Chelin Hu

GPER Deficiency in Male Mice Results in Insulin Resistance, Dyslipidemia, and a Proinflammatory State

Estrogen is an important regulator of metabolic syndrome, a collection of abnormalities including obesity, insulin resistance/glucose intolerance, hypertension, dyslipidemia, and inflammation, which together lead to increased risk of cardiovascular disease and diabetes. The role of the G protein-coupled estrogen receptor (GPER/GPR30), particularly in males, in these pathologies remains unclear. We therefore sought to determine whether loss of GPER contributes to aspects of metabolic syndrome in male mice. Although 6-month-old male and female GPER knockout (KO) mice displayed increased body weight compared with wild-type littermates, only female GPER KO mice exhibited glucose intolerance at this age. Weight gain in male GPER KO mice was associated with increases in both visceral and sc fat. GPER KO mice, however, exhibited no differences in food intake or locomotor activity. One-year-old male GPER KO mice displayed an abnormal lipid profile with higher cholesterol and triglyceride levels. Fasting blood glucose levels remained normal, whereas insulin levels were elevated. Although insulin resistance was evident in GPER KO male mice from 6 months onward, glucose intolerance was pronounced only at 18 months of age. Furthermore, by 2 years of age, a proinflammatory phenotype was evident, with increases in the proinflammatory and immunomodulatory cytokines IL-1β, IL-6, IL-12, TNFα, monocyte chemotactic protein-1, interferon γ-induced protein 10, and monokine induced by interferon gamma and a concomitant decrease in the adipose-specific cytokine adiponectin. In conclusion, our study demonstrates for the first time that in male mice, GPER regulates metabolic parameters associated with obesity and diabetes.

G Protein-Coupled Estrogen Receptor-Selective Ligands Modulate Endometrial Tumor Growth

Endometrial carcinoma is the most common cancer of the female reproductive tract. GPER/GPR30 is a 7-transmembrane spanning G protein-coupled receptor that has been identified as the third estrogen receptor, in addition to ERα and ERβ. High GPER expression is predictive of poor survival in endometrial and ovarian cancer, but despite this, the estrogen-mediated signaling pathways and specific estrogen receptors involved in endometrial cancer remain unclear. Here, employing ERα-negative Hec50 endometrial cancer cells, we demonstrate that GPER mediates estrogen-stimulated activation of ERK and PI3K via matrix metalloproteinase activation and subsequent transactivation of the EGFR and that ER-targeted therapeutic agents (4-hydroxytamoxifen, ICI182,780/fulvestrant, and Raloxifene), the phytoestrogen genistein, and the “ERα-selective” agonist propylpyrazole triol also function as GPER agonists. Furthermore, xenograft tumors of Hec50 cells yield enhanced growth with G-1 and estrogen, the latter being inhibited by GPER-selective pharmacologic antagonism with G36. These results have important implications with respect to the use of putatively ER-selective ligands and particularly for the widespread long-term use of “ER-targeted” therapeutics. Moreover, our findings shed light on the potential mechanisms of SERM/SERD side effects reported in many clinical studies. Finally, our results provide the first demonstration that pharmacological inhibition of GPER activity in vivo prevents estrogen-mediated tumor growth.

G Protein-coupled Estrogen Receptor Protects from Atherosclerosis

Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity.

Deletion of G Protein–Coupled Estrogen Receptor Increases Endothelial Vasoconstriction

