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Repression of the Putative Tumor |
1. INTRODUCTION
During the multistage process of mammary carcinogenesis, stepwise accumulation of genetic changes causes uncontrolled growth, disruption of normal glandular architecture, and invasion of epithelial cells into the adjacent stroma. While unscheduled proliferation also occurs in benign adenomas, subversion of orderly epithelial tissue organization is a hallmark of malignancy and plays a crucial role in tumor progression. Interestingly, the inability of carcinoma cells to correctly polarize and to generate histotypic three-dimensional structures is reflected in in vitro assays of morphogenesis. Thus, unlike cells derived from normal mammary epithelium, breast carcinoma cells are unable to form duct-like or alveolar- like structures when grown in collagen or basement membrane gels. Overexpression of normal or mutated proto-oncogenes or inactivation of tumor suppressor genes has been shown to result in mammary carcinogenesis and/or transformation of mammary epithelial cells in vitro. However, the potential role of oncogenes and tumor suppressor genes in the control of epithelial architecture has only recently begun to be elucidated. To address this question, we have investigated whether repression of the putative tumor suppressor gene Bard1 or expression of the Notch4 (int-3) oncogene in non-tumorigenic mammary epithelial cells affects their in vitro morphogenetic properties. For this purpose, we utilized TAC-2 clonal mammary epithelial cells, which when grown in three-dimensional collagen gels, have the ability to form branching tubules in response to HGF and alveolar-like cysts in response to hydrocortisone. The studies summarized below indicate that both repression of Bard1 and expression of Notch4(int-3) disrupt cyst morphogenesis and induce an invasive phenotype in TAC-2 mammary epithelial cells. 2. REPRESSION OF BARD1 (BRCA1-ASSOCIATED RING DOMAIN) GENE IN TAC-2 CELLS
BARD1 is a protein that interacts with the product of the breast cancer susceptibility gene BRCA1. The identification of BARD1 mutations in human breast carcinomas has suggested that this protein participate in BRCA1-mediated tumor suppression. However, whether BARD1 plays a role in mammary gland morphogenesis is not known. To address this question, we reduced levels of Bardl expression in TAC-2 mammary epithelial cells. By transfecting TAC-2 cells with an antisense sequence of murine Bard1, we obtained a partial reduction of Bard1 mRNA and protein expression levels. This resulted in marked phenotypic changes consisting of increased cell size and high frequency of multinucleated cells, when compared to mock-transfected TAC-2 cells.
Since disruption of normal glandular architecture is an early step in the process of mammary tumorigenesis, it was relevant to assess whether Bard1 repression would affect the morphogenetic properties of TAC-2 cells. When grown in collagen gels under control conditions, mock-transfected TAC-2 cells gave rise to small colonies with a morphology ranging from irregular cell aggregates to poorly branched structures.
Fig. 1. Repression of Bard1 abrogates hydrocortisone-induced lumen formation by TAC-2 cells. Cells suspended in collagen gels were incubated with 1 µg/ml hydrocortisone and 10 µg/ml insulin for 8 d. Under these conditions, mock-transfected cells form alveolar-like cystic structures containing a wide lumen (A) delimited by a palisade of cubic epithelial cells (D). In marked contrast, Bard1 repressed cells form solid ball-like structures (B) devoid of lumen (E), or irregular aggregates (C) containing small focal lumina (F). A-C, Bright-field microscopy; D-F, semi thin sections. Bars, 100 µm. [Reproduced from the J. of Cell Biol. 143, 1329-1339, by copyright permission of the Rockefeller University Press.] Addition of HGF to the cultures induced the formation of highly arborized branching cords, as previously observed with non-transfected TAC-2 cells. Addition of HGF to collagen gel cultures of Bard1 repressed cells induced branching tubulogenesis to a similar extent as in mock- transfected cells. These findings suggest that Bard1 does not play a major role in the elongation and branching of duct-like structures by TAC-2 cells. We next analyzed whether Bard1 repression could influence lumen formation by TAC-2 cells. When grown in collagen gels in the presence of hydrocortisone, mock-transfected cells formed spheroidal cysts enclosing a widely patent lumen (Fig. 1A) delimited by a palisade of cubic epithelial cells (Fig. 1D), as previously observed with non-transfected TAC-2 cells. In contrast, under the same experimental conditions, two different clones of Bard1 repressed cells formed either solid aggregates devoid of lumen (Fig. 1 B,E) or irregularly shaped cell aggregates occasionally containing small focal lumina (Fig. 1C,F). A quantitative analysis demonstrated a marked inhibition of lumen formation in Bard1 repressed cells, as evidenced by a significant 6-fold and 8-fold (p= 0.001) decrease in the mean percentage of cysts, when compared to mock-transfected cells. Taken together, these results demonstrate that repression of Bard1 expression inhibits the formation of alveolar-like cystic structures by TAC-2 cells.
