Angela H. Brodie

Obesity, energy balance, and cancer: New opportunities for prevention

Obesity is associated with increased risk and poor prognosis for many types of cancer. The mechanisms underlying the obesity-cancer link are becoming increasingly clear and provide multiple opportunities for primary to tertiary prevention. Several obesity-related host factors can influence tumor initiation, progression and/or response to therapy, and these have been implicated as key contributors to the complex effects of obesity on cancer incidence and outcomes. These host factors include insulin, insulin-like growth factor-I, leptin, adiponectin, steroid hormones, cytokines, and inflammation-related molecules. Each of these host factors is considered in the context of energy balance and as potential targets for cancer prevention. The possibility of prevention at the systems level, including energy restriction, dietary composition, and exercise is considered as is the importance of the newly emerging field of stem cell research as a model for studying energy balance and cancer prevention. Cancer Prev Res; 5(11); 1260–72. ©2012 AACR.

Pregnenolone stimulates LNCaP prostate cancer cell growth via the mutated androgen receptor

Pregnenolone (P(5)), a common precursor of many steroids, is present in the blood of normal adult men at concentrations of 1-3 nM. In vitro, P(5) was found to stimulate LNCaP-cell proliferation 7-8-fold at a physiological concentration (2 nM), and 3-4-fold at a subphysiological concentration (0.2 nM). Growth stimulation at the 2-nM concentration was comparable with that of the androgen, dihydrotestosterone at its physiological concentration (0.5 nM; 9-10-fold increase in cell number). To determine whether P(5) or its metabolites were mediating this growth response, LNCaP cells were incubated with [3H]P(5) and high-performance liquid chromatography (HPLC) was performed. After a 48-h exposure, two unidentified metabolites were detected. Although, the P(5) metabolites slightly increased LNCaP-cell growth in vitro, their effect was significantly less than P(5) alone, suggesting that the growth stimulation was mediated by P(5) itself. We further showed that P(5) sustained its proliferative activity in vivo and stimulated the growth of LNCaP-tumor xenografts in intact male SCID mice as well as in castrated animals. In order to determine whether P(5) was binding to a specific site in LNCaP cells, receptor binding studies were performed. Scatchard analysis predicted for a single class of binding sites with K(d)=1.4 nM. Studies were performed to determine the effects of P(5) on transcription mediated by wild-type and LNCaP androgen receptors. P(5) was shown to activate transcription through the LNCaP androgen receptor (AR), but not the wild-type AR. This implies that P(5) most likely stimulates LNCaP-cell proliferation through binding to the cellular mutated AR present in LNCaP cells. We have also demonstrated that drugs designed to be antagonists of the androgen, progesterone and estrogen receptors, and one of our novel compounds designed to be an inhibitor of androgen synthesis, were potent inhibitors of the AR-mediated transcriptional activity induced by P(5), and were able to inhibit LNCaP-cell proliferation. These findings suggest that some prostate cancer patients who appear to become hormone-independent may have tumors which are stimulated by P(5) via a mutated AR and that these patients could benefit from treatment with antiestrogens, antiprogestins, or with some of our novel androgen synthesis inhibitors.

Aromatase and its inhibitors—an overview☆

Estrogen synthesis by aromatase occurs in a number of tissues throughout the body. Strategies which reduce production of estrogen offer useful means of treating hormone-dependent breast cancer. Initially, several steroidal compounds were determined to be selective inhibitors of aromatase. The most potent of these, 4-hydroxyandrostenedione (4-OHA) inhibits aromatase competitively but also causes inactivation of the enzyme. A number of other steroidal inhibitors appear to act by this mechanism also. In contrast, the newer imidazole compounds are reversible, competitive inhibitors. In vivo studies demonstrated that 4-OHA inhibited aromatase activity in ovarian and peripheral tissues and reduced plasma estrogen levels in rat and non-human primate species. In rats with mammary tumors, reduction in ovarian estrogen production was correlated with tumor regression. 4-OHA was also found to inhibit gonadotropin levels in animals in a dose-dependent manner. The mechanism of this effect appears to be associated with the weak androgenic activity of the compound. Together with aromatase inhibition, this action may contribute to reducing the growth stimulating effects of estrogen. A series of studies have now been completed in postmenopausal breast cancer patients treated with 4-OHA either 500 mg/2 weeks or weekly, or 250 mg/2 weeks. These doses did not affect gonadotropin levels. Plasma estrogen concentrations were significantly reduced. Complete or partial tumor regression occurred in 26% of the patients and the disease was stabilized in 25% of the patients. The results suggest that 4-OHA is of benefit to postmenopausal patients who have relapsed from prior hormonal therapies. Several of the steroidal inhibitors are now entering clinical trials as well as non-steroidal compounds which are more potent and selective than aminoglutethimide. Aromatase inhibitors should provide several useful additions to the treatment of breast cancer.

