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Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.

Weaver VM, Petersen OW, Wang F, Larabell CA, Briand P, Damsky C, Bissell MJ - J. Cell Biol. (1997)

Bottom Line: A stimulatory beta1-integrin antibody proved to be ineffective.The observed phenotypes were reversible when the cells were disassociated and the antibodies removed.Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

View Article: PubMed Central - PubMed

Affiliation: Ernest Orlando Lawrence Berkeley National Laboratory, California 94720, USA.

ABSTRACT
In a recently developed human breast cancer model, treatment of tumor cells in a 3-dimensional culture with inhibitory beta1-integrin antibody or its Fab fragments led to a striking morphological and functional reversion to a normal phenotype. A stimulatory beta1-integrin antibody proved to be ineffective. The newly formed reverted acini re-assembled a basement membrane and re-established E-cadherin-catenin complexes, and re-organized their cytoskeletons. At the same time they downregulated cyclin D1, upregulated p21(cip,wat-1), and stopped growing. Tumor cells treated with the same antibody and injected into nude mice had significantly reduced number and size of tumors in nude mice. The tissue distribution of other integrins was also normalized, suggesting the existence of intimate interactions between the different integrin pathways as well as adherens junctions. On the other hand, nonmalignant cells when treated with either alpha6 or beta4 function altering antibodies continued to grow, and had disorganized colony morphologies resembling the untreated tumor colonies. This shows a significant role of the alpha6/beta4 heterodimer in directing polarity and tissue structure. The observed phenotypes were reversible when the cells were disassociated and the antibodies removed. Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

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Alteration of α6/β4-integrin signaling in S-1 cells leads to the formation of disorganized colonies. (a and a′) Confocal fluorescence microscopy of double immunostaining for α6- (Texas red) and β4-integrins (FITC): T4-β1 revertant acini (a′) showed basally polarized α6- and β4-integrins, while T4-2 mock–treated (T-4 IgG) colonies (a) demonstrated disorganized, nonpolarized expression. (b)  Cell surface expression of β4-integrin heterodimers using biotinylation: Tumor colonies had lower cell surface levels of β4-integrin heterodimers (60% lower) relative to the S-1 acini (see Fig. 3 d), which were restored in the T4-β1 reverted acini (40% higher) relative to  the T4-2 colonies. (c) Immunoblot of p21cip,waf-1 levels in T4-2 IgG and T4-β1 colonies: The level of p21 protein was clearly increased in  the T4-β1 reverted acini. (d and d′) Phase contrast micrographs of S-1 nonmalignant acini (d) and tumorigenic T4-2 colonies (d′) viewed  directly inside EHS: S-1 cells formed spherical structures (d), whereas T4-2 cells formed large, irregular colonies (d′). (e and e′) Phase contrast micrographs of S-1 nonmalignant cells treated with function altering β4-integrin antibodies (e) and tumorigenic T4-2 cells treated  with function blocking β1-integrin antibodies (e′) viewed directly inside EHS: S-1 cells treated with β4-integrin antibodies formed large,  irregular colonies (e), while T4-2 cells treated with β1-integrin function-blocking antibody (e′) formed spherical structures similar to S-1  acini (see Fig. 1 a). All cultures were analyzed after 10–12 d inside EHS. Bar, 16 μm.
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Figure 7: Alteration of α6/β4-integrin signaling in S-1 cells leads to the formation of disorganized colonies. (a and a′) Confocal fluorescence microscopy of double immunostaining for α6- (Texas red) and β4-integrins (FITC): T4-β1 revertant acini (a′) showed basally polarized α6- and β4-integrins, while T4-2 mock–treated (T-4 IgG) colonies (a) demonstrated disorganized, nonpolarized expression. (b) Cell surface expression of β4-integrin heterodimers using biotinylation: Tumor colonies had lower cell surface levels of β4-integrin heterodimers (60% lower) relative to the S-1 acini (see Fig. 3 d), which were restored in the T4-β1 reverted acini (40% higher) relative to the T4-2 colonies. (c) Immunoblot of p21cip,waf-1 levels in T4-2 IgG and T4-β1 colonies: The level of p21 protein was clearly increased in the T4-β1 reverted acini. (d and d′) Phase contrast micrographs of S-1 nonmalignant acini (d) and tumorigenic T4-2 colonies (d′) viewed directly inside EHS: S-1 cells formed spherical structures (d), whereas T4-2 cells formed large, irregular colonies (d′). (e and e′) Phase contrast micrographs of S-1 nonmalignant cells treated with function altering β4-integrin antibodies (e) and tumorigenic T4-2 cells treated with function blocking β1-integrin antibodies (e′) viewed directly inside EHS: S-1 cells treated with β4-integrin antibodies formed large, irregular colonies (e), while T4-2 cells treated with β1-integrin function-blocking antibody (e′) formed spherical structures similar to S-1 acini (see Fig. 1 a). All cultures were analyzed after 10–12 d inside EHS. Bar, 16 μm.

Mentions: The ability of T4-β1 cells to re-form a basally organized basement membrane (Fig. 5 g′) indicated the re-establishment of acinar polarity. Polarity has been shown to be associated with a basal localization of α6/β4-integrin heterodimers in keratinocytes (DeLuca et al., 1994). T4-β1 reverted acini had polar, basally localized α6- and β4-integrins (compare Fig. 7, a–7 a′), increased cell surface β4-integrin heterodimers (Fig. 7 b), and higher p21cip,waf-1 levels (Fig. 7 c). These findings showed that the β1-integrin signaling pathway was not only interconnected with cateninE-cadherin adherens junction assembly, but was also connected to the α6/β4-integrin signaling pathway.


Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.