Since loss of contact-mediated inhibition of proliferation is a hallmark of malignant transformation, we analyzed the growth properties of mock-transfected and Bard1 repressed TAC-2 cells. Although both cell lines showed a similar proliferation rate in subconfluent cultures, they had a markedly different behavior when grown to post-confluence. Whereas postconfluent mock-transfected cells formed regular, contact-inhibited monolayer, Bardl repressed cells overlapped each other and continued to grow in a disorganized criss-cross pattern. This differential behavior was further enhanced in the presence of exogenous growth factors. Thus, addition of either epidermal growth factor (EGF) or insulin-like growth factor-I (IGF-I) did not induce multilayering in mock-transfected cells, but resulted in a marked degree of stratification in Bard1 repressed cells (Fig. 2).
This indicates that Bard1 repressed cells have lost sensitivity to contact-inhibition of growth. However, Bard1 repressed cells lacked the ability to grow under anchorage-independent conditions, and were not tumorigenic in vivo. Altogether, our findings suggest that repression of Bard1 induces a partially transformed phenotype in TAC-2 cells, consisting of disruption of normal alveolar-like morphogenesis and loss of contact-inhibition of cell growth. It remains to be established whether complete inhibition of Bard1 expression could induce a fully malignant phenotype.
Fig. 2. Loss of contact-mediated inhibition of cell proliferation in Bard1- repressed cells. Cells were seeded in collagen-coated wells at saturating cell density and grown for 2 d, at which time the cultures were incubated in the absence or the presence of 20 ng/ml EGF for a further 5 d. In EGF-supplemented cultures, mock-transfected cells form a contact-inhibited cobblestone-like monolayer (a), whereas Bard1 repressed cells (b) grow in a disordered crisscross pattern. (c and d) Thin sections perpendicular to the plane of cultures shown in a and b demonstrate the lack of stratification in mock-transfected cells and the obvious multilayering in Bard1 repressed cells. Bars: (a and b) 100 µm; (c and d) 5 µm. [Reproduced from the Journal of Cell Biology, 143, 1329-1339, by copyright permission of the Rockefeller University Press.]
3. EXPRESSION OF AN ACTIVATED NOTCH4 (INT-3) ONCOPROTEIN IN TAC-2 CELLS
Genes of the Notch family encode transmembrane receptor proteins that regulate cell differentiation in a variety of cell types in both vertebrates and invertebrates. Notch4 (int-3) is an oncogenic, truncated form of Notch4 which has most of its extracellular domain deleted and behaves as a constitutively activated receptor. Expression of Notch4(int-3) as a transgene in the mouse impairs mammary gland ductal morphogenesis and promotes the formation of undifferentiated carcinoma. To gain insight into the mechanisms that might be responsible for the tumorigenicity of this oncogene, we studied the biological consequences of Notch4(int-3) expression on the in vitro morphogenetic properties of TAC-2 cells. We found that retroviral expression of Notch4(int-3) in TAC-2 mammary epithelial cells impairs HGF-induced formation of branching tubules in collagen gels. Interestingly, the structures formed by Notch4(int-3) expressing cells (TAC-2 int-3 cells) in collagen gels were not only less branched than those generated by mock-transfected TAC-2 cells, but were also frequently devoid of a central lumen. These observations led us to investigate whether expression of Notch4(int-3), in addition to inhibiting branching tubulogenesis, might specifically interfere with the process of lumen formation. When grown in collagen gels in the presence of hydrocortisone, mock-transfected TAC-2 cells formed alveolar-like cystic structures containing a wide lumen (cf. Fig. 1A,D), as previously observed with wild type TAC-2 cells. In contrast, TAC-2 int-3 cells were virtually unable to form alveolar-like cystic colonies, and generated instead solid cell aggregates devoid of a central lumen. Thin section electron microscopy revealed that the wall of the cysts formed by mock-transfected cells consisted of closely apposed, well polarized epithelial cells. Intercellular junctions were located between the apical portions of adjacent cells, and microvilli were mostly restricted to the surface of the cell facing the lumen of the cyst (Fig. 3A). In marked contrast, the colonies formed by TAC-2 int-3 cells consisted of loosely associated epithelial cells lacking obvious cell polarity. The cells were separated by a wide intercellular space bridged by cytoplasmic processes establishing focal cell-cell contacts. Abundant polymorphic microvilli projected from the entire cell surface (except for regions of cell-collagen interactions), and no clearly defined apical pole could be identified (Fig. 3B). Taken together with the finding that TAC-2 int-3 cells fail to undergo branching tubulogenesis in response to HGF, these results demonstrate that activated Notch4(int-3) subverts the normal morphogenetic properties of TAC-2 cells. They also suggest that formation of disorganized cell aggregates by TAC-2 int-3 cells recapitulates the architectural destructuring observed in anaplastic carcinomas induced by transgenic expression of Notch4(int-3).
