Reversal of tamoxifen resistance of human breast carcinomas in vivo by neutralizing antibodies to transforming growth factor-beta.

BACKGROUND: Overexpression of transforming growth factor (TGF)-beta has been reported in human breast carcinomas resistant to antiestrogen tamoxifen, but the role of TGF-beta in this resistant phenotype is unclear. We investigated whether inhibition of TGF-beta2, which is overexpressed in LCC2 tamoxifen-resistant human breast cancer cells, could modify antiestrogen resistance. METHODS: TGF-beta2 expression was evaluated in LCC2 cells and tamoxifen-sensitive LCC1 cells by northern blot analysis. Secreted TGF-beta activity was quantified by use of an 125I-TGF-beta competitive radioreceptor assay. Sensitivity to tamoxifen was measured in a soft agarose colony-forming assay and in a xenograft model in nude and beige/nude mice. Natural killer (NK) cell cytotoxicity was measured by 51Cr release from LCC1 and LCC2 cell targets coincubated with human peripheral blood mononuclear cells. Decrease in TGF-beta2 expression in LCC2 cells was achieved by treatment with antisense oligodeoxynucleotides and confirmed by TGF-beta2 immunoblot analysis. RESULTS AND CONCLUSIONS: The proliferative response of LCC2 cells to tamoxifen in vitro was not altered by TGF-beta neutralizing antibodies. However, established LCC2 tumors in nude mice treated with tamoxifen plus TGF-beta antibodies failed to grow, whereas tumors treated with tamoxifen plus a control antibody continued to proliferate. This reversal of tamoxifen resistance by TGF-beta antibodies did not occur in beige/nude mice, which lack NK-cell function, suggesting that immune mechanisms may be involved in the antitumor effects of tamoxifen. Antisense TGF-beta2 oligodeoxynucleotides enhanced the NK sensitivity of LCC2 cells in the presence of tamoxifen. Finally, LCC1 tumors were markedly more sensitive to tamoxifen in NK-active than in NK-deficient mice. IMPLICATIONS: These data suggest that host NK function mediates, in part, the antitumor effect of tamoxifen and that TGF-beta2 may abrogate this mechanism, thus contributing to tamoxifen resistance.

A kinase-defective transforming growth factor-beta receptor type II is a dominant-negative regulator for human breast carcinoma MCF-7 cells.

The role of transforming growth factor (TGF)-beta type II receptor (T beta RII) in TGF-beta resistance and tumor progression is now well recognized. To test the effects of T beta RII loss in determining malignancy, we transfected a T beta RII-expressing, TGF-beta-sensitive, MCF-7 cell strain (ME24) with a tetracycline-repressible truncated T beta RII (kdT beta RII) construct lacking the cytoplasmic domain of the receptor. Transfection of kdT beta RII into parental ME24 cells (designated ME24t6 after transfection) resulted in high expression levels of kdT beta RII mRNA and cell surface protein which were reversible by tetracycline treatment. ME24t6 cells did not respond to exogenous TGF-beta 1 as measured by inhibition of proliferation or fibronectin (FN) induction, indicating that the truncated T beta RII acted as a dominant-negative inhibitor of both the growth inhibitory and extracellular matrix (ECM) stimulatory TGF-beta effects. Furthermore, inhibition of kdT beta RII expression by tetracycline treatment led to TGF-beta 1-mediated cell growth arrest in the G1 phase of cell cycle and to the accumulation of the hypophosphorylated form of retinoblastoma (Rb) protein. However, compared to parental ME24 cells, transfectants failed to show increased tumorigenicity, indicating that loss of T beta RII itself is not sufficient to account for differences in the malignant properties of T beta RII-expressing and non-expressing MCF-7 cell strains.

Processing of the transforming growth factor beta type I and II receptors. Biosynthesis and ligand-induced regulation.

Three cell surface transforming growth factor beta (TGFbeta) receptor (R) proteins regulate the effects of TGFbeta isoforms on growth and differentiation. TGFbeta-IR and -IIR are transmembrane serine/threonine kinases directly mediating the signaling across the plasma membrane. Both TGFbeta and its receptors are ubiquitously expressed, hence the fine regulation of the multiplicity of responses most likely involves several levels of control including the regulation of expression, complex formation, and down-regulation of the receptor proteins. In mink lung epithelial cells, TGFbeta-IIR was first synthesized as a approximately 60-kDa endoglycosidase H-sensitive precursor protein, which was converted to a mature approximately 70-kDa protein. The half-life of metabolically labeled mature TGFbeta-IIR was estimated to be 60 min and was further reduced to approximately 45 min in the presence of exogenous TGFbeta1. Minimal internalization of 125I-TGFbeta1 at 37 degrees C was detected suggesting that the rapid turnover was not due to endocytosis and degradation of the ligand-receptor complexes. TGFbeta-IR was synthesized as a approximately 53-kDa precursor protein, which was processed to a mature approximately 55-kDa receptor protein. The half-life of TGFbeta-IR was >12 h. A fraction of tunicamycin-treated type I and II receptors that reach the cell surface was able to associate in the presence of ligand suggesting that heteromeric complexes can form in a post-endoplasmic reticulum compartment before full glycosylation is achieved. These results show differential processing and turnover of TGFbeta-IR and TGFbeta-IIR providing a potential additional mechanism for modulation of cellular responses to TGFbetas.