Endogenous estrogens mediate protective effects in the cardiovascular system, affecting both endothelium-dependent and endothelium-independent mechanisms. Previous studies have suggested that nonselective estrogen receptor agonists such as endogenous estrogens inhibit endothelium-dependent vasoconstriction; however, the role of estrogen receptors in this response has not yet been clarified. This study investigated whether the intracellular transmembrane G protein–coupled estrogen receptor (GPER) regulates vascular reactivity in mice. Effects of chronic deficiency (using mice lacking the GPER gene) and acute inhibition (using the GPER-selective antagonist G15) on endothelium-dependent and endothelium-independent vascular reactivity, and the effects of GPER deficiency on vascular gene expression and structure were investigated. We found that chronic GPER deficiency is associated with increased endothelial prostanoid-mediated vasoconstriction but has no effect on endothelial nitric oxide bioactivity, gene expression of endothelial nitric oxide synthase and thromboxane prostanoid (TP) receptor, or vascular structure. GPER deletion also increases TP receptor–mediated contraction. Acute GPER blockade enhances endothelium-dependent contractions and reduces endothelial nitric oxide bioactivity. Contractions in response to TP receptor activation are unaffected by G15. In conclusion, this study identifies GPER as the first estrogen receptor with inhibitory activity on endothelium-dependent contractility. These findings may be important for understanding and treating diseases associated with increased endothelial vasoconstrictor prostanoid activity such as hypertension and obesity.

G Protein–Coupled Estrogen Receptor Regulates Mammary Tumorigenesis and Metastasis

The role of 17β-estradiol (E2) in breast cancer development and tumor growth has traditionally been attributed exclusively to the activation of estrogen receptor-α (ERα). Although targeted inhibition of ERα is a successful approach for patients with ERα+ breast cancer, many patients fail to respond or become resistant to anti-estrogen therapy. The discovery of the G protein–coupled estrogen receptor (GPER) suggested an additional mechanism through which E2 could exert its effects in breast cancer. Studies have demonstrated clinical correlations between GPER expression in human breast tumor specimens and increased tumor size, distant metastasis, and recurrence, as well as established a proliferative role for GPER in vitro; however, direct in vivo evidence has been lacking. To this end, a GPER-null mutation [GPER knockout (KO)] was introduced, through interbreeding, into a widely used transgenic mouse model of mammary tumorigenesis [MMTV-PyMT (PyMT)]. Early tumor development, assessed by the extent of hyperplasia and proliferation, was not different between GPER wild-type/PyMT (WT/PyMT) and those mice harboring the GPER-null mutation (KO/PyMT). However, by 12 to 13 weeks of age, tumors from KO/PyMT mice were smaller with decreased proliferation compared with those from WT/PyMT mice. Furthermore, tumors from the KO/PyMT mice were of histologically lower grade compared with tumors from their WT counterparts, suggesting less aggressive tumors in the KO/PyMT mice. Finally, KO/PyMT mice displayed dramatically fewer lung metastases compared with WT/PyMT mice. Combined, these data provide the first in vivo evidence that GPER plays a critical role in breast tumor growth and distant metastasis. Implications: This is the first description of a role for the novel estrogen receptor GPER in breast tumorigenesis and metastasis, demonstrating that it represents a new target in breast cancer diagnosis, prognosis, and therapy. Mol Cancer Res; 12(11); 1644–54. ©2014 AACR.

Targeting the transposase domain of the DNA repair component Metnase to enhance chemotherapy.

Previous studies have shown that the DNA repair component Metnase (SETMAR) mediates resistance to DNA damaging cancer chemotherapy. Metnase has a nuclease domain that shares homology with the Transposase family. We therefore virtually screened the tertiary Metnase structure against the 550,000 compound ChemDiv library to identify small molecules that might dock in the active site of the transposase nuclease domain of Metnase. We identified eight compounds as possible Metnase inhibitors. Interestingly, among these candidate inhibitors were quinolone antibiotics and HIV integrase inhibitors, which share common structural features. Previous reports have described possible activity of quinolones as antineoplastic agents. Therefore, we chose the quinolone ciprofloxacin for further study, based on its wide clinical availability and low toxicity. We found that ciprofloxacin inhibits the ability of Metnase to cleave DNA and inhibits Metnase-dependent DNA repair. Ciprofloxacin on its own did not induce DNA damage, but it did reduce repair of chemotherapy-induced DNA damage. Ciprofloxacin increased the sensitivity of cancer cell lines and a xenograft tumor model to clinically relevant chemotherapy. These studies provide a mechanism for the previously postulated antineoplastic activity of quinolones, and suggest that ciprofloxacin might be a simple yet effective adjunct to cancer chemotherapy.

Erratum: G Protein-coupled Estrogen Receptor Protects from Atherosclerosis.

