Loss of PPARγ expression in mammary secretory epithelial cells creates a pro‐breast tumorigenic environment

Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator‐activated receptor (PPAR)γ^(+/?) mice, we showed normal expression of PPARγ was critical to stop 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced breast tumorigenesis. PPARγ is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE‐specific loss of PPARγ was hypothesized to enhance DMBA‐mediated breast tumorigenesis. To test this, MSE cell‐specific PPARγ knockout (PPARγ‐MSE KO) and control (PPARγ‐WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPARγ‐WT, n = 15; PPARγ‐MSE KO, n = 25) or one supplemented with a PPARγ activating drug (DMBA + ROSI: PPARγ‐WT, n = 17; PPARγ‐MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPARγ‐MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPARγ‐WT controls. PPARγ activation significantly reduced DMBA‐mediated malignant mammary tumor volumes irrespective of genotype. MSE‐specific PPARγ loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPARγ activation in MSEs delayed mammary tumor growth in part by down‐regulating Cox‐1, Cox‐2 and cyclin D1. Collectively, these studies highlight a protective role of MSE‐specific PPARγ during breast tumorigenesis, and support a novel chemotherapeutic role of PPARγ activation in breast cancer.     

Alpha‐eleostearic acid suppresses proliferation of MCF‐7 breast cancer cells via activation of PPARγ and inhibition of ERK 1 / 2

Alpha‐eleostearic acid (α‐ESA) is known to suppress the growth in cancer cells although its underlying molecular mechanisms have not been fully elucidated. The present study was designed to elucidate and evaluate the anticancer mechanism of α‐ESA on MCF‐7 breast cancer cells. Also, an attempt was made to better understand the anticancer mechanism by which α‐ESA activated PPARγ and attenuated the ERK1/2 MAPK phosphorylation state. The MCF‐7 breast cancer cell‐line and nontumorigenic MCF‐10A human mammary epithelial cells were treated with α‐ESA and compared with negative control (without treatment) and positive control groups (treated with rosiglitazone), and changes of apoptosis‐related molecules, PPARγ and pERK1/2 were examined. In MCF‐7 cells treated with α‐ESA, we found that the expression of p53, p21, and Bax was up‐regulated whereas expression of Bcl‐2 and procaspase‐9 was down‐regulated. Moreover, nuclear translocation of PPARγ by α‐ESA positively correlated with inhibition of ERK1/2 activation. Our data suggest that α‐ESA can be considered to be a PPARγ agonist and thus a candidate for a chemotherapeutic agent against breast cancer. (Cancer Sci 2009; 00: 000–000)     

Peroxisome proliferator‐activated receptor γ upregulates galectin‐9 and predicts prognosis in intestinal‐type gastric cancer

The importance of PPARγ (peroxisome proliferator‐activated receptor γ) in gastric cancer (GC) is unclear. We investigated the role of PPARγ in GC cell lines and an animal model, and its prognostic significance of PPARγ in GC patients. We controlled PPARγ and galectin‐9 expression by using siRNAs and lentiviral constructs. Interaction between PPARγ and galectin‐9 was evaluated using luciferase and chromatin immunoprecipitation assays. PPARγ expression in GCs was determined by immunohistochemical staining of tissue microarrays and survival analysis was done. Overexpression of PPARγ was accompanied by increased galectin‐9. Enhanced PPARγ or galectin‐9 expression increased E‐cadherin expression; decreased expression of N‐cadherin, fibronectin, snail, twist and slug and reduced cell invasion and migration. PPARγ bound to the galectin‐9 promoter region. Galectin‐9 activity increased in PPARγ‐overexpressing cells but decreased in PPARγ siRNA‐treated cells. In a zebrafish xenograft model, the number of migrated cancer cells and number of fish with AGS cells in the tail vein were reduced in PPARγ‐overexpressing GC cells. PPARγ was expressed in 462 of the 688 patients (69.2%) with GC. In 306 patients with intestinal‐type GC, those with PPARγ‐positive tumors had lower overall and cancer‐specific mortalities than those with PPARγ‐negative tumors. PPARγ expression was an independent prognostic factor for overall and GC‐specific mortality in patients with intestinal‐type GC (adjusted hazard ratio, 0.42; 95% CI, 0.22–0.81). PPARγ inhibits cell invasion, migration and epithelial–mesenchymal transition through upregulation of galectin‐9 in vitro and in vivo.     

