Background: In our previous studies, it was found protein kinase C alpha (PKCα) overexpression in a hormone dependent breast cancer cell line T47D results in autonomous growth and tamoxifen (TAM) resistance both in vitro and in vivo (Tonetti et. al., Br. J. Cancer 2000 6:782-91; Lin et. al., Carcinogenesis. 2006 27:1538-46). Furthermore, we find that PKCα overexpression is relevant in TAM-resistant breast cancer in the clinic (Tonetti et. al., Br. J. Cancer 2003 88:1400-02). Most notably, 17-β estradiol (E2) inhibits T47D:A18/PKCα cell growth in vivo but not on plastic (Chisamore et. al., Clin. Cancer Res. 2001 7:3156-3165). We observed that growth in Matrigel, a reconstituted basement membrane, was sufficient for the E2 inhbitory effect. Therefore we applied Matrigel 3-dimensional cell culture to study the underlying mechanisms of the E2-inhibitory effect. Previous studies have shown fulvestrant, a pure antiestrogen that induces estrogen receptor (ER) degradation, can abolish the inhibitory effect of E2 on T47D/PKCα tumor growth in vivo. This result suggests that the ER is likely to participate in the mechanism of E2 inhibitory effect. There is growing evidence that the cell membrane associated ER mediates many signaling actions of E2 and may contribute to TAM resistance in breast cancer. To determine whether the membrane associated ER is involved in E2-inhibited cell growth, we asked whether treatment of T47D/PKCα cells grown in Matrigel with the cell membrane impermeable bovine serum albumin conjugated E2 (E2-BSA, Sigma) was sufficient to inhibit colony formation as well as E2.

Methods: The hormone dependentT47D/neo cells were transfected with the ERE-tk-luc plasmid treated with E2 (10-9M), E2-BSA (10-9M) or fulvestrant (10-7M), and ERE-luciferase activity was determined. T47D/neo and T47D/PKCα cells were seeded in phenol-red free Matrigel containing E2, E2-BSA or vehicle in 6-well plates. Colonies were counted on the 20th day.

Results: Since unconjugated E2 is always an important concern in studies using E2-BSA, an ERE-luciferase reporter assay confirmed the absence of unconjugated E2 in the preparation. Both E2 and E2-BSA-treated T47D/PKCαcells form 40~50% less colonies as compared with untreated T47D/PKCα cells. Moreover, whereas E2 stimulated the T47D/neo cell growth in Matrigel, E2-BSA did not. These observations additionally confirmed the absence of E2-BSA dissociation or free E2 in E2-BSA preparation used in our experiments.

Conclusion: We conclude that both E2 and E2-BSA conjugate have a similar inhibitory effect on T47D:A18/PKCα cell growth in Matrigel. These results indicate that the membrane associated ER may mediate the observed E2 inhibitory effect on T47D/PKCα colony formation in Matrigel and in vivo. Our work suggests that activation of membrane associated ER may be a useful therapeutic approach for the treatment of TAM-resistant, PKCα overexpressing breast cancer.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA