Protein pathway activation mapping reveals molecular networks associated with antiestrogen resistance in breast cancer cell lines

Abstract

Previously, we have identified a panel of breast cancer antiestrogen resistance (BCAR) genes. Several of these genes have clinical relevance because mRNA or protein levels associate with tamoxifen resistance or tumor aggressiveness. We postulated that changes in activation status of protein signaling networks induced by BCAR genes may provide better insight into the mechanisms underlying antiestrogen resistance. Key signal transduction pathways were analyzed for changes in activation or expression using reverse-phase protein microarrays probed with 78 antibodies against signaling proteins with known roles in tumorigenesis. We used ZR-75-1-derived cell lines transduced with AKT1, AKT2, BCAR1, BCAR3, BCAR4, EGFR, GRB7, HRAS, HRAS v12 or HEF1 and MCF7-derived cell lines transduced with BCAR3, BCAR4 or EGFR. In the antiestrogenresistant cell lines, we observed increased phosphorylation of several pathways involved in cell proliferation and survival. All tamoxifen-resistant cell lines contained high levels of phosphorylated AKT and its biochemically linked substrates Forkhead box O1/3. The activation of ERBB2, ERBB3 and the downstream modulators focal adhesion kinase and SHC were activated in cells with overexpression of BCAR4. Remarkable differences were observed for the levels of activated AMPK alpha1, cyclins, STAT5, STAT6, ERK1/2 and BCL2. The comparison of the cell signaling networks in estrogen-dependent and -independent cell lines revealed biochemically linked kinase-substrate markers that comprised systemically activated signaling pathways involved in tamoxifen resistance. Our results show that this model provides insights into the molecular and cellular mechanisms of breast cancer progression and antiestrogen resistance. This knowledge may help the development of novel targeted treatments.Breast cancers are initially dependent on estrogens for growth and progression. Antiestrogens prevent estrogens from binding the estrogen receptor (ER) alpha and thereby inhibit cell proliferation. ERa is expressed in about two-thirds of primary breast cancers. For over 30 years, the estrogen antagonist tamoxifen has been the preferred endocrine treatment for patients with ERa-positive breast cancer in premenopausal and postmenopausal women. 1 Tamoxifen is effective in the treatment of metastatic breast cancer, ductal carcinoma in situ, as adjuvant therapy, and more recently in chemoprevention of the malignancy. 2,3 In patients with breast cancer with recurrent disease, tamoxifen induces an objective response in approximately more than half of the patients with primary cancers expressing the ER 4 and references herein. However, the treatment is palliative, and progression of the tumor cells to a hormone-independent phenotype is inevitable. Several mechanisms have been identified underlying primary endocrine resistance of breast cancer and the progression to acquired resistance. The most favored explanations for mechanisms causing tamoxifen resistance are based on pharmacology of the drug; alterations in the interactions bet...

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer

“…94 On the other hand, gene transcription analyses and pathway activation mapping have revealed that overexpression of BCAR3 or BCAR1 activates signaling networks that have not only common elements but also distinctive features. 109,120 Taken together, these findings highlight the complexities in the activities of NSP and CAS proteins and their modules, which remain to be further unraveled in future studies.…”

“…101,102 AKT activation downstream of PI3 kinase may also contribute to BCAR3-dependent antiestrogen resistance. 102,109 Another key role of BCAR3 likely contributing to estrogen-independent growth involves increasing the activity of SRC recruited to BCAR1, thus leading to not only phosphorylation of the BCAR1 substrate domain and subsequent CRK-RAC1 signaling 21,37,89,99,101,102 but likely also other SRC-dependent activities. 108,[110][111][112] Interestingly, endogenous BCAR3 has also been found to mediate some of the proliferative effects of the EGF receptor in the nontransformed MCF12A human breast cell line, suggesting that a similar cross-talk with the EGF receptor might also play a role in breast cancer cell proliferation and antiestrogen resistance.…”

Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth

“…These IC 50 values were similar to those needed to induce internalization and degradation of HER3 receptors. Given upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors (for example lapatinib, gefitinib, erlotinib) [12-14], we next assessed the effect of perhexiline combined with lapatinib, an agent commonly used to treat HER2-positive breast cancer. Treatment of MDA-MB-468 cells with 100 nM lapatinib for 2 hours led to decreased phosphorylation and activation of HER3 and downstream Akt signaling (Figure 4C).…”

“…Unfortunately, clinical efficacy is not satisfactory as drug resistance reduces durable responses. Upregulation of HER3 has been found as one of the major mechanisms underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors (for example lapatinib, gefitinib, erlotinib) and to endocrine therapy in the treatment of breast cancer [12-14]. For example, it has been shown that the expression of HER3 ligand heregulin (HRG) as well as activation of HER3 signaling is involved in resistance to anti-estrogen therapies in vitro and in vivo [15-17].…”

Proteomic Strategies to Characterize Signaling Pathways

“…Protein microarrays have been used extensively for characterizing signaling pathways in various cancers including prostate cancer [83], ovarian cancer [84], and T-cell acute lymphoblastic leukemia [85]. In addition, protein microarray technology has also been used to identify molecular networks associated with antiestrogen resistance in breast cancers [86], to monitor response to kinase inhibitors [87], and to study aberrant activation of signaling molecules in head and neck cancers [88]. Peptide microarrays are also being used for high-throughput screening of kinase substrates and also to characterize motifs recognized by specific kinases [33,89].…”