Stromal and epithelial TGF-β signaling in mammary tumorigenesis

There is compelling evidence from transgenic mouse studies and analysis of mutations in human carcinomas indicating that the TGF-β signal transduction pathway is tumor suppressive. We have shown that overexpression of TGF-β1 in mammary epithelial cells suppresses the development of carcinomas and that expression of a dominant negative type II TGF-β receptor (DNIIR) in mammary epithelial cells under control of the MMTV promoter/enhancer increases the incidence of mammary carcinomas. Studies of human tumors have demonstrated inactivating mutations in human tumors of genes encoding proteins involved in TGF-β signal transduction, including DPC4/Smad4, Smad2, and the type II TGF-β receptor (TβRII). There is also evidence that TGF-β can enhance the progression of tumors. This hypothesis is being tested in genetically modified mice. To attain complete loss of TβRII, we have generated mice with loxP sites flanking exon 2 of Tgfbr2 and crossed them with mice expressing Cre recombinase under control of the MMTV promoter/enhancer to obtain Tgfbr2^mgKO mice. These mice show lobuloalveolar hyperplasia. Mice are being followed for mammary tumor development. Tgfbr2^mgKO mice that also express polyoma virus middle T antigen under control of the MMTV promoter (MMTV-PyVmT) develop mammary tumors with a significantly shorter latency than MMTV-PyVmT mice and show a marked increase in pulmonary metastases. Our data do not support the hypothesis that TGF-β signaling in mammary carcinoma cells is important for invasion and metastasis, at least in this model system.

The importance of stromal-epithelial interactions in mammary gland development and tumorigenesis is well established. These interactions probably involve autocrine and paracrine action of multiple growth factors, including members of the TGF-β family, which are expressed in both stroma and epithelium. Again, to accomplish complete knockout of the type II TGF-β receptor gene in mammary stromal cells, FSP1-Cre and Tgfbr2 ^flox/flox mice were crossed to attain Tgfbr2^fspKO mice. The loss of TGF-β responsiveness in fibroblasts resulted in intraepithelial neoplasia in prostate and invasive squamous cell carcinoma of the forestomach with high penetrance by 6 weeks of age. Both epithelial lesions were associated with an increased abundance of stromal cells. Activation of paracrine hepatocyte growth factor (HGF) signaling was identified as one possible mechanism for stimulation of epithelial proliferation. TGF-β signaling in fibroblasts thus modulates the growth and oncogenic potential of adjacent epithelia in selected tissues.

More recently, we have examined the effects of Tgfbr2^fspKO fibroblasts on normal and transformed mammary epithelium. We analyzed the role of TGF-β signaling by stromal cells in mammary tumor progression. To avoid the possibility of endogenous wild-type fibroblasts masking potential effects of Tgfbr2^fspKO cells on tumor progression, we implanted PyVmT mammary carcinoma cells with Tgfbr2^fspKO or wild-type fibroblasts in the subrenal capsule of nude mice. Mammary tumor cells implanted with Tgfbr2^fspKO cells exhibited an increase in tumor growth and intravasation associated with an increase in tumor cell survival, proliferation and an increase in tumor angiogenesis compared with tumor cells implanted with control fibroblasts. We demonstrated increased expression of several growth factors by Tgfbr2^fspKO fibroblasts compared with control fibroblasts in primary culture. These included HGF, MSP and TGF-α. There was an increase in tumor cell activating phosphorylation of the cognate receptors, c-Met, RON, erbB1, and erbB2 in carcinomas accompanied by Tgfbr2^fspKO fibroblasts.

The Tgfbr2^fspKO mouse model illustrates that a signaling pathway known to suppress cell-cycle progression when activated in epithelial cells can also have an indirect inhibitory effect on epithelial proliferation when activated in adjacent stromal fibroblasts in vivo. Loss of this inhibitory effect can result in increased epithelial proliferation and may even progress to invasive carcinoma in some tissues.