Weaver VM, Petersen OW, Wang F, Larabell CA, Briand P, Damsky C, Bissell MJ - J. Cell Biol. (1997)

Alteration of α6/β4-integrin signaling in S-1 cells leads to the formation of disorganized colonies. (a and a′) Confocal fluorescence microscopy of double immunostaining for α6- (Texas red) and β4-integrins (FITC): T4-β1 revertant acini (a′) showed basally polarized α6- and β4-integrins, while T4-2 mock–treated (T-4 IgG) colonies (a) demonstrated disorganized, nonpolarized expression. (b)  Cell surface expression of β4-integrin heterodimers using biotinylation: Tumor colonies had lower cell surface levels of β4-integrin heterodimers (60% lower) relative to the S-1 acini (see Fig. 3 d), which were restored in the T4-β1 reverted acini (40% higher) relative to  the T4-2 colonies. (c) Immunoblot of p21cip,waf-1 levels in T4-2 IgG and T4-β1 colonies: The level of p21 protein was clearly increased in  the T4-β1 reverted acini. (d and d′) Phase contrast micrographs of S-1 nonmalignant acini (d) and tumorigenic T4-2 colonies (d′) viewed  directly inside EHS: S-1 cells formed spherical structures (d), whereas T4-2 cells formed large, irregular colonies (d′). (e and e′) Phase contrast micrographs of S-1 nonmalignant cells treated with function altering β4-integrin antibodies (e) and tumorigenic T4-2 cells treated  with function blocking β1-integrin antibodies (e′) viewed directly inside EHS: S-1 cells treated with β4-integrin antibodies formed large,  irregular colonies (e), while T4-2 cells treated with β1-integrin function-blocking antibody (e′) formed spherical structures similar to S-1  acini (see Fig. 1 a). All cultures were analyzed after 10–12 d inside EHS. Bar, 16 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139858&req=5

Figure 7: Alteration of α6/β4-integrin signaling in S-1 cells leads to the formation of disorganized colonies. (a and a′) Confocal fluorescence microscopy of double immunostaining for α6- (Texas red) and β4-integrins (FITC): T4-β1 revertant acini (a′) showed basally polarized α6- and β4-integrins, while T4-2 mock–treated (T-4 IgG) colonies (a) demonstrated disorganized, nonpolarized expression. (b) Cell surface expression of β4-integrin heterodimers using biotinylation: Tumor colonies had lower cell surface levels of β4-integrin heterodimers (60% lower) relative to the S-1 acini (see Fig. 3 d), which were restored in the T4-β1 reverted acini (40% higher) relative to the T4-2 colonies. (c) Immunoblot of p21cip,waf-1 levels in T4-2 IgG and T4-β1 colonies: The level of p21 protein was clearly increased in the T4-β1 reverted acini. (d and d′) Phase contrast micrographs of S-1 nonmalignant acini (d) and tumorigenic T4-2 colonies (d′) viewed directly inside EHS: S-1 cells formed spherical structures (d), whereas T4-2 cells formed large, irregular colonies (d′). (e and e′) Phase contrast micrographs of S-1 nonmalignant cells treated with function altering β4-integrin antibodies (e) and tumorigenic T4-2 cells treated with function blocking β1-integrin antibodies (e′) viewed directly inside EHS: S-1 cells treated with β4-integrin antibodies formed large, irregular colonies (e), while T4-2 cells treated with β1-integrin function-blocking antibody (e′) formed spherical structures similar to S-1 acini (see Fig. 1 a). All cultures were analyzed after 10–12 d inside EHS. Bar, 16 μm.
Mentions: The ability of T4-β1 cells to re-form a basally organized basement membrane (Fig. 5 g′) indicated the re-establishment of acinar polarity. Polarity has been shown to be associated with a basal localization of α6/β4-integrin heterodimers in keratinocytes (DeLuca et al., 1994). T4-β1 reverted acini had polar, basally localized α6- and β4-integrins (compare Fig. 7, a–7 a′), increased cell surface β4-integrin heterodimers (Fig. 7 b), and higher p21cip,waf-1 levels (Fig. 7 c). These findings showed that the β1-integrin signaling pathway was not only interconnected with cateninE-cadherin adherens junction assembly, but was also connected to the α6/β4-integrin signaling pathway.

Bottom Line: A stimulatory beta1-integrin antibody proved to be ineffective.The observed phenotypes were reversible when the cells were disassociated and the antibodies removed.Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

View Article: PubMed Central - PubMed

Affiliation: Ernest Orlando Lawrence Berkeley National Laboratory, California 94720, USA.

ABSTRACT
In a recently developed human breast cancer model, treatment of tumor cells in a 3-dimensional culture with inhibitory beta1-integrin antibody or its Fab fragments led to a striking morphological and functional reversion to a normal phenotype. A stimulatory beta1-integrin antibody proved to be ineffective. The newly formed reverted acini re-assembled a basement membrane and re-established E-cadherin-catenin complexes, and re-organized their cytoskeletons. At the same time they downregulated cyclin D1, upregulated p21(cip,wat-1), and stopped growing. Tumor cells treated with the same antibody and injected into nude mice had significantly reduced number and size of tumors in nude mice. The tissue distribution of other integrins was also normalized, suggesting the existence of intimate interactions between the different integrin pathways as well as adherens junctions. On the other hand, nonmalignant cells when treated with either alpha6 or beta4 function altering antibodies continued to grow, and had disorganized colony morphologies resembling the untreated tumor colonies. This shows a significant role of the alpha6/beta4 heterodimer in directing polarity and tissue structure. The observed phenotypes were reversible when the cells were disassociated and the antibodies removed. Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

Show MeSH
Related in: MedlinePlus