Linda A. deGraffenried

Inhibition of mTOR Activity Restores Tamoxifen Response in Breast Cancer Cells with Aberrant Akt Activity

The Akt kinase is a serine/threonine protein kinase that has been implicated in mediating a variety of biological responses. Studies show that high Akt activity in breast carcinoma is associated with a poor pathophenotype, as well as hormone and chemotherapy resistance. Additionally, high Akt activity is associated with other features of poor prognosis. Thus, a chemotherapeutic agent directed specifically toward tumors with high Akt activity could prove extremely potent in treating those breast tumors with the most aggressive phenotypes. Several studies have demonstrated that rapamycin, which inhibits mammalian target of rapamycin (mTOR), a downstream target of Akt, sensitizes certain resistant cancer cells to chemotherapeutic agents. This study evaluated the efficacy of mTOR inhibition in the treatment of tamoxifen-resistant breast carcinoma characterized by high Akt activity. We found that MCF-7 breast cancer cell lines expressing a constitutively active Akt are able to proliferate under reduced estrogen conditions and are resistant to the growth inhibitory effects of tamoxifen, both in vitro as well as in vivo in xenograft models. Cotreatment with the mTOR inhibitor rapamycin in vitro, or the ester of rapamycin, CCI-779 (Wyeth) in vivo, inhibited mTOR activity and restored sensitivity to tamoxifen, suggesting that Akt-induced tamoxifen resistance is mediated in part by signaling through the mTOR pathway. Although the mechanism underlying the synergism remains to be understood, the results were associated with rapamycin’s ability to block transcriptional activity mediated by estrogen receptor α, as assessed by reporter gene assays with estrogen-responsive element luciferase. These data corroborate prior findings indicating that Akt activation induces resistance to tamoxifen in breast cancer cells. Importantly, these data indicate a novel mechanism for tamoxifen resistance and suggest that blockage of the phosphatidylinositol 3′-kinase/Akt signaling pathway by mTOR inhibition effectively restores the susceptibility of these cells to tamoxifen. These data may have implication for future clinical studies of mTOR inhibition in breast carcinoma.

NSAID Use Reduces Breast Cancer Recurrence in Overweight and Obese Women: Role of Prostaglandin–Aromatase Interactions

Obesity is associated with a worse breast cancer prognosis and elevated levels of inflammation, including greater cyclooxygenase-2 (COX-2) expression and activity in adipose-infiltrating macrophages. The product of this enzyme, the proinflammatory eicosanoid prostaglandin E2 (PGE2), stimulates adipose tissue aromatase expression and subsequent estrogen production, which could promote breast cancer progression. This study demonstrates that daily use of a nonsteroidal anti-inflammatory drug (NSAID), which inhibits COX-2 activity, is associated with reduced estrogen receptor α (ERα)-positive breast cancer recurrence in obese and overweight women. Retrospective review of data from ERα-positive patients with an average body mass index of >30 revealed that NSAID users had a 52% lower recurrence rate and a 28-month delay in time to recurrence. To examine the mechanisms that may be mediating this effect, we conducted in vitro studies that utilized sera from obese and normal-weight patients with breast cancer. Exposure to sera from obese patients stimulated greater macrophage COX-2 expression and PGE2 production. This was correlated with enhanced preadipocyte aromatase expression following incubation in conditioned media (CM) collected from the obese-patient, sera-exposed macrophages, an effect neutralized by COX-2 inhibition with celecoxib. In addition, CM from macrophage/preadipocyte cocultures exposed to sera from obese patients stimulated greater breast cancer cell ERα activity, proliferation, and migration compared with sera from normal-weight patients, and these differences were eliminated or reduced by the addition of an aromatase inhibitor during CM generation. Prospective studies designed to examine the clinical benefit of NSAID use in obese patients with breast cancer are warranted.

The enhancing effects of obesity on mammary tumor growth and Akt/mTOR pathway activation persist after weight loss and are reversed by RAD001

The prevalence of obesity, an established risk and progression factor for postmenopausal breast cancer, remains high in US women. Activation of Akt/mammalian target of rapamycin (mTOR) signaling plays a key role in the obesity–breast cancer link. However, the impact of weight normalization in obese postmenopausal women on breast tumorigenesis and/or Akt/mTOR activation is poorly characterized. To model this, ovariectomized female C57BL/6 mice were fed a control diet (n = 20), a calorie restriction (CR) regimen (n = 20), or a diet‐induced obesity (DIO) diet (n = 30). At week 17, DIO mice were switched to control diet, resulting in formerly obese (FOb) mice with weights identical to the controls by week 20. MMTV‐Wnt‐1 mammary tumor cells were injected at 20 wk into each mouse. Two weeks post‐injection, vehicle or the mTOR inhibitor RAD001 at 10 or 15 mg/kg body weight (n = 10/diet group) was administered by gavage twice/week until termination. Relative to controls, CR mice had decreased (and DIO mice had increased) serum insulin‐like growth factor‐1 (IGF‐1) and phosphorylation of Akt/mTOR pathway components. RAD001 decreased tumor growth in the CR, control, and FOb mice. Wnt‐1 tumor cells treated in vitro with serum from mice from each group established that diet‐dependent circulating factors contribute to tumor growth and invasiveness. These findings suggest weight normalization in obese mice does not immediately reverse tumor progression or Akt/mTOR activation. Treatment with RAD001 blocked mammary tumor development and mTOR activation observed in the FOb mice, suggesting combination of lifestyle and pharmacologic strategies may be effective for breaking the obesity–breast cancer link.

