A novel function for the nm23-H1 gene: overexpression in human breast carcinoma cells leads to the formation of basement membrane and growth arrest

Abstract

Background: We have developed a culture system using reconstituted basement membrane components in which normal human mammary epithelial cells exhibit several aspects of the development and differentiation process, including formation of acinar-like structures, production and basal deposition of basement membrane components, and production and apical secretion of sialomucins. Cell lines and cultures from human breast carcinomas failed to recapitulate this process. The data indicate the importance of cellular interactions with the basement membrane in the regulation of normal breast differentiation and, potentially, its loss in neoplasia.

Purpose: Our purpose was to use this assay to investigate the role of the putative metastasis suppressor gene nm23-H1 in mammary development and differentiation.

Methods: The metastatic human breast carcinoma cell line MDA-MB-435, clones transfected with a control pCMVBamneo vector, and clones transfected with pCMVBamneo vector containing nm23-H1 complementary DNA (the latter of which exhibited a substantial reduction in spontaneous metastatic potential in vivo) were cultured within a reconstituted basement membrane. Clones were examined for formation of acinus-like spheres, deposition of basement membrane components, production of sialomucin, polarization, and growth arrest.

Results: In contrast to the parental cell line and control transfectants, MDA-MB-435 breast carcinoma cells overexpressing Nm23-H1 protein regained several aspects of the normal phenotype within reconstituted basement membrane. Nm23-H1 protein-positive cells formed organized acinus-like spheres, deposited the basement membrane components type IV collagen and, to some extent, laminin to the outside of the spheres, expressed sialomucin, and growth arrested. Growth arrest of Nm23-H1 protein-positive cells was preceded by and correlated with formation of a basement membrane, suggesting a causal relationship.

Conclusion: The data indicate a previously unidentified cause-and-effect relationship between nm23-H1 gene expression and morphological-biosynthetic-growth aspects of breast differentiation in this model system.

Implications: While the basement membrane microenvironment is capable of directing the differentiation of normal human breast cells, neoplastic transformation abrogates this relationship, suggesting that intrinsic cellular events are also critical to this process. The data identify nm23-H1 gene expression as one of these events, suggesting an important role in the modulation of cellular responsiveness to the microenvironment. The data also identify previously unknown growth inhibitory effects of nm23-H1 gene overexpression.

Fig. 1

Immunohistochemical staining of Nm23 protein to show morphology and Nm23 protein expression in 12-day cultures of MDA-MB-435 cells in monolayer (A, C, and E) and in EHS matrix (B, D, and F). Panels A and B show nm23-H1 gene-transfected clone H1-177; Panels C and D show control-transfected clone C-100; panels E and F show untransfected parent MDA-MB-435 cells. Note the intense cytoplasmic staining for exogenous Nm23 protein in H1-177 cells (A and B) and the capacity of these cells to form spheres in EHS (B). Panels A, C,and E (original magnification ×440; panels B, D, and F original magnification ×550).

Fig. 2

Immunohistochemical staining of type IV collagen and laminin expressed by nm23-H1 gene-transfected MDA-MB-435 clone H1-177 cells (A) and normal HMT-3522 breast epithelial cells (B). Arrows show localization of type IV collagen at the basal surface of spheres formed by nm23 gene-transfected cells and normal breast cells. Insets show similar localization but less intense staining of laminin for H1-177 cells (inset of panel A) and for reference HMT-3522 cells (inset of panel B). Note the absence of collagen IV deposition by the untransfected parental MDA-MB-435 cells (C) and control transfectants clone C-100 (D) (original magnification ×400; inset original magnification ×320).

Fig. 3

Immunohistochemical localization of sialomucin (arrows) expressed by MDA-MB-435 clone H1-177 cells (A), normal control HMT-3522 cells (B), control MDA-MB-435 clone C-100 cells (C), and control untransfected parental MDA-MB-435 cells (D). Note the apical and lateral accumulation of sialomucin by normal HMT-3522 spheres (B) and the nonpolar expression of sialomucin by the MDA-MB-435 cells (A, C, and D) (original magnification ×400).