Prostaglandin d synthase is a potential novel therapeutic agent for the treatment of gastric carcinomas expressing PPARγ

The antitumor activity of prostaglandin (PG) D₂ has been demonstrated against some types of cancer, including gastric cancer. However, exogenous PGD₂ is not useful from a clinical point of view because it is rapidly metabolized in vivo. The aim of this study was to clarify the antitumor efficacy of an alternative, PGD synthase (PGDS), on gastric cancer cells. The effects of PGD₂ and PGDS on the proliferation of gastric cancer cells were examined in vivo and in vitro. The expression levels of PGD₂ receptors and peroxisome proliferator‐activated receptor γ (PPARγ) were evaluated by RT‐PCR. The effects of a PPARγ antagonist or siPPARγ on the proliferation of cancer cells and the c‐myc and cyclin D1 expression were examined in the presence or absence of PGD₂ or PGDS. PPARγ was expressed in gastric cancer cell lines, but PGD₂ receptors were not. PGD₂ and PGDS significantly decreased the proliferation of gastric cancer cells that highly expressed PPARγ. PGDS increased the PGD₂ production of gastric cancer cells. A PPARγ antagonist and siPPARγ transfection significantly suppressed the growth‐inhibitory effects of PGD₂ and PGDS. Expression of c‐myc and cyclin D1 was significantly decreased by PGD₂; this inhibitory effect was suppressed by PPARγ antagonist. Both PGD₂ and PGDS significantly decreased subcutaneous tumor growth in vivo. Tumor volume after PGDS treatment was significantly less than PGD₂ treatment. These findings suggest that PGDS and PGD₂ decrease the proliferation of gastric cancer cells through PPARγ signaling. PGDS is a potentially promising therapeutic agent for gastric cancers that express PPARγ.     

Effect of α‐tocopherol,N‐acetylcysteine and omeprazole on esophageal adenocarcinoma formation in a rat surgical model

We previously demonstrated that oxidative stress subsequent to gastroesophageal reflux is an important driving force of esophageal adenocarcinoma (EAC) formation in the esophagogastroduodenal anastomosis (EGDA) rat model. This study investigated the possible tumor inhibitory effects of 2 antioxidants, α‐tocopherol (389 and 778 ppm), N‐acetylcysteine (NAC, 500 and 1,000 ppm), and their combination (389 and 500 ppm, respectively), as well as an antacid therapeutic agent, omeprazole (1,400 ppm). The rats were fed experimental diets 2 weeks after EGDA. All the animals were sacrificed 40 weeks after EGDA and the esophagi were harvested for histopathological examination. α‐Tocopherol dose‐dependently decreased the incidence of EAC (p = 0.03), with 778 ppm α‐tocopherol reducing the incidence of EAC to 59% (16/27) in comparison with 84% (26/31) in the control group (p = 0.04). Supplementation of α‐tocopherol also increased the serum concentration of α‐tocopherol. NAC at 500 and 1,000 ppm did not significantly decrease EAC incidence; however, the combination of α‐tocopherol 389 ppm and NAC 500 ppm significantly reduced the incidence of EAC to 55% (15/27) (p = 0.02). α‐Tocopherol alone or in combination with NAC significantly reduced the number of infiltrating cells positively stained for 4‐hydroxynonenal. Omeprazole showed only a slight nonsignificant inhibitory effect at the dose given. Our results suggest that supplementation with α‐tocopherol inhibits the development of EAC in the rat EGDA model and similar inhibitory effect can be achieved when a lower dose of α‐tocopherol is used in combination with NAC. © 2008 Wiley‐Liss, Inc.     

Effects of estrogen on breast cancer development: Role of estrogen receptor independent mechanisms

Development of breast cancer involves genetic factors as well as lifetime exposure to estrogen. The precise molecular mechanisms whereby estrogens influence breast tumor formation are poorly understood. While estrogen receptor α (ERα) is certainly involved, nonreceptor mediated effects of estradiol (E₂) may also play an important role in facilitating breast tumor development. A “reductionist” strategy allowed us to examine the role of ERα independent effects of E₂ on mammary tumor development in ERα knockout (ERKO) mice bearing the Wnt‐1 oncogene. Exogenous E₂ “clamped” at early follicular and midluteal phase levels (i.e., 80 and 240 pg/ml) accelerated tumor formation in a dose‐related fashion in ERKO/Wnt‐1 animals (p = 0.0002). Reduction of endogenous E₂ by oophorectomy (p < 0.001) or an aromatase inhibitor (AI) (p = 0.055) in intact ERKO/Wnt‐1 animals delayed tumorigenesis as further evidence for an ER‐independent effect. The effects of residual ERα or β were not involved since enhancement of tumor formation could not be blocked by the antiestrogen fulvestrant. 17α‐OH‐E₂, a metabolizable but ER‐impeded analogue of E₂ stimulated tumor development without measurable uterine stimulatory effects. Taken together, our results suggest that ER‐independent actions of E₂ can influence breast tumor development in concert with ER dependent effects. These observations suggest 1 mechanism whereby AIs, which block E₂ synthesis, would be more effective for breast cancer prevention than use of antiestrogens, which only block ER‐mediated effects.     