Obesity-related systemic factors promote an invasive phenotype in prostate cancer cells

Background:Obesity is associated with larger tumors, shorter time to PSA failure, and higher Gleason scores. However, the mechanism(s) by which obesity promotes aggressive prostate cancer remains unknown. We hypothesize that circulating factors related to obesity promote prostate cancer progression by modulating components of the metastatic cascade.Methods:Male C57BL/6 mice (6 weeks) were fed an ad libitum diet-induced obesity (60% fat) or control diet (10% fat) for 12 weeks. Serum was collected, metabolic and inflammatory proteins were measured by an antibody array. Sera were used to measure, in vitro, characteristics of a metastatic phenotype.Results:Comparable to obese men, obese sera contained higher levels or leptin, vascular endothelial growth factor, PAI-1, interleukin-6 (IL-6) and lower levels of testosterone. In prostate cells, serum was used to assess: proliferation, invasion, migration, epithelial-mesenchymal-transition (EMT) and matrix metalloproteinase (MMP) activity. LNCaP and PacMetUT1 cells exposed to obese sera increased proliferation, whereas PrEC and DU145 were unaffected. LNCaP, PacMetUT1 and DU145 cancer cells exposed to obese sera resulted in increased invasion, migration and MMP-9 activity. Prostate cancer cells exposed to obese sera showed increased vimentin, dispersion of e-cadherin and β-catenin from the plasma membrane.Conclusion:We report, prostate cancer cells exposed to sera from obese mice increases proliferation, invasion, migration, MMP activity and induces changes in proteins critical for EMT.

Regulation of the Estrogen Receptor α Minimal Promoter by Sp1, USF-1 and ERα

The exact molecular mechanisms regulating estrogen receptor α (ERα) expression in breast tumors are unclear, but studies suggest that they are partly at the level of transcription. We have focused on the transcription factors that regulate the ERα minimal promoter, which we have previously shown to reside within the first 245 bp of the 5′-flanking region of the gene. Within this region are several elements essential for full ERα promoter transcriptional activity, including a GC box and an imperfect E box. In earlier studies we demonstrated an essential function for the Sp1 family of transcription factors in the regulation of ERα expression. We have now identified both USF-1 and ERα itself as components of a multi-protein complex of transcription factors that interacts at the ERα minimal promoter and is essential for its full transcriptional activity. Electrophoretic mobility shift assays demonstrated that Sp1 and USF-1, but not ERα, bind directly to the ERα minimal promoter. We showed by GST pull-down assays that ERα is able to interact in vitro with USF-1, suggesting, in addition to a possible interaction between ERα and Sp1, a mechanism whereby ERα is able to interact with the protein complex. Combined exogenous expression of the components of the complex in MCF-7 breast cancer cells resulted in a synergistic effect on transactivation of the ERα minimal promoter, suggesting that the importance of the protein complex is in the interactions among the components. Based upon these findings, we propose a possible model for transcription from the ERα minimal promoter.

Omega-3 Fatty Acid Inhibition of Prostate Cancer Progression to Hormone Independence Is Associated With Suppression of mTOR Signaling and Androgen Receptor Expression

Currently, progression of prostate cancer to androgen independence remains the primary obstacle to improved survival. In order to improve overall survival, novel treatment strategies that are based upon specific molecular mechanisms that prolong the androgen-dependent state and that are useful for androgen-independent disease need to be identified. Both epidemiological as well as preclinical data suggest that omega-3 fatty acids are effective primary tumor prevention agents; however, their efficacy at preventing and treating refractory prostate cancer has not been as thoroughly investigated. We used an in vitro model of androgen ablation to determine the effect of treatment with omega-3 fatty acids on the progression to an androgen-independent state. The omega-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were able to prevent progression of LNCaP cells while the omega-6 fatty acid arachidonic acid (AA) actually promoted cell growth under conditions of hormone depletion. These results correlated with a decrease in the expression of the androgen receptor as well as suppression of the Akt/mTOR signaling pathway. Connecting the mechanisms by which omega-3 fatty acids affect phenotypic outcome is important for effective exploitation of these nutrient agents as a therapeutic approach. Understanding these processes is critical for the development of effective dietary intervention strategies that improve overall survival.

Docosahexaenoic acid selectively induces human prostate cancer cell sensitivity to oxidative stress through modulation of NF‐κB

Oxidative burden is strongly implicated in the pathogenesis of age‐related diseases, including prostate cancer tumor formation. As omega‐3 fatty acids possess known antioxidant properties, we investigated the effects of docosahexaenoic acid (DHA‐22:6n‐3), one component of fish oil, in modulating the effects of oxidative DNA damage in LNCaP and PacMetUT1 human prostate adenocarcinoma cells and in a normal human prostate cell line, PrEC.