Predominant cytosolic localization of type II transforming growth factor beta receptors in human breast carcinoma cells.

It is proposed that loss of a growth-inhibitory response to transforming growth factor beta (TGFbeta) contributes to breast cancer progression. Because cellular TGFbeta responsiveness often correlates with TGFbeta type II receptor (TGFbeta-IIR) expression, we have examined the cellular distribution of TGFbeta-IIRs in tumor and nontumor mammary epithelial cells. By immunoblot analysis, TGFbeta-IIR was detected both in membrane and cytosolic fractions of MDA-231 tumor cells as well as in normal human breast epithelial cells. The cytosolic protein appeared to be more abundant and was detected as a clear perinuclear staining by immunocytochemistry. The glycosylation patterns of the cytosolic and membrane form were different, indicating distinct receptor pools. The cytosolic TGFbeta-IIR did not bind 125I-labeled TGFbeta1 but had a detectable in vitro and in vivo kinase activity. MCF-7 breast cancer cells express the TGFbeta-IIR mRNA but show undetectable cell surface TGFbeta-IIR protein by affinity cross-linking. However, low levels of TGFbeta-IIR were observed in MCF-7 cytosol. Sequencing of the coding region of TGFbeta-IIR from MCF-7 cells indicated a point mutation (A439V) in a nonconserved region of the kinase domain. When MCF-7 cells were treated with sublethal doses of Adriamycin that induce cell differentiation, the membrane localization of TGFbeta-IIR and TGFbeta response were restored. Our results indicate the presence of a prominent, kinase-active TGFbeta-IIR in the cytosol of several mammary cell lines. This cytosolic pool of receptors is the only detectable one in MCF-7 cells. Loss of wild-type membrane receptors due to defects in trafficking presents a potential new mechanism for escape from negative growth control.

Blockade of transforming growth factor-beta signaling does not abrogate antiestrogen-induced growth inhibition of human breast carcinoma cells.

We have studied the role of autocrine transforming growth factor-beta (TGF-beta) signaling on antiestrogen-mediated growth inhibition of hormone-dependent T47D and MCF-7 human breast carcinoma cells. Tamoxifen treatment increased the secretion of TGF-beta activity into serum-free cell medium and the cellular content of affinity cross-linked type I and III TGF-beta receptors in both cell lines. Anti-pan-TGF-beta antibodies did not block anti-estrogen-induced recruitment in G1 and inhibition of anchorage-dependent and -independent growth of both cell lines. Early passage MCF-7 cells, which exhibit detectable type II TGF-beta receptors at the cell surface and exquisite sensitivity to exogenous TGF-beta1, were transfected with a tetracycline-controllable dominant-negative TGF-betaRII (DeltaRII) construct. Although the TGF-beta1 response was blocked by removal of tetracycline in MCF-7/DeltaRII cells, tamoxifen-mediated suppression of Rb phosphorylation, recruitment in G1, and inhibition of cell proliferation were identical in the presence and absence of tetracycline. TGF-beta1 treatment up-regulated the Cdk inhibitor p21 and induced its association with Cdk2 in MCF-7 cells; these responses were blocked by the DeltaRII transgene product. In MCF-7 cells with a functional TGF-beta signaling pathway, tamoxifen did not up-regulate p21 nor did it induce association of p21 with Cdk2, suggesting alternative mechanisms for antiestrogen-mediated cytostasis. Finally, transfection of late-passage, TGF-beta1 unresponsive MCF-7 cells with high levels of TGF-betaRII restored TGF-beta1-induced growth inhibition but did not enhance tamoxifen response in culture. Taken together these data strongly argue against any role for TGF-beta signaling on tamoxifen-mediated growth inhibition of hormone-dependent breast cancer cells.

Complex role of tumor cell transforming growth factor (TGF)-beta s on breast carcinoma progression.

Growth inhibition by the TGF-beta s has been extensively studied in both normal and transformed mammary epithelial cells. It has been proposed that loss of autocrine TGF-beta mediated growth regulation is a critical event in breast tumorigenesis and several lines of in vitro and in vivo data support this hypothesis. However, a positive association between the expression of TGF-beta s by tumor cells and the progression or maintenance of breast cancinoma cells has been observed in many studies in in vivo tumor models. Possible mechanisms for these growth enhancing effects of TGF-beta include immunosuppression mediated by tumor TGF-beta s, enhanced angiogenesis, increased peritumoral stroma formation, and cell adhesion. The net effect of tumor cell TGF-beta on the biology of breast carcinogenesis would depend on the balance between autocrine growth inhibition of mammary epithelial cells and these growth enhancing effects.