TamR Breast Cancer Research Articles

Abstract IA17: Targeting downstream effectors of growth factor signaling

Abstract Transmembrane growth factor receptors mediate signaling through multiple intracellular pathways. In breast cancer cells, the type I insulin-like growth factor receptor (IGF1R) has been implicated in the malignant phenotype. However, clinical trials with anti-IGF1R antibodies have been disappointing, in part, due to adaptive feedback pathways stimulated when IGF1R is blocked. To determine whether IGF1R inhibition could be enhanced by disrupting other pathways, we evaluated gene expression induced by receptor activation. In previous work, we found that xCT (SLC7A11) mRNA expression was increased by IGF-I in estrogen receptor (ER) positive breast cancer cell lines (MCF-7, T47D, and ZR-75-1) in an insulin receptor substrate-1 (IRS-1) dependent manner. xCT encodes the functional subunit of the heterodimeric plasma membrane transport system xC- critical for the cellular uptake of cystine to generate glutathione to modulate cellular redox control. IGF-I increased xC- transporter expression and function to control cellular redox levels. In MCF-7 cells, IGF-I-stimulated monolayer and anchorage-independent growth was suppressed by infecting cells with xCT shRNA or by treating cells with the xC- chemical inhibitor sulfasalazine (SASP). Anchorage-independent growth assays showed that disruption of xC- function by SASP sensitized cellular response of MCF-7 cells to anti-IGF-IR inhibitors (monoclonal antibody huEM164 and tyrosine kinase inhibitor NVP-AEW-541). IGF1R also activates PI3K/Akt/mTORC1 signaling to affect ER phosphorylation and mRNA cap dependent translation. In tamoxifen resistant cells, IGF1R is lost yet PI3K signaling is maintained. Since mRNA cap dependent translation is increased by PI3K signaling, we determined if inhibition of the eIF4F translation pathway would affect endocrine responsive and tamoxifen resistant cells. The eIF4F translation pathway is activated by IGF1R in wild-type cells and hyperactive in tamoxifen resistant MCF-7L (TamR) breast cancer cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of parental and TamR cells. Use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, pharmacologic inhibition of the eIF4E-eIF4G interaction also decreased the proliferation and anchorage dependent colony formation in TamR cells. Taken together, these data show that IGF1R activation stimulates multiple downstream effectors important for breast cancer cell biology. Inhibition of selected downstream signaling molecules is likely to have synergy with anti-IGF-IR drugs. Citation Format: Yuzhe Yang, Dedra Fagan, Lynsey Fettig Anderson, Kelly LaPara, Xihong Zhang, Aleksandra Ochnik, Jie Ying Chan, Heather Beckwith, Douglas Yee. Targeting downstream effectors of growth factor signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA17.

Re-expression of microRNA-375 reverses both tamoxifen resistance and accompanying EMT-like properties in breast cancer

Epithelial-mesenchymal transition (EMT) is an initiating event in tumor cell invasion and metastasis. It has been shown to occur in resistance to a range of cancer therapies, including tamoxifen. MicroRNAs (miRNAs) have been associated with EMT as well as resistance to standard therapies. To investigate the role of miRNAs in the development of resistance to tamoxifen as well as accompanying EMT-like properties, we established a tamoxifen-resistant (TamR) model by continually exposing MCF-7 breast cancer cells to tamoxifen. In addition to the molecular changes known to be involved in acquired tamoxifen resistance, TamR cells displayed mesenchymal features and had increased invasiveness. Genome-wide miRNA microarray analysis revealed that miRNA-375 was among the top downregulated miRNAs in resistant cells. Re-expression of miR-375 was sufficient to sensitize TamR cells to tamoxifen and partly reversed EMT. A combination of mRNA profiling, bioinformatics analysis and experimental validation identified metadherin (MTDH) as a direct target of miR-375. Knockdown of MTDH partially phenocopied the effects of miR-375 on the sensitivity to tamoxifen and the reversal of EMT. We observed an inverse correlation between the expression of miR-375 and its target MTDH in primary breast cancer samples, implying the pathological relevance of targeting. Finally, tamoxifen-treated patients with higher expression of MTDH had a shorter disease-free survival and higher risk of relapse. As most cancer-related deaths occur because of resistance to standard therapies and metastasis, re-expression of miR-375 or targeting MTDH might serve as potential therapeutic approaches for the treatment of TamR breast cancer.