Effects of α‐tocopherol and β‐carotene supplementation on cancer incidence and mortality: 18‐Year postintervention follow‐up of the Alpha‐Tocopherol,Beta‐Carotene Cancer Prevention Study

In the Alpha‐Tocopherol, Beta‐Carotene Cancer Prevention Study among 29,133 Finnish male smokers aged 50–69 years, daily α‐tocopherol (50 mg) for a median of 6.1 years decreased the risk of prostate cancer, whereas β‐carotene (20 mg) increased risk of lung cancer and overall mortality. To determine the postintervention effects of α‐tocopherol and β‐carotene, 25,563 men were followed 18 years for cancer incidence and all causes of mortality through national registers. Neither supplement had significant effects on post‐trial cancer incidence. Relative risk (RR) for lung cancer (n = 2,881) was 1.04 (95% confidence interval [CI], 0.96–1.11) among β‐carotene recipients compared with nonrecipients. For prostate cancer (n = 2,321), RR was 0.97 (95% CI, 0.89–1.05) among α‐tocopherol recipients compared with nonrecipients with the preventive effect of α‐tocopherol continuing ～8 years postintervention. Body mass index significantly modified the effect of α‐tocopherol on prostate cancer (p for interaction = 0.01) RR 1.00 (95% CI, 0.88–1.14) in normal‐weight men, 0.87 (95% CI, 0.77–0.98) in overweight men, and 1.25 (95% CI, 1.01–1.55) in obese men. The post‐trial relative mortality (based on 16,686 deaths) was 1.02 (95% CI, 0.98–1.05) for α‐tocopherol recipients compared with nonrecipients and 1.02 (95% CI, 0.99–1.05) for β‐carotene recipients compared with nonrecipients. α‐Tocopherol decreased post‐trial prostate cancer mortality (RR, 0.84; 95% CI, 0.70–0.99), whereas β‐carotene increased it (RR, 1.20; 95% CI, 1.01–1.42). In conclusion, supplementation with α‐tocopherol and β‐carotene appeared to have no late effects on cancer incidence. The preventive effect of moderate‐dose α‐tocopherol on prostate cancer continued several years post‐trial and resulted in lower prostate cancer mortality.     

MicroRNA ‘signature’ during estrogen-mediated mammary carcinogenesis and its reversal by ellagic acid intervention

Dysregulated miRNA expression has been associated with the development and progression of cancers, including breast cancer. The role of estrogen (E₂) in regulation of cell proliferation and breast carcinogenesis is well-known. Recent reports have associated several miRNAs with estrogen receptors in breast cancers. Investigation of the regulatory role of miRNAs is critical for understanding the effect of E₂ in human breast cancer, as well as developing strategies for cancer chemoprevention. In the present study we used the well-established ACI rat model that develops mammary tumors upon E₂ exposure and identified a ‘signature’ of 33 significantly modulated miRNAs during the process of mammary tumorigenesis. Several of these miRNAs were altered as early as 3 weeks after initial E₂ treatment and their modulation persisted throughout the mammary carcinogenesis process, suggesting that these molecular changes are early events. Furthermore, ellagic acid, which inhibited E₂-induced mammary tumorigenesis in our previous study, reversed the dysregulation of miR-375, miR-206, miR-182, miR-122, miR-127 and miR-183 detected with E₂ treatment and modulated their target proteins (ERα, cyclin D1, RASD1, FoxO3a, FoxO1, cyclin G1, Bcl-w and Bcl-2). This is the first systematic study examining the changes in miRNA expression associated with E₂ treatment in ACI rats as early as 3 week until tumor time point. The effect of a chemopreventive agent, ellagic acid in reversing miRNAs modulated during E₂-mediated mammary tumorigenesis is also established. These observations provide mechanistic insights into the new molecular events behind the chemopreventive action of ellagic acid and treatment of breast cancer.     

Synergistic actions of atorvastatin with γ‐tocotrienol and celecoxib against human colon cancer HT29 and HCT116 cells

The synergistic actions of atorvastatin (ATST) with γ‐tocotrienol (γ‐TT) and celecoxib (CXIB) were studied in human colon cancer cell lines HT29 and HCT116. The synergistic inhibition of cell growth by ATST and γ‐TT was demonstrated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and isobologram analysis. δ‐TT exhibited a similar inhibitory action when combined with ATST. Mevalonate and geranylgeranyl pyrophosphate eliminated most of the growth inhibitory effect of ATST, but only marginally decreased that of γ‐TT; whereas farnesyl pyrophosphate and squalene exhibited little effect on the inhibitory action of ATST and γ‐TT, indicating protein geranylgeranylation, but not farnesylation are involved in the inhibition of colon cancer cell growth. Both mevalonate and squalene restored the cellular cholesterol level that was reduced by ATST treatment, but only mevalonate eliminated the cell growth inhibitory effect, suggesting that the cholesterol level in cells does not play an essential role in inhibiting cancer cell growth. Protein level of HMG‐CoA reductase increased after ATST treatment, and the presence of γ‐TT attenuated the elevated level of HMG‐CoA reductase. ATST also decreased membrane‐bound RhoA, possibly due to a reduced level of protein geranylgeranylation; addition of γ‐TT enhanced this effect. The mediation of HMG‐CoA reductase and RhoA provides a possible mechanism for the synergistic action of ATST and γ‐TT. The triple combination of ATST, γ‐TT and CXIB showed a synergistic inhibition of cancer cell growth in MTT assays. The synergistic action of these three compounds was also illustrated by their induction of G₀/G₁ phase cell cycle arrest and apoptosis.     

