Ectopic expression of an oncogenic Ras has been shown to induce senescence instead of transformation in human mammary epithelial cells (HMEC) that are immortalized with ectopic expression of the human telomerase reverse transcriptase (hTERT) suggesting that additional molecular alterations are needed for these mammary cells to escape senescence and initiate tumorigenesis. The signaling pathway of transforming growth factor beta (TGFβ) has been shown to inhibit mammary tumorigenesis in various cell and animal models. Reduced or heterogenous expression of the type II TGFβ receptor (RII) was observed in a subset of patients with breast epithelial hyperplasia without atypia and was associated with an increased risk of subsequently developing invasive breast cancer. In this study, we investigated the effects of autocrine TGFβ and oncogenic ras signaling on the tumorigenic potential of HMEC. We generated two cell lines from HMEC/hTERT cells, one with attenuated TGFβ signaling expressing a dominant-negative RII receptor (DNRII) and the other co-expressing both DNRII and H-ras-V12 oncogene (DNRII-ras). The expression of DNRII blocked H-ras-V12-induced senescence. DNRII cells were found to be significantly less invasive in comparison to the DNRII-ras cells in an in vitro Matrigel invasion assay. Orthotopic inoculation of DNRII-ras cells (4 x 106 cells in 1:1 Matrigel) into the mammary fat pads of nude mice produced large tumors in 10 weeks (mean volume 1307mm3, n=6) with extensive lung metastasis while inoculation of DNRII cells (10x 106 cells in 1:1 Matrigel) did not produce any tumor. Histological examinations of the primary tumors and lung metastases formed by DNRII-ras cells revealed poorly differentiated areas that are spindled and epithelioid. Some areas showed squamous differentiation with keratin pearl formation. cDNA-microarray analysis showed up- or down-regulation of the expression of several significant and novel genes relevant to cancer progression in the DNRII-ras cells in comparison to the non-tumorigenic DNRII cells. Detailed analysis is underway. Thus, our study indicates that while attenuation of TGFβ signaling alone does not lead to the transformation, it can block senescence and promote malignant transformation induced by H-ras-V12 in HMEC/hTERT cells.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]