF cells were assayed for type IV collagenolytic activity using [3H]proline-labeled type IV collagen purified from Engelbreth-Holm-Swarm tumor as a substrate. We found excellent correlation between the type IV collagenolytic activities of living cells and their "spontaneous" lung metastatic potentials (r = 0.993). The levels of type IV collagenolytic activity in the conditioned medium depended on the cell culture conditions. In the presence or absence of acid-treated fetal bovine serum, highly metastatic cells secreted higher amounts of type IV collagenolytic enzymes in active and latent forms than did poorly metastatic cells. Incubation of procollagen type IV with medium conditioned by highly metastatic 13762NF cells and treated with trypsin resulted in the production of several large fragments characteristic of type IV collagen. The results suggest that enzymatic degradation of basement membrane type IV collagen is important in lung metastasis of 13762NF mammary adenocarcinoma cells.