Development of antiestrogen resistance is a major clinical problem, and therefore it is crucial to elucidate the mechanisms involved. To investigate whether gain-of-function or loss-of-function mechanisms was most likely to be involved, cell fusion between the antiestrogen-sensitive MCF-7 and the ICI 164384-and ICI 182780-resistant MCF-7/164 R -5 cell lines was performed. Furthermore, a fusion cell line between the tamoxifen-resistant MCF-7/TAM R -1 and the MCF-7/164 R -5 cell line was established. A thorough investigation of growth parameters and expression of selected proteins (estrogen receptor-α (ERα), progesterone receptor (PR), Bcl-2, IGF-binding protein-2 (IGFBP2) and IGF receptor Iα (IGF-IRα)) in the fusion partners and fusion cells revealed that both gain-and loss-of-function changes occurred, and that the mechanisms resulting in resistance to the two antiestrogens were different. This multi-factoriality of antiestrogen resistance is promising in relation to sequential treatment of breast cancer patients with different types of endocrine therapy. Furthermore, we found an association between antiestrogen resistance and reduced IGF-IRα expression. Overall, the data presented in this report support the usefulness of cell fusion to clarify the mechanisms involved in development of resistance to the pure antiestrogens ICI 182780 and ICI 164384 and the selective ER modulator tamoxifen and suggest IGF-IRα as a new sensitive marker for response to antiestrogen treatment.

Breast cancer is the most common cancer disease in women in the western world. Tamoxifen has been the standard first line endocrine therapy for patients with estrogen receptor (ER) positive tumors. Unfortunately, almost all patients with advanced disease develop tamoxifen resistance. This has lead to a search for new potent antiestrogens. One of the new compounds under development is the pure antiestrogen RU 58,668. To study the mechanisms behind acquired resistance to RU 58,668, the RU 58,668-resistant cell line MCF-7/RU58(R)-1 (RU58(R)-1) was developed. The RU58(R)-1 cell line was responsive to tamoxifen, but cross-resistant to ICI 182,780 and the estrogen-sensitivity was reduced compared to the parental MCF-7 cell line. The protein levels of ERalpha, IGF-I Receptor (IGF-IR) and Bcl-2 were severely reduced, when RU58(R)-1 cells were cultured with RU 58,668 and the expression of progesterone receptor (PR) was lost. The ERalpha level increased upon withdrawal of RU 58,668 and the ERalpha protein was destabilized by RU 58,668 in both cell lines. Regulation of most of the investigated estrogen-sensitive mRNAs was found to be normal in the resistant cells. The protein levels of IGF-IR, Bcl-2 and the IGF Binding Protein 2 (IGFBP2) reverted towards MCF-7 levels upon RU 58,668 withdrawal, but the resistant phenotype was maintained. Thus, it appears as acquired resistance to RU 58,668 is not a result of loss of the ERalpha expression or function and we suggest that in the presence of RU 58,668, the RU58(R)-1 cell line probably uses other mitogenic pathways than the ERalpha pathway for growth and survival.

While advances in science and technology have increased options for treating breast cancer, current social trends have changed the way people deal with this disease. Women in the United States are no longer simply passive patients, but rather they are survivors, advocates and activists who are speaking up for themselves and speaking out for issues relevant to the treatment and prevention of breast cancer. As the discoveries of basic science have been translated to better clinical treatment, a new sense of hope has emerged. Quality of life now shares the spotlight with quantity of life as breast cancer has shifted from an acute to a chronic condition and as the numbers of long-term survivors increase. While this new population tends to have more optimistic expectations for survival, they are also expressing concerns about issues affecting their lives through and beyond treatment. These issues include, but are not limited to, such concerns as efficient and accurate diagnosis, the complexity of treatment decisions, access to quality cancer care, informed consent, privacy issues, availability of supportive care treatments, and effective communication skills, especially with their physicians. Survivors are also concerned about the impact of their disease on spouses and family, on fertility and sexuality issues, on their employment and (in the USA) insurability, and on their long-term survival. The identification of these increasing issues has given rise to a consumer movement that encourages a shift away from powerless victim to empowered survivor.

Introduction: Estrogen is the main driver of growth of the majority of breast cancers and many patients benefit from endocrine therapy. However, both innate and acquired resistance is a major problem. In order to study resistance mechanisms and to explore targeted treatment options for endocrine resistant breast cancer, we have developed in vitro cell culture models based on the estrogen receptor (ER) positive and estrogen responsive human breast cancer cell lines MCF-7 and T47D. Methods: Panels of MCF-7 and T47D sublines with acquired resistant to tamoxifen, fulvestrant and aromatase inhibitors were established by selection of colonies surviving long term treatment. Long-term estrogen deprived (LTED) MCF-7/S9 cells were developed by step-wise serum reduction until transfer to serum-free medium. The endocrine resistant sublines were analysed with respect to expression and function of the ER and the HER system, with respect to response to 195 kinase inhibitors and to second-line endocrine therapy. Results: ER expression was reduced in tamoxifen and fulvestrant resistant MCF-7 sublines, unchanged in tamoxifen resistant T47D sublines, whereas fulvestrant resistant T47D sublines were ER negative. In general, ER was functional in the resistant MCF-7 cells. Both tamoxifen and fulvestrant resistant MCF-7 sublines expressed increased level of HER1, the activated form of HER3 and activated ERK1/2, whereas HER4 was severely decreased. In fulvestrant resistant T47D sublines, HER1 and HER4 expression were decreased whereas HER2 expression was highly upregulated. The kinase inhibitor screen disclosed HER receptors as common hits in the tamoxifen and fulvestrant resistant MCF-7 sublines. Regarding T47D, c-Src and Aurora were common hits for tamoxifen and fulvestrant resistant sublines. Tamoxifen resistant MCF-7 and T47D sublines were severely growth inhibited by ER knockdown with either siRNA or fulvestrant, but complete growth arrest required combined treatment with e.g ERK1/2 inhibitor. LTED MCF-7 cells were resistant to aromatase inhibitor treatment and sensitive to both tamoxifen and fulvestrant. Conclusions: In our large panels of MCF-7 and T47D derived endocrine resistant sublines, it was a general finding that ER is the main driver of growth of tamoxifen resistant tumor cells, and also of growth of LTED MCF-7 cells. The ER down modulator fulvestrant inhibited growth of tamoxifen resistant sublines but complete growth arrest required combined treatment with e.g. a kinase inhibitor targeting the activated growth promoting pathways in the resistant cells. In fulvestrant resistant sublines, ER played only a minor role in MCF-7 sublines and no role in T47D sublines. The HER system played a major role in fulvestrant resistant MCF-7 cells, whereas Aurora B and c-Src were new candidate targets for fulvestrant resistant T47D cells. These cell culture models reflect clinical findings and will be available for collaborative studies, e.g. to discover new predictive markers to select targeted therapy for resistant tumors. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-02.

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 wildtype fibroblasts in the subrenal capsule of nude mice. Mamm...