Cardiovascular disease risk is associated with excess body weight and elevated plasma free fatty acid (FFA) concentrations. This study examines how an alternate-day fasting (ADF) diet high (HF) or low (LF) in fat affects plasma FFA profiles in the context of weight loss, and changes in body composition and lipid profiles. After a 2-week weight maintenance period, 29 women (BMI 30–39.9 kg/m2) 25–65 years old were randomized to an 8-week ADF-HF (45% fat) diet or an ADF-LF (25% fat) diet with 25% energy intake on fast days and ad libitum intake on feed days. Body weight, BMI and waist circumference were assessed weekly and body composition was measured using dual x-ray absorptiometry (DXA). Total and individual FFA and plasma lipid concentrations were measured before and after weight loss. Body weight, BMI, fat mass, total cholesterol, LDL-C and triglyceride concentrations decreased (P < 0.05) in both groups. Total FFA concentrations also decreased (P < 0.001). In the ADF-LF group, decreases were found in several more FFAs than in the ADF-HF group. In the ADF-HF group, FFA concentrations were positively correlated with waist circumference. Depending on the macronutrient composition of a diet, weight loss with an ADF diet decreases FFA concentrations through potentially different mechanisms.

Reduced expression of the ferroptosis inhibitor GPx4 in multiple sclerosis and experimental autoimmune encephalomyelitis

Glutathione peroxidase 4 (GPx4) is the only enzyme capable of reducing toxic lipid hydroperoxides in biological membranes to the corresponding alcohols using glutathione as the electron donor. GPx4 is the major inhibitor of ferroptosis, a non‐apoptotic and iron‐dependent programmed cell death pathway, which has been shown to occur in various neurological disorders with severe oxidative stress. In this study, we investigate whether GPx4 expression is altered in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). The results clearly show that mRNA expression for all three GPx4 isoforms (cytoplasmic, mitochondrial and nuclear) decline in multiple sclerosis gray matter and in the spinal cord of MOG35–55 peptide‐induced EAE. The amount of GPx4 protein is also reduced in EAE, albeit not in all cells. Neuronal GPx4 immunostaining, mostly cytoplasmic, is lower in EAE spinal cords than in control spinal cords, while oligodendrocyte GPx4 immunostaining, mainly nuclear, is unaltered. Neither control nor EAE astrocytes and microglia cells show GPx4 labeling. In addition to GPx4, two other negative modulators of ferroptosis (γ‐glutamylcysteine ligase and cysteine/glutamate antiporter), which are critical to maintain physiological levels of glutathione, are diminished in EAE. The decrease in the ability to eliminate hydroperoxides was also evidenced by the accumulation of lipid peroxidation products and the reduction in the proportion of the docosahexaenoic acid in non‐myelin lipids. These findings, along with presence of abnormal neuronal mitochondria morphology, which includes an irregular matrix, disrupted outer membrane and reduced/absent cristae, are consistent with the occurrence of ferroptotic damage in inflammatory demyelinating disorders.

Evaluating mononuclear cells as nanoparticle delivery vehicles for the treatment of breast tumors

In breast cancer, certain types of circulating immune cells respond to long-range chemical signals from tumors by leaving the blood stream to actively infiltrate tumor tissue. The aim of this study was to evaluate whether immune cells could be used to deliver therapeutic nanoparticles into breast tumors in mice. Mononuclear splenocytes (MS) were harvested from donor mice, labeled with Indium-111, injected intravenously into immune-competent recipient mice (3 tumor-bearing and 3 control), and imaged longitudinally by SPECT/CT. For comparison, the biodistribution of bonemarrow derived macrophages (BMDM) in one pair of mice was also imaged. Quantitative analysis of the SPECT images demonstrates that, after 24 hours, the concentration of MS detected in mammary tumors is more than 3-fold higher than the concentration detected in normal mammary glands. The ratio of MS concentration in mammary tissue to MS concentration in non-target tissues (muscle, lung, heart, liver, spleen, and kidney) was enhanced in tumor-bearing mice (compared to controls), with statistical significance achieved for mammary/muscle (p<0.01), mammary/lung (p<0.05), and mammary/kidney (p<0.05). By contrast, BMDM did not show a different affinity for tumors relative to normal mammary tissue. MS were incubated with 100 nm red fluorescent nanoparticles, and flow cytometry demonstrated that ~35% of the MS population exhibited strong phagocytic uptake of the nanoparticles. After intravenous injection into tumor-bearing mice, fluorescence microscopy images of tumor sections show qualitatively that nanoparticle-loaded MS retain the ability to infiltrate mammary tumors. Taken together, these results suggest that MS carriers are capable of actively targeting therapeutic nanoparticles to breast tumors.

