EpH4 Cells Research Articles

Oncogenic Ras/Her-2 mediate hyperproliferation of polarized epithelial cells in 3D cultures and rapid tumor growth via the PI3K pathway.

Carcinogenesis by oncogenic Ras and Her-2 involves enhanced proliferation of epithelial cells in vivo. However, hyperproliferation induced by these oncogenes, or their downstream pathways in vitro has mainly been studied in cultured, fibroblastic cell lines. Here, we demonstrate that oncogenic Ha-Ras or constitutively active Her-2 cause increased proliferation and cyclin D1 upregulation in fully polarized, mammary epithelial cells (EpH4), if cultivated as organotypic structures in three-dimensional collagen/matrigel matrices. Under standard culture conditions, however, these oncogenes failed to induce hyperproliferation. Using both specific low molecular weight inhibitors and Ras-effector-specific mutants, we dissected signaling pathways downstream of oncogenic Ras (PI3K, Mek1/MAPK) with respect to (i) hyperproliferation in collagen gels and tumorigenesis in mice and (ii) epithelial/mesenchymal transition (EMT). We show that the Ras-activated PI3K pathway is required to induce rapid tumor growth and enhanced proliferation of EpH4 cells in collagen gels, but fails to cause EMT in vitro and in vivo. On the other hand, Ras-dependent activation of the Mek1/MAPK pathway in EpH4 cells (previously shown to cause EMT and metastasis) did not induce hyperproliferation in collagen gels and caused only slow tumor growth. Our data thus indicate that Ras-dependent signaling through the PI3K- and MAPK pathways fulfil distinct, but complementary functions during carcinogenesis.

Inhibition of proliferation and expression of T1 and cyclin D1 genes by thyroid hormone in mammary epithelial cells.

The relationship between thyroid hormone (triiodothyronine, T(3)) and breast cancer is unclear. We studied the effect of the c-erbA/TR alpha proto-oncogene encoding a functional T(3) receptor (TR alpha 1), of its ligand T(3), and of its retroviral, mutated counterpart, the v-erbA oncogene, on the proliferation capacity of nontumorigenic mammary epithelial cells (EpH4). We found that EpH4 cells expressing ectopically TR (EpH4 + TR alpha 1) or v-erbA (EpH4 + v-erbA) proliferated faster than parental EpH4 cells that contained low levels of endogenous TR. T(3) inhibited DNA synthesis and proliferation in EpH4 + TR alpha 1 cells but not EpH4 or EpH4 + v-erbA cells. The study of cell-cycle genes showed that T(3) decreased cyclin D1 RNA and protein levels in EpH4 + TR alpha 1 cells. In addition, T(3) downregulated the expression of T1, a gene that is overexpressed in human breast adenocarcinomas and is induced by mitogens, serum, and several oncogenes and cytokines. Inhibition of the T1 gene by T(3) required both de novo mRNA and protein synthesis. Furthermore, T(3) abolished the induction of T1 by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and inhibited the activity of an activation protein 1-dependent promoter (-73-Col-CAT) in EpH4 + TR alpha 1 cells, suggesting that interference with activation protein 1 transcription factor plays a part in the inhibition of the T1 gene. Our results showed that T(3) reduced the proliferation of mammary epithelial cells and inhibited the expression of cyclin D1 and T1 genes.

Cripto-1 Enhances Migration and Branching Morphogenesis of Mouse Mammary Epithelial Cells

Cripto-1 is an EGF-CFC protein that performs an important role during early vertebrate development and is overexpressed in several types of human cancer. In the present study mouse EpH4, NMuMG, and TAC-2 mammary epithelial cells that are negative for endogenous cripto-1 expression were transfected with the murine cripto-1 cDNA. Cripto-1-transfected cell lines exhibited functional and physiological differences from the original cell lines including enhanced anchorage-independent growth in soft agar (EpH4 cells), growth in serum-free medium, increased proliferation, and formation of branching, duct-like structures when grown in a three-dimensional collagen type I matrix. Furthermore, cripto-1-expressing cell lines showed elevated migration in vitro in Boyden chamber and wound-healing assays. These results indicate that cripto-1 can function through an autocrine pathway that enables mammary epithelial cells to undergo an epithelial to mesenchymal transition.

Inhibition of tenascin-C expression in mammary epithelial cells by thyroid hormone.

Multiple data suggest a relationship between thyroid hormone (triiodothyronine (T3)) and carcinogenesis. Studies on breast cancer have been inconclusive, suggesting contradictory effects of thyroid status and diseases. Recently, we reported that expression of the extracellular matrix glycoprotein tenascin-C is modulated by T3 during rat brain development. Because tenascin-C has been reported to have growth-, motility-, and angiogenic-promoting activities and to become upregulated during tumorigenesis in breast carcinoma and stromal cells, we analyzed the effects of T3 on tenascin-C expression in mammary epithelial cells. In this study, we showed that tenascin-C RNA expression was inhibited by T3 in normal un-transformed EpH4 mouse mammary epithelial cells expressing appropriate receptors. T3's action appeared to be due to a decreased half-life of the tenascin-C mRNA, with a maximum effect (85% at 100 nM) 48 h after addition. T3 also downregulated tenascin-C in the human mammary tumor cell line SKBR-3, which expresses endogenous thyroid receptors. Immunoprecipitation experiments confirmed that tenascin-C protein content was also decreased by T3 in EpH4 cells (70% reduction at 100 nM). Dexamethasone had a similar inhibitory effect (70% at 100 nM), whereas estradiol, the antiestrogen ICI 164,384, progesterone, and all-trans retinoic acid did not alter tenascin-C expression. Our data demonstrate an inhibitory action of T3 on tenascin-C expression in mammary epithelial cells that may play a role in the physiological regulation of this gene and in neoplastic processes.