Targeting abnormal DNA repair in therapy-resistant breast cancers

Although hereditary breast cancers have defects in the DNA damage response that result in genomic instability, DNA repair abnormalities in sporadic breast cancers have not been extensively characterized. Recently, we showed that, relative to nontumorigenic breast epithelial MCF10A cells, estrogen receptor–positive (ER+) MCF7 breast cancer cells and progesterone receptor–positive (PR+) MCF7 breast cancer cells have reduced steady-state levels of DNA ligase IV, a component of the major DNA–protein kinase (PK)-dependent nonhomologous end joining (NHEJ) pathway, whereas the steady-state level of DNA ligase IIIα, a component of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, is increased. Here, we show that tamoxifen- and aromatase-resistant derivatives of MCF7 cells and ER−/PR− cells have even higher steady-state levels of DNA ligase IIIα and increased levels of PARP1, another ALT NHEJ component. This results in increased dependence upon microhomology-mediated ALT NHEJ to repair DNA double-strand breaks (DSB) and the accumulation of chromosomal deletions. Notably, therapy-resistant derivatives of MCF7 cells and ER−/PR− cells exhibited significantly increased sensitivity to a combination of PARP and DNA ligase III inhibitors that increased the number of DSBs. Biopsies from ER−/PR− tumors had elevated levels of ALT NHEJ and reduced levels of DNA–PK-dependent NHEJ factors. Thus, our results show that ALT NHEJ is a novel therapeutic target in breast cancers that are resistant to frontline therapies and suggest that changes in NHEJ protein levels may serve as biomarkers to identify tumors that are candidates for this therapeutic approach. Mol Cancer Res; 10(1); 96–107. ©2011 AACR.

Challenges in the endocrine management of breast cancer

The goal of endocrine therapy in breast cancer is to block the action of estrogen on the tumor cells either by inhibiting estrogen from binding to the specific estrogen receptor or by inhibiting its synthesis. Tamoxifen, a selective estrogen receptor modulator, is the standard endocrine treatment for hormone receptor-positive breast cancer, both in the adjuvant and metastatic settings. Tamoxifen inhibits the binding of estrogen to the receptor, resulting in inhibition of hormone action. However, as tamoxifen is also weakly estrogenic, it may not be optimally effective and increases the risk of endometrial cancer and stroke. Furthermore, patients may be refractory or may become resistant to tamoxifen treatment. Since aromatase inhibitors (AI) block the synthesis of estrogen and have no intrinsic estrogenic activity, they have the potential to be more effective than tamoxifen. Their different mechanism of action and chemical structures may also circumvent tamoxifen resistance. Consequently, AIs are currently being evaluated as an alternative to tamoxifen treatment. A preclinical model has recently been developed to compare the efficacy of AIs and antiestrogens in different treatment schemes and to assist in the design of clinical trials. Current studies with the MCF-7Ca xenograft model are exploring the effects of combination and sequential therapy on tumor growth. The efficacy of AIs in the treatment of hormone receptor-positive breast cancer was first demonstrated in five multicenter second-line trials enrolling several hundreds of postmenopausal patients with metastatic breast cancer who had failed tamoxifen treatment. More recently, anastrozole demonstrated efficacy at least equivalent to that of tamoxifen in first-line randomized, phase III clinical trials in postmenopausal women with hormone receptor-positive or unknown metastatic breast cancer, whereas letrozole demonstrated superiority. The steroidal AI exemestane is currently under evaluation. Letrozole is the only AI to have been studied in a randomized, phase III trial in the neoadjuvant setting. In this trial, more patients underwent breast-conserving surgery with letrozole than with tamoxifen. Smaller phase II studies also suggest that both anastrozole and exemestane are active in the neoadjuvant setting. Because neoadjuvant trials permit temporal sampling of breast tissue, substudies in the phase III trial with letrozole have examined the impact of such biomarkers as estrogen receptor, progesterone receptor and epidermal growth factor receptor family members, HER-1 and HER-2, on patient response. AIs are currently under evaluation in the adjuvant setting, and preliminary results of the Arimidex, Tamoxifen Alone or in Combination (ATAC) trial have been reported. AIs have proven as safe as tamoxifen in trials in patients with metastatic breast cancer. Ongoing clinical trials in the adjuvant setting include companion studies of end-organ effects, particularly bone metabolism and lipid metabolism evaluations. Quality-of-life assessments are also parts of major clinical trials. A head-to-head quality-of-life assessment of anastrozole compared with letrozole demonstrated patient preference for letrozole. These assessments also clearly indicated the eagerness of patients to participate actively in treatment decisions