Pregnancy-associated glycoprotein (α2-pag) - A possible marker in breast cancer?

Serum levels of pregnancy-associated α-glycoprotein (α²-PAG) and estradiol-17β (E₂) and the estrogen receptor content (ER) in the tumor cytosol were measured in 174 women aged 24 to 94 years with primary breast cancer and the values were related to patient age, tumor size and clinical stage. While E₂, levels were significantly lower and ER values significantly higher in older women, no significant correlation was found between α-PAG on one hand and the patients' age, E₂, or ER values or tumor size on the other. A significant positive correlation (p= 0.016, Pearson correlation test) was found between α₂-PAG values and the clinical stage of the tumor. The results suggest the possible use of α₂-PAG as a marker for the monitoring of disseminated breast cancer during systemic therapy.     

Vitamin E intake and risk of esophageal and gastric cancers in the NIH‐AARP Diet and Health Study

We investigated the association of dietary α‐tocopherol, γ‐tocopherol and supplemental vitamin E intake with the risk of esophageal squamous cell carcinoma (n = 158), esophageal adenocarcinoma (n = 382), gastric cardia adenocarcinoma (n = 320) and gastric noncardia adenocarcinoma (GNCA; n = 327) in the NIH‐AARP Diet and Health Study, a cohort of approximately 500,000 people. Data on dietary and supplemental vitamin E intake were collected using a validated questionnaire at baseline and were analyzed using Cox regression models. Intakes were analyzed as continuous variables and as quartiles. For dietary α‐tocopherol, we found some evidence of association with decreased esophageal squamous cell carcinoma and increased esophageal adenocarcinoma risk in the continuous analyses, with adjusted hazard ratios and 95% confidence intervals of 0.90 (0.81–0.99) and 1.05 (1.00–1.11), respectively, per 1.17 mg (half the interquartile range) increased intake. However, in quartile analyses, the p value for trend was nonsignificant for both these cancers. There was no association between dietary α‐tocopherol and gastric cardia adenocarcinoma or GNCA. We observed no statistically significant associations with γ‐tocopherol. For supplemental vitamin E, the results were mainly null, except for a significantly lower risk of GNCA with higher doses of supplemental vitamin E. An increase of 71 mg/day (half the interquartile range) in supplemental vitamin E had an hazard ratio (95% confidence interval) of 0.92 (0.85–1.00) and the p value for trend in the quartile analysis was 0.015. © 2009 UICC     

Estradiol downregulation of the tumor suppressor gene BTG2 requires estrogen receptor‐α and the REA corepressor

B‐cell Translocation Gene 2 (BTG2/TIS21/PC3) is an anti‐proliferative tumor suppressor gene whose expression is significantly reduced in breast carcinomas, and in MCF‐7 and T‐47D breast cancer cell lines treated with estradiol (E2). In this study the mechanisms involved in E2 down regulation of BTG2 gene expression were examined. Depletion of ERα by siRNA indicated that the receptor is required for E2 down regulation of BTG2 mRNA levels, and cycloheximide experiments indicated that the effect of E2 on BTG2 expression was independent of intermediary protein synthesis. Chromatin immunoprecipitation analyses revealed that ERα interacts with the BTG2 promoter in a ligand‐independent fashion whereas transfection experiments indicated that ERα's DNA and ligand binding domains are required for E2 repression of BTG promoter activity. Surprisingly, histone deacetylase (HDACs) activity is essential for basal expression as evidenced by trichostatin A inhibition of BTG2 mRNA levels. Estradiol treatment did not alter histone H3 acetylation although it did induce displacement of RNA polymerase II from the BTG2 gene. Depletion of the ER specific corepressor REA (Repressor of Estrogen Receptor Activity) significantly abrogated E2‐mediated BTG2 repression. Taken together, our results reveal a requirement of HDAC activity for basal BTG2 expression and the ERα‐REA interaction for estrogen repression of the BTG2 gene. The ability of E2‐bound ERα and REA to suppress BTG2 expression indicates a positive role for this corepressor in regulation of breast cancer cell proliferation. © 2008 Wiley‐Liss, Inc.