Abstract 3176: The EGFR pathway as the Achilles’ heel for human papillomavirus-induced tumors: EGFR/MAPK pathway inhibitors exhibit antiviral activities and limit tumor growth in vivo

Human papillomaviruses (HPVs) are etiological agents of many anogenital and oropharyngeal cancers. HPV[+] oropharyngeal squamous cell carcinomas (OPSCCs) typically respond more favorably to current treatment regimens (including radiotherapy combined with cisplatin or cetuximab, an EGFR monoclonal antibody), than do HPV-negative OPSCCs. The discrepancy in patient outcomes has been attributed, in part, to substantially fewer genetic mutations present in HPV[+] cancers. Paradoxically, an increasing body of literature notes lower EGFR expression in HPV[+] vs. HPV[-] OPSCC. Yet, HPV oncoproteins E5, E6 and E7 each work to increase EGFR signaling, and MAPK-regulated AP-1 transcription factors direct HPV early gene expression. The goal of this work is to test the hypothesis that upon infection, HPV establishes a feed-forward loop with the EGFR pathway to drive viral gene expression independent of EGFR levels. We modeled early neoplasia with cell lines maintaining episomal HPV16 genomes, and cancers using SCC lines with integrated HPV16 genomes. Cells treated with EGF, EGFR inhibitors (cetuximab, erlotinib), or MEK antagonists (PD98059, trametinib) were evaluated for signaling and viral responses. Consistent with our hypothesis, we found increased EGF-dependent EGFR activation levels in HPV[+] cell lines, as well as heightened MEK1/2 activity independent of EGFR activation in HPV[+] cell lines compared to uninfected cells. RT-qPCR revealed that HPV oncogene transcription was enhanced by EGFR activation, whereas EGFR and MEK inhibitors led to significant declines in HPV transcription. In cells with episomal HPV genomes, lower viral genome levels accompanied inhibitor-reduced viral transcription. These data suggest EGFR/MAPK pathway interference leads to anti-viral affects, which are expected to restore p53 and pRb function and prevent tumorigenesis in vivo. As predicted, cell proliferation and tumor growth in NOD/SCID-gamma mouse HPV[+] SCC xenografts were significantly reduced with EGFR and MEK inhibitors. Experiments are underway to quantify the restored function of p53 and pRb in drug-treated cells and determine if these cells become sensitized to lower doses of cisplatin or radiation. In summary, our results reveal HPV infection creates a feed-forward loop with the EGFR/MAPK proliferation pathway, and EGFR and MEK antagonists have anti-viral effects, including reduction in the E6 and E7 oncoproteins needed to maintain the transformed phenotype in vitro and in vivo. Additionally, the ability of the drugs to reduce episomal viral genome levels suggests a potential curative effect if administered prior to viral genome integration. Lastly, this work reveals an anti-viral molecular mechanism which may account for the favorable outcomes of patients with HPV[+] OPSCC treated with cetuximab-radiotherapy. Citation Format: Anastacia M. Griego, Pamela Barraza, Chelin Hu, Agnieszka Dziduszko, Brianna K. Crowley, Helen J. Hathaway, Julie E. Bauman, Michelle A. Ozbun. The EGFR pathway as the Achilles’ heel for human papillomavirus-induced tumors: EGFR/MAPK pathway inhibitors exhibit antiviral activities and limit tumor growth in vivo . [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3176. doi:10.1158/1538-7445.AM2014-3176