Upregulation of ER Signaling as an Adaptive Mechanism of Cell Survival in HER2-Positive Breast Tumors Treated with Anti-HER2 Therapy

Abstract

Purpose: To investigate the direct effect and therapeutic consequences of epidermal growth factor receptor 2 (HER2)-targeting therapy on expression of estrogen receptor (ER) and Bcl2 in preclinical models and clinical tumor samples.

Experimental design: Archived xenograft tumors from two preclinical models (UACC812 and MCF7/HER2-18) treated with ER and HER2-targeting therapies and also HER2+ clinical breast cancer specimens collected in a lapatinib neoadjuvant trial (baseline and week 2 posttreatment) were used. Expression levels of ER and Bcl2 were evaluated by immunohistochemistry and Western blot analysis. The effects of Bcl2 and ER inhibition, by ABT-737 and fulvestrant, respectively, were tested in parental versus lapatinib-resistant UACC812 cells in vitro.

Results: Expression of ER and Bcl2 was significantly increased in xenograft tumors with acquired resistance to anti-HER2 therapy compared with untreated tumors in both preclinical models (UACC812: ER P = 0.0014; Bcl2 P < 0.001 and MCF7/HER2-18: ER P = 0.0007; Bcl2 P = 0.0306). In the neoadjuvant clinical study, lapatinib treatment for 2 weeks was associated with parallel upregulation of ER and Bcl2 (Spearman coefficient: 0.70; P = 0.0002). Importantly, 18% of tumors originally ER-negative (ER(-)) converted to ER(+) upon anti-HER2 therapy. In ER(-)/HER2(+) MCF7/HER2-18 xenografts, ER reexpression was primarily observed in tumors responding to potent combination of anti-HER2 drugs. Estrogen deprivation added to this anti-HER2 regimen significantly delayed tumor progression (P = 0.018). In the UACC812 cells, fulvestrant, but not ABT-737, was able to completely inhibit anti-HER2-resistant growth (P < 0.0001).

Conclusions: HER2 inhibition can enhance or restore ER expression with parallel Bcl2 upregulation, representing an ER-dependent survival mechanism potentially leading to anti-HER2 resistance.

Figure 1. Expression of ER and its downstream gene products Bcl2 and PR increases at the time of acquired resistance to HER2-targeting therapy in two different HER2+/ER+ pre-clinical models in vivo

A. Expression levels of ER, Bcl2, and PR in UACC812 tumor xenografts grown only in the presence of estrogen (E₂; n=6) and treated with lapatinib in the presence of E₂ (E₂ + L; n=6). L-treated tumors showed a significant increase in the expression of ER, PR, and Bcl2, compared with tumors treated with vehicle. B. Expression levels of ER, Bcl2, and PR in MCF7 HER2-18 tumor xenografts grown in presence of E₂ with (n=9) or without (n=9) HER2-targeted therapy. Tumors treated with lapatinib plus trastuzumab (LT) were harvested either when still sensitive to the treatment (n=4) or at the time of acquired resistance (n=5)(8). LT-sensitive tumors showed a non-significant trend toward increase in ER expression, as well as in Bcl2 and PR levels, compared with control tumors (E₂). LT-resistant tumors showed a significant increase in ER, Bcl2, and PR expression compared with control.

Figure 2. ER and Bcl2 changes upon neoadjuvant treatment with lapatinib

A. Each line represents changes in ER expression levels from baseline to week 2. ER levels are expressed as Allred scores. Solid red lines indicate tumors with ER-positive status at baseline (Allred score ≥ 3); dashed red lines indicate those three tumors that were ER-negative at baseline (Allred score < 3) and became positive at week 2; solid black lines indicate tumors with ER-negative status at baseline that remained negative at week 2 (14 tumors). B. Each line represents Bcl2 expression changes from baseline to week 2. Bcl2 levels are expressed as percentage of positive cells. Solid red lines indicate Bcl2 changes in the 6 tumors with ER-positive status at baseline; dashed red lines indicate Bcl2 changes in the 3 tumors converting from ER-negative to positive; solid black lines indicate Bcl2 changes in the 14 tumors that were ER-negative at both baseline and week 2. C. Percentage of tumors with a Bcl2 increase over time, according to ER status. Bcl2 increased at week 2 in 8 out of 9 of the ER-positive tumors, (including the 3 tumors converting to ER-positive), and in 1 out the 14 tumors that remained ER-negative.

Figure 3. Effects of pharmacologic inhibition of Bcl2 and ER in ER+/HER2+ cells with acquired resistance to lapatinib in vitro

A. Tumor cell growth relative to control (DMSO) evaluated by methylene blue assay after 6 days of treatment with DMSO, ABT-737 (1 μM), fulvestrant (Ful; 0.1 μM), or the combination of ABT-737 with Ful, in parental and lapatinib (L)-resistant UACC812 cells. B. Evaluation of apoptosis by annexin V assay after 24 hours of treatment with either DMSO (control), ABT-737 (1 μM), Ful (0.1 μM), or the combination of ABT-737 with Ful, in parental and L-resistant UACC812 cells. C. Western blot evaluation of phosphorylated (p)-HER2 and total (t)-HER2, and ER and its downstream gene products PR and Bcl2, in parental and L-resistant UACC812 cells after 72 hours of treatment with either DMSO, ABT-737 (1 μM), Ful (0.1 μM), or the combination of ABT-737 with Ful.

Figure 4. Endocrine therapy delays tumor progression in presence of restored ER expression in tumor xenografts treated with anti-HER therapy

A. Mice bearing MCF7 HER2-18 tumor xenografts were subjected to estrogen deprivation (ED) until development of resistance, which was associated with complete loss of ER expression (18). At that time, the combination of trastuzumab, pertuzumab, and gefitinib (TPG) was added to ED. Anti-HER therapy induced tumor regression in 53% of mice (responders), whereas the remaining tumors continued to grow despite the inhibition of both ER and HER pathways. Restoration of ER expression was significantly associated with tumor response. B. Mice bearing MCF7 HER2-18 tumor xenografts were randomized at the time of ED-resistance to receive TPG with or without continuing endocrine therapy (ED). The combination of TPG + ED was able to significantly delay tumor growth compared to TPG + E2 in the responders, but not in the non-responders.