Isolation of EpH4 mammary epithelial cell subpopulations which differ in their morphogenetic properties.

EpH4 is a nontumorigenic cell line derived from spontaneously immortalized mouse mammary gland epithelial cells (Fialka et al., 1996). When grown in collagen gels, EpH4 cells give rise to different types of structures, e.g., solid cords or branching tubes. By removing and subsequently dissociating single three-dimensional colonies of defined morphology, we have isolated six clonal subpopulations of EpH4 cells which display distinct morphogenetic properties in collagen gel cultures. Thus, cells from the H1B clone form branching cords devoid of a central lumen, K3A3 cells form cords enclosing small multifocal lumina, and J3B1 cells form large cavitary structures containing a wide lumen. I3G2 cells form either cords or tubes, depending on the type of serum added to the culture medium. Finally, when grown in serum-free medium, Bela cells form spherical cysts, whereas Be4a cells form long, extensively branched tubes. In additional assays of morphogenesis, i.e., cell sandwiching between two collagen gels or culture on a thick layer of Matrigel (a laminin-rich extracellular matrix), all clones form epithelial-cell lined cavitary structures, except H1B cells which are unable to generate lumina under these conditions. The EpH4 sublines we have isolated provide an in vitro system for studying the mechanisms responsible for lumen formation and branching morphogenesis, as well as for identifying the factors which subvert these developmental processes during mammary carcinogenesis.

1,25-Dihydroxyvitamin D3 inhibits tenascin-C expression in mammary epithelial cells

Tenascin-C is an extracellular matrix protein with growth-, invasion- and angiogenesis-promoting activities. Tenascin-C is upregulated in breast carcinoma and stromal cells, and in many other cell types during tumorigenesis. We demonstrate that tenascin-C RNA expression is inhibited by 1,25-dihydroxyvitamin D3 (1,25-D3) in a variety of mouse and human mammary epithelial cell lines exhibiting normal or malignant phenotype. In EpH4 cells, the inhibition is maximum 24 h after 1,25-D3 treatment and correlates with a dose-dependent reduction in the synthesis of tenascin-C protein. Furthermore, 1,25-D3 also abolishes the induction of tenascin-C by serum or the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate. The inhibition of tenascin-C expression may be relevant for the anticancer activity of 1,25-D3.

Rab17 regulates membrane trafficking through apical recycling endosomes in polarized epithelial cells.

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.

Mammary epithelial cell differentiation in vitro is regulated by an interplay of egf action and tenascin-c downregulation

Expression of the extracellular matrix glycoprotein tenascin-C in the mammary gland is associated with cellular proliferation and cell motility during organogenesis and tumorigenesis. Because the source and the regulation of tenascin-C in these tissues are unclear, we have used tenascin-C cDNA, FITC-immunofluorescence and immuno-precipitation to examine tenascin-C expression of mammary epithelial cells. Using several mammary epithelial cell lines we could show that tenascin-C can be produced and secreted by epithelial cells. However it was found that tenascin-C synthesis was inversely correlated with the polarized epithelial phenotype. Among three mouse mammary epithelial cell clones, tenascin-C expression was most abundant in HC-11 cells, the least differentiated cell type. Expression levels were high during the growth phase but were nearly abolished when cells were grown to confluence and induced to express milk proteins. Downregulation of tenascin-C by EGF apparently commits HC-11 cells to respond to lactogenic hormones and consequently, hormone induced levels of beta-casein mRNA decreased significantly when HC-11 cells were grown on a tenascin-C substrate. On the other hand, TGF-beta, another growth factor involved in coordinated growth and differentiation of the mammary gland in vivo was found to be a very potent inducer of tenascin-C. The generation of fully polarized and tight epithelium affected the levels of tenascin-C expression. In contrast to HC-11 cells, which do not form epithelial domes in vitro, highly polarized and dome forming EpH4 and Fos-ER cells nearly lacked tenascin-C. Similarly, induction of dome formation in the rat mammary stem cell line Rama 25 by the differentiation inducer dimethylsulfoxide caused a loss of TN-C-transcripts. The inability of Fos-ER cells to develop domes in the presence of soluble tenascin-C also suggests its interference with induction and maintenance of mammary epithelial cell differentiation.

Thyroid hormone regulates stromelysin expression, protease secretion and the morphogenetic potential of normal polarized mammary epithelial cells.

Stromelysins are a group of proteases which degrade the extracellular matrix and activate other secreted proteases. Stromelysin (ST)-1 and ST-2 genes are induced by tumor promoters, oncogenes and growth factors, and have been involved in acquisition of the malignant phenotype. We show here that the thyroid hormone (T3) increases ST-1 and ST-2 expression in a non-transformed mouse mammary epithelial cell line (EpH4) in a way that is dependent on the level of thyroid receptor/c-erbA (TR alpha-1) expression. In agreement with this, T3 increases the secreted stromelysin activity and enhances the gelatinolytic activity of type IV collagenase. We have also demonstrated that T3 affects the epithelial polarity of EpH4 cells, diminishing the transepithelial electrical resistance of monolayers cultured on permeable filters, causing an abnormal distribution of polarization markers and the disruption of the organized 3-D structures formed by these cells in type I collagen gels. These results indicate that the ligand-activated TR alpha-1 plays an important role in regulating the morphogenetic and invasive capacities of mammary epithelial cells. Because the c-erbA locus is altered in several types of carcinoma, an altered or deregulated TR alpha-1 expression may also be important for breast cancer development and metastasis.