Despite significant improvement in the treatment outcome of hormone responsive postmenopausal breast cancer, some patients eventually acquire resistance to aromatase inhibitors (AIs). Using our MCF-7Ca xenograft model, we observed that although AIs such as anastrozole initially inhibit tumor growth effectively, tumors eventually began to grow. Our previous data show that anastrozole-resistant tumors upregulate growth factor receptor pathways as they adapt to grow in the low estrogen environment. Therefore, in the current study, we investigated the effect of inhibiting the growth factor receptor pathways with a MEK-1/2 inhibitor selumetinib (AZD6244, ARRY-142866). We treated the mice with anastrozole-resistant tumors with selumetinib alone or in combination with anastrozole. MCF-7Ca cells were inoculated sc into ovariectomized athymic nude mice supplemented throughout the experiment with androstenedione (100 μg/day), the substrate for aromatase conversion to estrogen. Once the tumors reached a measurable size (~300 mm(3)), the mice were treated with anastrozole (200 μg/day), supplemented with androstenedione (Δ(4)A). The tumors in the anastrozole group doubled in volume after 6 weeks, at which time the animals were regrouped to receive the following treatments: (i) anastrozole, (ii) anastrozole withdrawal (Δ(4)A alone), (iii) selumetinib (25 mg/kg/d, bid, po), and (iv) selumetinib + anastrozole, (n = 10 mice/group). The treatments were given for 6 weeks (till week 12) and then the mice were euthanized, the tumors were collected and analyzed. The tumors of mice treated with selumetinib + anastrozole had significantly lower growth rates than those treated with single agents (p = 0.008). Western blot analysis of the tumors showed that treatment with anastrozole resulted in upregulation of proteins in the growth factor receptor cascade such as p-mTOR, pAkt, pMEK, and pMAPK. This was accompanied by downregulation of ERα protein, consistent with previous findings. The treatment of mice with selumetinib resulted in downregulation of activated MAPK, along with p-mTOR, which likely resulted in upregulation of ERα. Our results suggest that inhibition of the growth factor receptor pathway with selumetinib can reverse anastrozole resistance.

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20050 Background: The 88 kDa autocrine growth factor PC-Cell Derived Growth Factor (GP88) plays a critical role in breast tumorigenesis. GP88 expression was low in estrogen receptor positive cells, whereas in ER negative cells, it was constitutively overexpressed. Increased GP88 expression was associated with anti-estrogen therapy resistance in ER+ cells and Herceptin resistance in Her-2 overexpressing breast tumors. Antisense inhibition of GP88 expression in human breast adenocarcinoma lead to inhibition of tumor growth in vivo. Immunohistochemical studies have shown that GP88 was expressed in 80% of invasive ductal carcinomas in correlation with expression of poor prognosis markers whereas normal tissues and benign breast lesions were negative. Since GP88 is secreted by breast cancer cells, we examined whether GP88 was found in the circulation at an elevated level in the sera of breast cancer patients when compared to healthy individuals. Methods: A blood sampling study was conducted to determine the serum level of GP88 in healthy volunteers (HV) and breast cancer patients (BC pts). Ten ml of blood was drawn every three months to obtain serum. GP88 serum concentration was determined in triplicate by quantitative enzyme immunoassay using human GP88 as standard. 126 BC pts were accrued. . In addition, sera from 53 healthy volunteers were obtained to establish a GP88 baseline in HV. BC pts characteristics: race: Caucasian- 61, African American-60, Asian-5; median age: 52.5 (range 26–84), stage I-32, II-34, III-18, IV-42. Results: Circulating GP88 was measurable in the serum. Median level of GP88 was 32.8 ng/ml (range 15.3–42.8) in HV and 43.8 ng/ml (range 15.4–158.4) in BC pts, (p-value = 0.0007). Conclusions: GP88 is measurable in the sera of HV and BC pts. Comparison between the two groups indicates that GP88 level is significantly higher in the sera of BC pts. These studies are important since it identifies GP88 as a measurable biomarker that is also a therapeutic target of malignant transformation or malignant progression of breast carcinoma (BC). Future studies will examine the correlation of GP88 level with BC prognostic factors. Correlation between the serum level of GP88 and therapeutic response to systemic therapy in breast cancer patients will also be assessed. [Table: see text]

Treatment of hormone sensitive breast tumors with endocrine therapy such as antiestrogens or aromatase inhibitors has improved their clinical outcomes. However, not all tumors respond and the ones that do respond may eventually acquire resistance. One of the proposed mechanisms of resistance to endocrine therapy is overexpression of ERα and cross-talk of ERα with growth factor receptors. Studies including our own have shown that downregulation of ER with pure antiestrogen fulvestrant in combination with AIs may prolong responsiveness of the tumors to endocrine agents. Fulvestrant has been employed as either first or second line treatment for ER positive breast cancers alone or in combination with AIs. Studies have suggested that further escalation of dose may provide further benefit. However, dose escalation of fulvestrant which is administered via intramuscular injection is difficult due to its poor solubility. To overcome this shortcoming of an injectable drug, a novel orally active SERD (selective estrogen receptor downregulator), AZD9496 was developed. In addition to being orally active, AZD9496 is selective for mammary ERα. In the current study, we compared the effect of AZD9496 and fulvestrant on the growth of MCF-7Ca (human estrogen receptor positive MCF-7 cells stably transfected with human placental aromatase gene) xenografts grown in ovariectomized athymic nude mice. Tumors were allowed to form with androstenedione (aromatizable source of estrogen) supplement. When the tumors reached ~250 mm3, mice were grouped such that the mean tumor volumes were not significantly different (p>0.99). Mice bearing xenografts of MCF-7Ca were then treated with fulvestrant (1 mg/d-sc) or AZD9496 (5 mg/kg/d-po), alone or in combination with anastrozole (200μg/d-sc) for 23 weeks. Tumors were measured weekly and growth rate was calculated. AZD9496 was significantly better at inhibiting the growth of tumors compared to control (p<0.001) and anastrozole (p=0.04). AZD9496 was equally effective in inhibiting the growth of MCF-7Ca xenografts as fulvestrant (growth rate, p>0.99 and tumor volume on week 23, p=0.99). In the second study, efficacy of AZD9496 was evaluated on against anastrozole resistant MCF-7Ca xenografts. Tumors were treated with anastrozole (200μg/d) for 13 weeks. During this time, the tumors initially regressed but eventually began to grow and had doubled in volume. At this time-point, they were regrouped to receive second line treatment. Single agent AZD9496 was marginally significant compared to continued anastrozole treatment (p=0.07). Nevertheless, second line treatment with AZD9496 was equally effective as fulvestrant (p=0.36). The combination of anastrozole with AZD9496/fulvestrant was more effective in reducing tumor growth compared to continued anastrozole treatment. Next, we measured the effect of AZD9496 on the mouse uterus. Uterine weight of mice treated with AZD9496 was not significantly different from mice that were treated with androstenedione (p=0.99). These results suggest that AZD9496 was selective for tumor ERα and had no effect on the uterine ERα. These results suggest that AZD9496 may be a better alternative to fulvestrant due to its selectivity for mammary ER and same efficacy as fulvestrant obtained upon oral administration. Citation Format: Sabnis GJ, Kazi A, Schech A, Yu S, Golubeva O, Weir H, Brodie A. A new oral SERD AZD9496 for treatment of hormone dependent postmenopausal breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-09-10.