Dual IGF-1R/InsR inhibitor BMS-754807 synergizes with hormonal agents in treatment of estrogen-dependent breast cancer.

Insulin-like growth factor (IGF) signaling has been implicated in the resistance to hormonal therapy in breast cancer. Using a model of postmenopausal, estrogen-dependent breast cancer, we investigated the antitumor effects of the dual IGF-1R/InsR tyrosine kinase inhibitor BMS-754807 alone and in combination with letrozole or tamoxifen. BMS-754807 exhibited antiproliferative effects in vitro that synergized strongly in combination with letrozole or 4-hydroxytamoxifen and fulvestrant. Similarly, combined treatment of BMS-754807 with either tamoxifen or letrozole in vivo elicited tumor regressions not achieved by single-agent therapy. Notably, hormonal therapy enhanced the inhibition of IGF-1R/InsR without major side effects in animals. Microarray expression analysis revealed downregulation of cell-cycle control and survival pathways and upregulation of erbB in response to BMS-754807 plus hormonal therapy, particularly tamoxifen. Overall, these results offer a preclinical proof-of-concept for BMS-754807 as an antitumor agent in combination with hormonal therapies in hormone-sensitive breast cancer. Cooperative cell-cycle arrest, decreased proliferation, and enhanced promotion of apoptosis may contribute to antitumor effects to be gauged in future clinical investigations justified by our findings.

Anti-HER2 immunoliposomes for selective delivery of electron paramagnetic resonance imaging probes to HER2-overexpressing breast tumor cells.

Electron paramagnetic resonance (EPR) imaging is an emerging modality that can detect and localize paramagnetic molecular probes (so-called spin probes) in vivo. We previously demonstrated that nitroxide spin probes can be encapsulated in liposomes at concentrations exceeding 100 mM, at which nitroxides exhibit a concentration-dependent quenching of their EPR signal that is analogous to the self-quenching of fluorescent molecules. Therefore, intact liposomes encapsulating high concentrations of nitroxides exhibit greatly attenuated EPR spectral signals, and endocytosis of such liposomes represents a cell-activated contrast-generating mechanism. After endocytosis, the encapsulated nitroxide is liberated and becomes greatly diluted in the intracellular milieu. This dequenches the nitroxides to generate a robust intracellular EPR signal. It is therefore possible to deliver a high concentration of nitroxides to cells while minimizing background signal from unendocytosed liposomes. We report here that intracellular EPR signal can be selectively generated in a specific cell type by exploiting its expression of Human Epidermal Growth Factor Receptor 2 (HER2). When targeted by anti-HER2 immunoliposomes encapsulating quenched nitroxides, Hc7 cells, which are novel HER2-overexpressing cells derived from the MCF7 breast tumor cell line, endocytose the liposomes copiously, in contrast to the parent MCF7 cells or control CV1 cells, which do not express HER2. HER2-dependent liposomal delivery enables Hc7 cells to accumulate 750 µM nitroxide intracellularly. Through the use of phantom models, we verify that this concentration of nitroxides is more than sufficient for EPR imaging, thus laying the foundation for using EPR imaging to visualize HER2-overexpressing Hc7 tumors in animals.

ETAR antagonist ZD4054 exhibits additive effects with aromatase inhibitors and fulvestrant in breast cancer therapy, and improves in vivo efficacy of anastrozole.