P3-01-05: Wnt Signaling: A New Target for Treatment and Prevention of Endocrine Resistant Breast Cancer?

Abstract Background: Wnt signaling is altered in nearly half of all breast cancers. Both up regulation of Wnt pathway activators and down regulation of pathway inhibitors have been identified in breast cancer. Previously we have reported that activity of β-catenin is altered in acquired tamoxifen-resistant (TamR) breast cancer cells compared to endocrine-sensitive parental MCF7 cells, with an increase in β-catenin-mediated gene transcription. Data is lacking on the role of Wnt signaling in acquired endocrine resistance in breast cancer. Endocrine relapse predicts a poor clinical outcome and remains an important target for future drug development. In this study we investigated the role of Wnt signaling in acquired tamoxifen resistance and its contribution to the associated aggressive phenotype in vitro. Materials/Methods: We performed exploratory Affymetrix HGU-133A gene microarray analysis of key Wnt pathway components in log-phase TamR vs. MCF7 cell lines, as well as comparing MCF7 cells +/− 10 day tamoxifen exposure. This was paralleled by western blot (WB) analysis. Effects of Wnt signaling inhibition on growth and cell migration were explored using two commercially available inhibitors: IWP2 (inhibits Wnt production, pLRP6) and PNU74654 (interferes with β-catenin-Tcf/Lef binding). MTT cell proliferation assays (6 days) and cell migration assays (24hrs) were carried out. Results: Microarray analysis of key Wnt pathway components revealed up regulation of wnts 3, 4 and 6; up regulation of Wnt pathway activators LRP5 and LRP6; down regulation of pathway inhibitors (DKK1, DKK4); and down regulation of Axinl (destruction complex component). Changes are >1.5-fold. WB analysis confirmed up regulation of total β-catenin and LRP6 in TamR versus MCF7 cells as well as activity of LRP6. In accordance with this, inhibition of Wnt signaling using IWP2 10uM or PNU74654 10uM, whilst having no effect on MCF7 cell proliferation, significantly suppressed the growth of TamR cells (p<0.001). Inhibition of Wnt signaling in TamR cells also suppressed cell migration (p<0.001). Gene microarray data for MCF7 cells treated with tamoxifen for 10 days showed up regulation of LRP6 and wnt4 and down regulation of LRP5, wnt6, Axin1 and DKK4 compared to control. Discussion: These observations suggest that deregulated Wnt signaling may play a role in acquired tamoxifen resistance in breast cancer where it may act to promote growth and the development of a more aggressive phenotype. Its interaction with other pathways is important in the prevention of resistance with co-treatment strategies. Wnt1 can rescue MCF7 cells from growth suppression caused by 4-hydroxytamoxifen. Given the early changes in the Wnt pathway in MCF7 cells treated with tamoxifen, further exploration in the context of earlier Wnt targeting alongside antihormone treatment is required. Monitoring of Wnt signaling within clinical samples from patients who have relapsed after hormone treatment is urgently warranted given that the Wnt inhibitors showed promise in controlling this state in vitro. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-05.

Study and application of epidermal growth factor receptor signaling pathway in tamoxifen resistance of breast cancer

In clinical it has been demonstrated that a lot of malignant tumors exist over-expression of the epidermal growth factor receptor (EGFR) , and the phenomena in the breast cancer which is resistante to Tamoxifen(TAM-R) is associated with poor survival. It is very important to understand the underlying mechanisms of EGFR in the TAM-R breast cancer, and major efforts are now being made to overcome the condition.In some preclinical trials,it has been proved that mechanisms of tamoxifen resistance to endocrine therapy is associated with EGFR signaling activity in hormone-dependent breast cancer cells. There have been reports of the inhibitors to prevent EGFR signaling to treatment of the TAM-R breast cancer in clinical. However, the clinical data demonstrated that not all patients responded to the inhibitor therapy, indicating the existence of an intrinsic or de novo resistance to the drug, which is a new problem. Key words: Receptor,epidermal growth factor ; Estrogen receptor alpha ; Tamoxifen ; Trug resistance,neoplasm ; Protein-tyrosine kinases ;