Fig. 3. Disruption of cyst morphogenesis and of apico-basal polarity by Notch4 (int-3). Thin sections of colonies formed in collagen gels by mock- and Notch4(int-3)-transfected TAC-2 cells incubated with hydrocortisone. (A) Wall of a cyst formed by mock-transfected cells. (B) Portion of a colony formed by TAC-2 int-3 cells. Bars, 5 µm. See text for details.
To determine whether expression of Notch4 (int-3) in TAC-2 cells induces additional changes that may be relevant to the process of mammary carcinogenesis, we evaluated the ability of TAC-2 cells to invade three-dimensional collagen gels. When grown on a collagen gel under control conditions, mock-transfected cells formed a cobblestone-like monolayer and remained exclusively confined to the surface of the gel. Addition of EGF to the cultures did not induce appreciable invasion (Fig. 4A,C). In contrast, TAC-2 int-3 cells invaded the underlying collagen matrix as thick branched cords. Invasion was enhanced by the addition of EGF to the cultures (Fig. 4B,D) and was suppressed in a dose-dependent manner by a synthetic inhibitor of matrix metalloproteinases. These results demonstrate that expression of Notch4(int-3) in TAC-2 cells is associated with the acquisition of invasive properties. 
Fig. 4. Expression of Notch4 (int-3) confers an invasive phenotype to TAC-2 cells. TAC-2 cells were seeded onto the surface of a collagen gel and grown for 6 days in the presence of 20 ng/ml EGF (A,B) or 20 ng/ml EGF and 10µg/ml insulin (C,D). Whereas mock-transfected cells (A,C) form a monolayer exclusively confined to the surface of the gel, TAC-2 int-3 cells (B,D) invade the underlying matrix as thick branched cords. (A,B) Phase-contrast microscopy (focus is 20µm beneath the surface of the gel); bars, 150 µm. (C,D) Semi-thin sections perpendicular to the surface of the gel; bars, 100 µm.
We next investigated whether Notch4 (int-3) expression may alter contact-inhibition of cell proliferation. We found that whereas mock-transfected cells form cobblestone-like, contact-inhibited monolayers, TAC-2 int-3 cells grow in a disorganized criss-cross pattern and overlap each other in post-confluent cultures. These findings suggest that Notch4(int-3) blunt the responsiveness of TAC-2 cells to the normal regulatory mechanisms responsible for contact-inhibition of proliferation, thereby maintaining them in a hyperproliferative state. However, we found that TAC-2 int-3 cells are unable to form colonies when grown under anchorage-independent conditions. Thus, while exhibiting some properties of transformed cells (i.e., disorganized growth in collagen gels and cell multilayering in two-dimensional cultures), TAC-2 int-3 cells are still dependent on cell-substratum adhesion for proliferation.
Taken together, our results demonstrate that expression of Notch4 (int-3) in TAC-2 cells disrupts their ability to form organized lumen-containing structures, confers upon them an invasive phenotype, and suppresses contact-inhibition of cell proliferation. These alterations may represent important contributing factors in the development of anaplastic carcinomas observed in Notch4(int-3) transgenic mice.
4. CONCLUSION The studies we have summarized above suggest that subversion of the normal morphogenetic properties of mammary epithelial cells represent an important mechanism by which loss of tumor suppressors or expression of oncogenes promotes malignant progression of epithelial tumors.
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Repression