Anethole suppressed cell survival and induced apoptosis in human breast cancer cells independent of estrogen receptor status.

BACKGROUND Many traditional Chinese medicines target the treatment of inflammation which is emerging to be a critical component to cancer development and progression. The key aromatic compound in star anise anethole has demonstrated both anti and pro-cancerous effects depending on the estrogen receptor statuses in individual cell lines. In this study, we investigated the effect of anethole on the physiological responses and specific apoptotic pathways in human breast cancer MCF-7 and MDA-MB-231 cells that are well-characterized to represent estrogen receptor (ER) positivity and its counterpart in breast cancer respectively. How anethole affects the activity and expression of apoptotic caspases, the function of transcriptional factor NF-kB and the relative influence ER exerts on these events are areas of significant research relevance and results may impact the pharmaceutical development of anethole and its use as dietary supplementation. METHODOLOGY/PRINCIPAL FINDINGS Initial analyses of physiological responses using MTT and colony formation assays had demonstrated a preferentially suppression of cell survival to cell proliferation in both ER+ and ER- cells when cells were exposed to anethole. Western blot analysis has demonstrated induction of caspase 9 and PARP1/2 cleavage in parallel with elevated expression of c-FLIP (s) and p53. The transcriptional activity of NF-kB, an upstream activator of p53 is suppressed in both cell lines when treated with anethole. CONCLUSIONS In conclusion, anethole in an ER independent manner suppresses cell survival and induces apoptotic events in MCF-7 and MDA-MB-231 at an optimal concentration of 1 × 10(-3)M. In search of alternative compounds for therapeutic development, this study has demonstrated that anethole may be viable as an anti-cancer agent through the modulation of apoptosis, cell survival and proliferation in breast cancer cells.

Obesity enhances nongenomic estrogen receptor crosstalk with the PI3K/Akt and MAPK pathways to promote in vitro measures of breast cancer progression

IntroductionEpidemiological and clinical studies indicate that obesity is associated with a worse postmenopausal breast cancer prognosis and an increased risk of endocrine therapy resistance. However, the mechanisms mediating these effects remain poorly understood. Here we investigate the molecular pathways by which obesity-associated circulating factors in the blood enhance estrogen receptor alpha (ERα) positive breast cancer cell viability and growth.MethodsBlood serum was collected from postmenopausal breast cancer patients and pooled by body mass index (BMI) category (Control: 18.5 to 24.9 kg/m2; Obese: ≥30.0 kg/m2). The effects of patient sera on MCF-7 and T47D breast cancer cell viability and growth were examined by MTT and colony formation assays, respectively. Insulin-like growth factor receptor 1(IGF-1R), Akt, and ERK1/2 activation and genomic ERα activity were assessed to determine their possible contribution to obese patient sera-induced cell viability and growth. To further define the relative contribution of these signaling pathways, cells grown in patient sera were treated with various combinations of ERα, PI3K/Akt and MAPK targeted therapies. Comparisons between cells exposed to different experimental conditions were made using one-way analysis of variance (ANOVA) and Students t test.ResultsCells grown in media supplemented with obese patient sera displayed greater cell viability and growth as well as IGF-1R, Akt and ERK1/2 activation relative to control sera. Despite the lack of a significant difference in genomic ERα activity following growth in obese versus control patient sera, we observed a dramatic reduction in cell viability and growth after concurrent inhibition of the ERα and PI3K/Akt signaling pathways. Further, we demonstrated that ERα inhibition was sufficient to attenuate obese serum-induced Akt and ERK1/2 activation. Together, these data suggest that obesity promotes greater ERα positive breast cancer cell viability and growth through enhanced crosstalk between nongenomic ERα signaling and the PI3K/Akt and MAPK pathways.ConclusionsCirculating factors in the serum of obese postmenopausal women stimulate ERα positive breast cancer cell viability and growth by facilitating non-genomic ERα crosstalk with the PI3K/Akt and MAPK signaling pathways. These findings provide valuable insight into one mechanism by which obesity may promote ERα positive postmenopausal breast cancer progression and endocrine therapy resistance.

The Role of the Insulin/IGF System in Cancer: Lessons Learned from Clinical Trials and the Energy Balance-Cancer Link

Numerous epidemiological and pre-clinical studies have demonstrated that the insulin/insulin-like growth factor (IGF) system plays a key role in the development and progression of several types of cancer. Insulin/IGF signaling, in cooperation with chronic low-grade inflammation, is also an important contributor to the cancer-promoting effects of obesity. However, clinical trials for drugs targeting different components of this system have produced largely disappointing results, possibly due to the lack of predictive biomarker use and problems with the design of combination therapy regimens. With careful attention to the identification of likely patient responders and optimal drug combinations, the outcome of future trials may be improved. Given that insulin/IGF signaling is known to contribute to obesity-associated cancer, further investigation regarding the efficacy of drugs targeting this system and its downstream effectors in the obese patient population is warranted.