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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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Biochemical characterization of integrins in the HMT3522 cells after culture in 3-dimensions. (a and b) Western analysis of β1- and β4-integrins: Total cell lysates of S-1 and T4-2 colonies showed elevated expression of β1- (a) and β4-integrins (b) in  the tumor cell colonies. (c and d) Cell surface expression of β1-  and β4-integrin heterodimers using biotinylation: Tumor colonies  had 12.5% higher plasma membrane levels of β1- (c) but 60%  lower levels of β4-integrin heterodimers (d) than S-1 acini. (e)  Relative scan units (RSU) showing relative colony surface levels  of β1- and β4-integrin heterodimers: There was a 2.8-fold relative  increase in the ratio of β1-integrins to β4-integrin on the colony  cell surface of T4-2 colonies as compared to the S-1 acini. All cells  were analyzed after 10–12 d inside EHS.
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Figure 3: Biochemical characterization of integrins in the HMT3522 cells after culture in 3-dimensions. (a and b) Western analysis of β1- and β4-integrins: Total cell lysates of S-1 and T4-2 colonies showed elevated expression of β1- (a) and β4-integrins (b) in the tumor cell colonies. (c and d) Cell surface expression of β1- and β4-integrin heterodimers using biotinylation: Tumor colonies had 12.5% higher plasma membrane levels of β1- (c) but 60% lower levels of β4-integrin heterodimers (d) than S-1 acini. (e) Relative scan units (RSU) showing relative colony surface levels of β1- and β4-integrin heterodimers: There was a 2.8-fold relative increase in the ratio of β1-integrins to β4-integrin on the colony cell surface of T4-2 colonies as compared to the S-1 acini. All cells were analyzed after 10–12 d inside EHS.

Mentions: We have postulated previously that tumor cells have lost their ability to respond correctly to ECM-induced signals (Petersen et al., 1992; Howlett et al., 1995). To determine if the loss of structural organization and growth control exhibited by the tumor cells was related to an alteration in their integrins, we characterized the integrin receptors for laminin in the two cell lines. While both S-1 and T4-2 cells expressed integrins β1, β4, α3, and α6, their distribution patterns were radically different in 3-D cultures (Fig. 2, a and a′ through d and d′). S-1 acini had basally distributed β1-, β4-, and α6-integrins and basolateral α3-integrin, consistent with their polarized phenotype. In contrast, all of these integrins were found to be randomly distributed and disorganized in the T4-2 colonies. Western blot analysis of the tumor colonies showed over-expression of both β1- and β4-integrins relative to the levels observed in the S-1 acini (Fig. 3, a and b). Cell surface labeling and immunoprecipitation experiments revealed that the surface levels of β1-integrins were slightly higher (12.5%) in the T4-2 cells (Fig. 3 c) whereas the surface β4-integrin levels were much lower (60%) in T4-2 than in S-1 colonies (Fig. 3 d). However, the ratio of β1- to β4-integrins at the cell surface was increased by more than 2.8-fold in the T4-2 cell colonies than in the S-1 acini (Fig. 3 e).


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)

Biochemical characterization of integrins in the HMT3522 cells after culture in 3-dimensions. (a and b) Western analysis of β1- and β4-integrins: Total cell lysates of S-1 and T4-2 colonies showed elevated expression of β1- (a) and β4-integrins (b) in  the tumor cell colonies. (c and d) Cell surface expression of β1-  and β4-integrin heterodimers using biotinylation: Tumor colonies  had 12.5% higher plasma membrane levels of β1- (c) but 60%  lower levels of β4-integrin heterodimers (d) than S-1 acini. (e)  Relative scan units (RSU) showing relative colony surface levels  of β1- and β4-integrin heterodimers: There was a 2.8-fold relative  increase in the ratio of β1-integrins to β4-integrin on the colony  cell surface of T4-2 colonies as compared to the S-1 acini. All cells  were analyzed after 10–12 d inside EHS.
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Related In: Results  -  Collection

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Figure 3: Biochemical characterization of integrins in the HMT3522 cells after culture in 3-dimensions. (a and b) Western analysis of β1- and β4-integrins: Total cell lysates of S-1 and T4-2 colonies showed elevated expression of β1- (a) and β4-integrins (b) in the tumor cell colonies. (c and d) Cell surface expression of β1- and β4-integrin heterodimers using biotinylation: Tumor colonies had 12.5% higher plasma membrane levels of β1- (c) but 60% lower levels of β4-integrin heterodimers (d) than S-1 acini. (e) Relative scan units (RSU) showing relative colony surface levels of β1- and β4-integrin heterodimers: There was a 2.8-fold relative increase in the ratio of β1-integrins to β4-integrin on the colony cell surface of T4-2 colonies as compared to the S-1 acini. All cells were analyzed after 10–12 d inside EHS.
Mentions: We have postulated previously that tumor cells have lost their ability to respond correctly to ECM-induced signals (Petersen et al., 1992; Howlett et al., 1995). To determine if the loss of structural organization and growth control exhibited by the tumor cells was related to an alteration in their integrins, we characterized the integrin receptors for laminin in the two cell lines. While both S-1 and T4-2 cells expressed integrins β1, β4, α3, and α6, their distribution patterns were radically different in 3-D cultures (Fig. 2, a and a′ through d and d′). S-1 acini had basally distributed β1-, β4-, and α6-integrins and basolateral α3-integrin, consistent with their polarized phenotype. In contrast, all of these integrins were found to be randomly distributed and disorganized in the T4-2 colonies. Western blot analysis of the tumor colonies showed over-expression of both β1- and β4-integrins relative to the levels observed in the S-1 acini (Fig. 3, a and b). Cell surface labeling and immunoprecipitation experiments revealed that the surface levels of β1-integrins were slightly higher (12.5%) in the T4-2 cells (Fig. 3 c) whereas the surface β4-integrin levels were much lower (60%) in T4-2 than in S-1 colonies (Fig. 3 d). However, the ratio of β1- to β4-integrins at the cell surface was increased by more than 2.8-fold in the T4-2 cell colonies than in the S-1 acini (Fig. 3 e).

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