Endothelin-1 (ET-1) and endothelin A receptor (ETAR) contribute to the development and progression of breast carcinomas by modulating cell proliferation, angiogenesis, and anti-apoptosis. We investigated antitumoral effects of the specific ETAR antagonist ZD4054 in breast cancer cells and xenografts, and assessed antitumoral efficacy of the combinations of ZD4054 with aromatase inhibitors and fulvestrant. Gene expression changes were assessed by quantitative real-time PCR. Cell proliferation was measured using alamarBlue; migration and invasion assays were performed using modified Boyden chambers. Evaluating the antitumoral efficacy of ZD4054 in vivo, different breast cancer models were employed using nude mice xenografts. ZD4054 reduced ET-1 and ETAR expression in MCF-7, MDA-MB-231, and MDA-MB-468 breast cancer cells in a concentration-dependent manner. ZD4054 inhibited invasion by up to 37.1% (P = 0.022). Combinations of ZD4054 with either anastrozole or letrozole produced significant reductions in migration of aromatase-overexpressing MCF-7aro cells (P < 0.05). Combination of ZD4054 with fulvestrant reduced MCF-7 cell migration and invasion by 36.0% (P = 0.027) and 56.7% (P < 0.001), respectively, with effects significantly exceeding those seen with either compound alone. Regarding tumor volume reduction in vivo, ZD4054 (10 mg/kg) was equipotent to fulvestrant (200 mg/kg) and exhibited additive effects with anastrozole (0.5 mg/kg). These data are the first indicating that selective ETAR antagonism by ZD4054 displays antitumoral activity on breast cancer cells in vitro and in vivo. Our data strongly support a rationale for the clinical use of ZD4054 in combination with endocrine therapies.

Stopping treatment can reverse acquired resistance to letrozole.

Using the intratumoral aromatase xenograft model, we have observed that despite long-lasting growth inhibition, tumors eventually begin to grow during continued letrozole treatment. In cells isolated from these long-term letrozole-treated tumors (LTLT-Ca), estrogen receptor-alpha (ERalpha) levels were decreased, whereas signaling proteins in the mitogen-activated protein kinase cascade were up-regulated along with human epidermal growth factor receptor 2 (Her-2). In the current study, we evaluated the effect of discontinuing letrozole treatment on the growth of letrozole-resistant cells and tumors. The cells formed tumors equally well in the absence or presence of letrozole and had similar growth rates. After treatment was discontinued for 6 weeks, letrozole was administered again. Marked tumor regression was observed with this second course of letrozole treatment. Similarly, in MCF-7Ca xenografts, a 6-week break in letrozole treatment prolonged the responsiveness of the tumors to letrozole. To understand the mechanisms of this effect, LTLT-Ca cells were cultured in the absence of letrozole for 16 weeks. The resulting cell line (RLT-Ca) exhibited properties similar to MCF-7Ca cells. The cell growth was inhibited by letrozole and stimulated by estradiol. The expression of phosphorylated mitogen-activated protein kinase (MAPK) was reduced and ERalpha and aromatase levels increased compared with LTLT-Ca cells and were similar to levels in MCF-7Ca cells. These results indicate that discontinuing treatment can reverse letrozole resistance. This could be a beneficial strategy to prolong responsiveness to aromatase inhibitors for patients with breast cancer.

Combination of anastrozole with fulvestrant in the intratumoral aromatase xenograft model.

Although the aromatase inhibitor anastrozole has been shown to be very effective in the treatment of hormone-dependent postmenopausal breast cancer, some patients with advanced disease will develop resistance to treatment. To investigate therapeutic strategies to overcome resistance to anastrozole treatment, we have used an intratumoral aromatase model that simulates postmenopausal breast cancer patients with estrogen-dependent tumors. Growth of the tumors in the mice was inhibited by both anastrozole and fulvestrant compared with the control tumors. Nevertheless, tumors had doubled in size at 5 weeks of treatment. We therefore investigated whether switching the original treatments to anastrozole or fulvestrant alone or the combination of anastrozole plus fulvestrant would reduce tumor growth. The results showed that the best strategy to reverse the insensitivity to anastrozole or fulvestrant is to combine the two agents. Additionally, the tumors treated with anastrozole plus fulvestrant from the beginning had only just doubled their size after 14 weeks of treatment, whereas the anastrozole and fulvestrant treatments alone resulted in 9- and 12-fold increases in tumor size, respectively, in the same time period. Anastrozole plus fulvestrant from the beginning or in sequence was associated with down-regulation of signaling proteins involved in the development of hormonal resistance such as insulin-like growth factor type I receptor beta, mitogen-activated protein kinase (MAPK), p-MAPK, AKT, mammalian target of rapamycin (mTOR), p-mTOR, and estrogen receptor alpha compared with tumors treated with anastrozole or fulvestrant alone. These results suggest that blocking the estrogen receptor and aromatase may delay or reverse the development of resistance to aromatase inhibitors in advanced breast cancer patients.