AROMATASE AND BREAST CANCER

Several aromatase inhibitors and also new antiestrogens are now available for treating breast cancer. We have developed a model to compare the antitumor efficacy of these agents and to explore strategies for their optimal use. Results from the model have been predictive of clinical outcome. In this model, tumors are grown in ovariectomized, immunodeficient mice from MCF-7 human breast cancer cells transfected with the aromatase gene (MCF-7Ca). The possibility that blockade of estrogen action and estrogen synthesis may be synergistic was explored by treating mice with the aromatase inhibitor letrozole and the antiestrogen tamoxifen alone and in combination. The results indicated that letrozole alone was better than all other treatments. In addition, when tamoxifen treatment was no longer effective, tumor growth was significantly reduced in mice switched to letrozole treatment. However, tumors ultimately began to grow during continued treatment. To investigate the mechanisms by which tumors eventually adapt and grow during letrozole treatment, we determined the expression of signaling proteins in tumors during the course of letrozole treatment compared to the tumors of control mice. Tumors initially up-regulated the ER while responding to treatment, but subsequently receptor levels decreased in tumors unresponsive to letrozole. Also, Her-2 and adapter proteins (p-Shc and Grb-2) as well as all of the signaling proteins in the MAPK cascade (p-Raf, p-Mekl/2, and p-MAPK), but not in the Pl3/Akt pathway, were increased in tumors no longer responsive to letrozole. To investigate whether sensitivity to letrozole could be regained, cells were isolated from the letrozole resistant tumors (LTLT) and treated with inhibitors of the MAPKinase pathway (PD98059 and UO126). These compounds reduced MAPK activity and increased ER expression. EGFR/Her-2 inhibitors, gefitinib and AEE78S although not effective in the parental MCF-70a cells, restored the sensitivity of LTLT cells to letrozole. In xenografts, beginning treatment with letrozole and faslodex to down regulate the ER prevented increases in Her-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results suggest that blocking both ER- and growth factor-mediated transcription may delay development of resistance and maintain growth inhibition of ER+ breast cancer.

HDAC inhibitor entinostat restores responsiveness of letrozole-resistant MCF-7Ca xenografts to aromatase inhibitors through modulation of Her-2.

We previously showed that in innately resistant tumors, silencing of the estrogen receptor (ER) could be reversed by treatment with a histone deacetylase (HDAC) inhibitor, entinostat. Tumors were then responsive to aromatase inhibitor (AI) letrozole. Here, we investigated whether ER in the acquired letrozole-resistant tumors could be restored with entinostat. Ovariectomized athymic mice were inoculated with MCF-7Ca cells, supplemented with androstenedione (Δ4A), the aromatizable substrate. When the tumors reached about 300 mm3, the mice were treated with letrozole. After initial response to letrozole, the tumors eventually became resistant (doubled their initial volume). The mice then were grouped to receive letrozole, exemestane (250 μg/d), entinostat (50 μg/d), or the combination of entinostat with letrozole or exemestane for 26 weeks. The growth rates of tumors of mice treated with the combination of entinostat with letrozole or exemestane were significantly slower than with the single agent (P < 0.05). Analysis of the letrozole-resistant tumors showed entinostat increased ERα expression and aromatase activity but downregulated Her-2, p-Her-2, p-MAPK, and p-Akt. However, the mechanism of action of entinostat in reversing acquired resistance did not involve epigenetic silencing but rather included posttranslational as well as transcriptional modulation of Her-2. Entinostat treatment reduced the association of the Her-2 protein with HSP-90, possibly by reducing the stability of Her-2 protein. In addition, entinostat also reduced Her-2 mRNA levels and its stability. Our results suggest that the HDAC inhibitor may reverse letrozole resistance in cells and tumors by modulating Her-2 expression and activity. Mol Cancer Ther; 12(12); 2804–16. ©2013 AACR.

