Acquisition of Letrozole Resistance Through Activation of the p38/MAPK Signaling Cascade

Abstract

Background/Aim: Previous reports identified a global proteomic signature of estrogen-independent letrozole resistant breast cancer cells, however, it remains unclear how letrozole-resistance is impacted when cells remain estrogen receptor positive (ER+). Materials and Methods: To capture the protein expression profile associated with ER+ Aromatase inhibitor (AI) resistance, a global proteomic analysis was conducted using the letrozole-sensitive (T47Darom cells) and letrozole-resistant cells (T47DaromLR cells). To examine the molecular features associated with this phenotype Kaplan-Meier analysis, phospho-antibody arrays, proliferation and apoptosis assays were conducted. Results: MAP3K6 was upregulated in the T47DaromLR cells by 3.2-fold (p<0.01) which was associated with a decrease in relapse-free survival among breast cancer patients (p=0.0019). Members of the MAPK/p38 pathway (i.e., and p70S6K MAPK) were also increased in the T47DaromLR cells, while inhibiting p38 led to decreased proliferation and induction of apoptosis. Conclusion: Activation of the p38/MAPK pathway leads to ER+ AI-resistance.Aromatase inhibitors, like letrozole, are the first-line therapy for postmenopausal women with estrogen-dependent breast cancer. These agents block the biosynthesis of estrogen through aromatase inhibition and inhibit the proliferation of estrogen-dependent breast tumors. Unfortunately, after several years some patients develop therapeutic resistance, and the tumors continue to proliferate. To study aromatase inhibitor resistance in the laboratory setting, a letrozole resistant cell line was developed by inoculating the MCF-7Ca cells (MCF-7 cells stably transfected into the human aromatase gene) into immunocompromised mice and treating the mice with letrozole for 56 weeks (1). After long-term treatment, the tumors acquired resistance to letrozole, were cultured in vitro and termed long term letrozole treated (LTLT)-Ca cells. Previous studies in our lab have demonstrated that as the letrozole-sensitive cells (AC-1) transition to the letrozoleresistant phenotype (LTLT-Ca) they are associated with estrogen independence, up-regulation of growth factor signaling cascades, and increased motility (2). However, the changes that occur as cells acquire resistance to letrozole while maintaining estrogen dependence remains unclear.To this end, an in vitro model of letrozole resistance was developed whereby the T47D parental cell line was stably transfected with the human aromatase gene and termed the T47Darom cells. The T47Darom cells were treated with letrozole for 75 weeks until they became resistant to letrozole and were called T47DaromLR cells. When the T47DaromLR cells were characterized, they retained both aromatase activity and ER expression (3). As such, this was an ideal model to test the hypothesis that there are distinct proteomic profiles between estrogen-dependent and estrogen-independent letrozole resistant breast cancer cells, 583 This article is freely accessible online.

Novel Therapeutic Combination Targets the Growth of Letrozole-Resistant Breast Cancer through Decreased Cyclin B1

“…Given that the resistant cells were associated with an enhanced growth factor signaling signature and increased aromatase expression, we were interested in determining how proliferation would be impacted by treating the cells with an anti-estrogen (i.e., fulvestrant), the dual HER2 and EGFR inhibitor (i.e., lapatinib), as well as androstenedione (i.e., the substrate for the aromatase enzyme) for 24, 48, and 72 h. We also chose to treat both cell lines with 10 µM glyceollin ± lapatinib, since previous studies from our lab indicated that glyceollin inhibited the growth of estrogen-independent LTLT-Ca cells [ 23 ] and combination therapy inhibited the growth of estrogen-dependent T47DaromLR cells [ 11 ]. Stimulating the AC-1 cells with androstenedione dramatically increased cellular proliferation while only modest effects were observed in the LTLT-Ca cells ( Figure 2 ).…”

“…To date, few studies investigate targeted approaches for AI-resistant breast cancer such as the use of lapatinib [ 5 , 11 ], intermittent letrozole treatment [ 12 , 13 ], histone deacetylase (HDAC) inhibitors [ 12 , 14 ], cdk4/6 inhibitors [ 15 ], or co-targeting cdk2 and cdk4/6 [ 16 ]. While co-targeting cdk2 and cdk4/6 with endocrine therapy is promising in the pre-clinical setting, most of these approaches are associated with continued tumor proliferation and tumor relapse, and in most cases, failure to respond to treatment can be fatal.…”

“…When letrozole-resistant cells were treated with glyceollin I, cell proliferation and cell motility were attenuated and the EMT was reversed [ 23 ]. Although glyceollins as monotherapy are promising in the endocrine-refractory preclinical setting [ 11 , 23 , 24 ], it is unlikely that monotherapy could significantly decrease tumorigenesis and metastasis in the clinical setting. Therefore, it is plausible that combinatorial therapeutic approaches may be more efficacious.…”

Glyceollins Trigger Anti-Proliferative Effects in Hormone-Dependent Aromatase-Inhibitor-Resistant Breast Cancer Cells through the Induction of Apoptosis

“…Previous research demonstrated that as hormone-dependent, letrozole-sensitive breast cancer cells transition to a hormone-independent and letrozole-resistant phenotype, they utilize growth factor signaling pathways such as EGFR [ 5 ], p38/MAPK [ 4 , 15 , 16 ], and HER2 [ 17 ] as a mechanism of survival. This transition was associated with migration, EMT, and a more aggressive phenotype.…”

“…Since acquisition of resistance is associated with increased MAPK and HER2 ( Figure 2 ) and activation of p38/MAPK signaling cascade [ 15 ], we were interested in examining if the glyceollin + lapatinib induced growth suppression was due to decreased activation of MAPK and/or HER. When the cells were treated with glyceollin and/or lapatinib, there was not a significant change in HER2 and MAPK activation ( Figure A2 ), suggesting this may represent one mechanism of resistance but may not indicate the causative inhibitory mechanism by glyceollin even though our previous report demonstrates that the T47DaromLR cells have higher levels of activated phospho-p38 compared to the T47Darom cells [ 15 ].…”