Preclinical modeling of endocrine response and resistance: focus on aromatase inhibitors.

The authors developed a breast cancer intratumoral aromatase model system to compare the antitumor efficacy of several aromatase inhibitors (AIs) and antiestrogens (AEs). Although the AI letrozole caused sustained growth inhibition, tumors eventually began to grow, even when treatment was maintained. For the current study, the mechanisms of resistance to letrozole during the course of treatment were investigated. Estrogen receptor alpha (ER-alpha) levels decreased below control levels in letrozole-resistant tumors. The decrease was simultaneous to an increase in phosphorylation of ER-alpha and an unaltered expression of progesterone receptor (PgR). Expression levels of HER-2, activated (phosphorylated) SHC-adaptor protein (p-Shc), growth factor receptor-bound protein 2 (Grb-2), p-Raf, phosphorylated mitogen-activated protein kinase kinase 1/2 (p-Mekl/2), and phosphorylated mitogen-activated protein kinase (p-MAPK) were increased. When cells isolated from letrozole-resistant tumors (LTLTCa cells) were treated with inhibitors of the HER-2 signaling pathway, ER-alpha expression and estradiol-stimulated transactivation was restored. The HER-2 blocker trastuzumab also restored the sensitivity of LTLTCa cells to AIs and AEs. These findings suggested that there is crosstalk between ER and HER-2 signaling. To prevent activation of the HER-2 pathway and resistance to AIs, mice were treated with a combination of AIs and the ER down-regulator fulvestrant. There was no increase in HER-2 or p-MAPK expression, and tumor growth was inhibited significantly. When trastuzumab was added to unresponsive tumors under letrozole treatment, it significantly inhibited tumors growth compared with switching to trastuzumab alone. However, the trastuzumab plus letrozole combination was more effective than letrozole alone only in refractory breast tumors. These results suggested that blocking both ER and HER-2 signaling may delay the development of resistance to AIs in patients with recurrent breast cancer.

Role of androgens on MCF-7 breast cancer cell growth and on the inhibitory effect of letrozole.

Previous work has shown that androgens inhibit breast cancer cells and tumor growth. On the other hand, androgens can be converted to mitogenic estrogens by aromatase in breast cancer cells. Here, we report that androgens, such as the aromatizable androstenedione and the non-aromatizable 5alpha-dihydrotestosterone, inhibit MCF-7 cell proliferation. This effect is observed only in the absence or at a low concentration of estrogens and is evident in cells with low aromatase activity. Growth of a new aromatase stably transfected MCF-7 cell line (Ac1) was stimulated by conversion of androstenedione into estrogens and was sensitive to aromatase inhibitors. We show that blockade of the androgen receptor (AR) in these cells by the antiandrogen casodex or by the anti-AR small interfering RNA inhibited the antiproliferative effect of dihydrotestosterone and letrozole (aromatase inhibitor). We also show that suppression of the estrogen-induced antiapoptotic protein Bcl-2 may be involved in the antiproliferative effects of androgens and letrozole. These effects can be reversed by casodex. In conclusion, the results suggest that aromatase inhibitors may exert their antiproliferative effect not only by reducing the intracellular production of estrogens but also by unmasking the inhibitory effect of androgens acting via the AR.

Synthesis and preliminary pharmacological evaluation of new (+/-) 1,4-naphthoquinones structurally related to lapachol.

Seven new 1,4-naphthoquinones structurally related to lapachol were synthesized from lawsone and oxygenated arylmercurials. These compounds can also be seen as pterocarpan derivatives where the A-ring was substituted by the 1,4-naphthoquinone nucleus. Pharmacological screening provided evidence of significant biological activities, including effects against proliferation of the MCF-7 human breast cancer cell line, against Herpes Simplex Virus type 2 infection, and against snake poison-induced myotoxicity. One derivative displaced flunitrazepam binding and showed benzodiazepine-like activity, suggesting novel neuroactive structural motifs.