Xenograft models for aromatase inhibitor studies

As several aromatase inhibitors are now available for treating breast cancer, we developed a model system to compare their antitumor efficacy and to explore strategies for their optimal use. Tumors are grown in ovariectomized, immunodeficient mice from MCF-7 human breast cancer cells transfected with the aromatase gene (MCF-7Ca) and can therefore synthesize as well as respond to estrogen. Results from this model have been predictive of clinical outcome. Thus, inhibiting estrogen action and estrogen synthesis by treating mice with the aromatase inhibitor letrozole and the antiestrogen tamoxifen in combination did not result in synergy. Moreover, when tamoxifen treatment was no longer effective, tumor growth was significantly reduced in response to sequential letrozole treatment. However, our findings indicate that letrozole alone was better than all other treatments. Although letrozole resulted in long sustained growth inhibition, tumors eventually grew despite continued treatment. Mechanisms of resistance to letrozole were investigated during the course of treatment. ER was initially upregulated in responding tumors, but subsequently decreased below control levels in tumors no longer responsive to letrozole. Her-2 as well as adapter proteins (p-Shc and Grb-2) and signaling proteins in the MAPK cascade (p-Raf, p-Mekl/2, and p-MAPK), were all increased in letrozole resistant tumors. In LTLT cells, isolated from the letrozole resistant tumors and treated with inhibitors of the MAPKinase pathway, MAPK activity was decreased and ER expression restored to control levels. Inhibitors of EGFR/Her-2 also restored the sensitivity of LTLT cells to letrozole. These results suggest that crosstalk occurs between ER and tyrosine kinase receptor signaling. Therefore, to investigate whether down-regulating ER would prevent activation of MAPK and resistance to letrozole, xenografts were treated with letrozole and faslodex in combination. Her-2 and MAPK were not increased and tumor growth was inhibited throughout 29 weeks of treatment. These results suggest that blocking both ER and growth factor mediated transcription may delay development of resistance to letrozole and maintain its growth inhibition of breast cancer.

Nonhypoxic regulation and role of hypoxia-inducible factor 1 in aromatase inhibitor resistant breast cancer

IntroductionAlthough aromatase inhibitors (AIs; for example, letrozole) are highly effective in treating estrogen receptor positive (ER+) breast cancer, a significant percentage of patients either do not respond to AIs or become resistant to them. Previous studies suggest that acquired resistance to AIs involves a switch from dependence on ER signaling to dependence on growth factor-mediated pathways, such as human epidermal growth factor receptor-2 (HER2). However, the role of HER2, and the identity of other relevant factors that may be used as biomarkers or therapeutic targets remain unknown. This study investigated the potential role of transcription factor hypoxia inducible factor 1 (HIF-1) in acquired AI resistance, and its regulation by HER2.MethodsIn vitro studies using AI (letrozole or exemestane)-resistant and AI-sensitive cells were conducted to investigate the regulation and role of HIF-1 in AI resistance. Western blot and RT-PCR analyses were conducted to compare protein and mRNA expression, respectively, of ERα, HER2, and HIF-1α (inducible HIF-1 subunit) in AI-resistant versus AI-sensitive cells. Similar expression analyses were also done, along with chromatin immunoprecipitation (ChIP), to identify previously known HIF-1 target genes, such as breast cancer resistance protein (BCRP), that may also play a role in AI resistance. Letrozole-resistant cells were treated with inhibitors to HER2, kinase pathways, and ERα to elucidate the regulation of HIF-1 and BCRP. Lastly, cells were treated with inhibitors or inducers of HIF-1α to determine its importance.ResultsBasal HIF-1α protein and BCRP mRNA and protein are higher in AI-resistant and HER2-transfected cells than in AI-sensitive, HER2- parental cells under nonhypoxic conditions. HIF-1α expression in AI-resistant cells is likely regulated by HER2 activated-phosphatidylinositide-3-kinase/Akt-protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway, as its expression was inhibited by HER2 inhibitors and kinase pathway inhibitors. Inhibition or upregulation of HIF-1α affects breast cancer cell expression of BCRP; AI responsiveness; and expression of cancer stem cell characteristics, partially through BCRP.ConclusionsOne of the mechanisms of AI resistance may be through regulation of nonhypoxic HIF-1 target genes, such as BCRP, implicated in chemoresistance. Thus, HIF-1 should be explored further for its potential as a biomarker of and therapeutic target.

Applicability of the intratumor aromatase preclinical model to predict clinical trial results with endocrine therapy

Preclinical models and clinical studies have shown that aromatase inhibitors (AIs) are powerful inhibitors of estrogen synthesis and significantly suppress estrogen in vivo. For more than 20 years, standard first-line treatment for postmenopausal women with metastatic breast cancer has been the antiestrogen tamoxifen, a selective estrogen receptor modulator (SERM) with differential effects on breast, endometrial, bone, and vascular tissues. The estrogenic activity of tamoxifen is associated with deleterious clinical side effects, including vaginal bleeding, endometrial cancer, and thromboembolism. AIs are established second-line treatments in patients who progress with tamoxifen. Compared with progestins, such as megestrol acetate, or the earlier AIs aminoglutethimide and fadrozole, the new AIs, including exemestane, anastrozole, and letrozole, have increased efficacy and clinical benefit and cause fewer side effects in patients with metastatic breast cancer. Letrozole and anastrozole are approved first-line therapy for patients with metastatic breast cancer and as second-line treatment after tamoxifen failure. Studies in the intratumoral aromatase xenograft preclinical model have shown better responses with AIs than with antiestrogens in first-line therapy, and these data are consistent with the results from clinical trials. This model is now being used to assess whether combined or sequential administration of AIs with other agents may provide additional benefit.