Mark Cline

Effect of progestin on the ovarian epithelium of macaques : Cancer prevention through apoptosis?

Objective: The apoptosis pathway is a vital mechanism in vivo that functions to eradicate genetically damaged cells prone to malignancy. The purpose of this study was to determine whether oral contraceptives, which confer significant protection against subsequent epithelial ovarian cancer, induce apoptosis in the ovarian epithelium. Methods: Female cynomologus macaques (N = 75) were randomized to receive a diet for 35 months containing either no hormones, the oral contraceptive Triphasil (Wyeth-Ayerst Laboratories, Philadelphia, PA), the estrogenic component of Triphasil (ethinyl estradiol) alone, or the progestin component of Triphasil (levonorgestrel) alone, each administered in a cyclic fashion. At study termination, the animals underwent ovariectomy and the ovarian epithelium was examined morphologically and immunihistochemically for apoptosis. The percentage of ovarian epithelial cells undergoing apoptosis was measured in each animal and compared between the treatment groups. Results: The median percentage of ovarian epithelial cells undergoing apoptosis by treatment was control (3.8%), ethinyl estradiol (1.8%), Triphasil (14.5%), and levonorgesrel (24.9%). Compared with control and ethinyl estradiol-treated monkeys, a statistically significant increase in the proportion of apoptotic cells was noted in the ovarian epithelium of monkeys treated with the oral contraceptive Triphasil (P ≤ .01) or levonorgestrel (P < .001), with a maximal effect (six-fold) seen in the group treated with levonorgestrel alone. Conclusion: Oral contraceptive progestin induces apoptosis in the ovarian epithelium. Given the importance of the apoptosis pathway for cancer prevention, an effective chemopreventive strategy may be possible using progestins or other agents that selectively induced apoptosis in the ovarian epithelium to prevent the development of ovarian cancer.

Lack of mitochondrial trifunctional protein in mice causes neonatal hypoglycemia and sudden death

Mitochondrial trifunctional protein (MTP) is a hetero-octamer of four alpha and four beta subunits that catalyzes the final three steps of mitochondrial long chain fatty acid beta-oxidation. Human MTP deficiency causes Reye-like syndrome, cardiomyopathy, or sudden unexpected death. We used gene targeting to generate an MTP alpha subunit null allele and to produce mice that lack MTP alpha and beta subunits. The Mtpa(-/-) fetuses accumulate long chain fatty acid metabolites and have low birth weight compared with the Mtpa(+/-) and Mtpa(+/+) littermates. Mtpa(-/-) mice suffer neonatal hypoglycemia and sudden death 6-36 hours after birth. Analysis of the histopathological changes in the Mtpa(-/-) pups revealed rapid development of hepatic steatosis after birth and, later, significant necrosis and acute degeneration of the cardiac and diaphragmatic myocytes. This mouse model documents that intact mitochondrial long chain fatty acid oxidation is essential for fetal development and for survival after birth. Deficiency of MTP causes fetal growth retardation, neonatal hypoglycemia, and sudden death.

Induction of Fatal Inflammation in LDL Receptor and ApoA-I Double-Knockout Mice Fed Dietary Fat and Cholesterol

Atherogenic response to dietary fat and cholesterol challenge was evaluated in mice lacking both the LDL receptor (LDLr(-/-)) and apoA-I (apoA-I(-/-)) gene, LDLr(-/-)/apoA-I(-/-) or double-knockout mice. Gender- and age-matched LDLr(-/-)/apoA-I(-/-) mice were fed a diet consisting of 0.1% cholesterol and 10% palm oil for 16 weeks and compared to LDLr(-/-) mice or single-knockout mice. The LDLr(-/-) mice showed a 6- to 7-fold increase in total plasma cholesterol (TPC) compared to their chow-fed mice counterparts, while LDLr(-/-)/apoA-I(-/-) mice showed only a 2- to 3-fold increase in TPC compared to their chow-fed controls. This differential response to the atherogenic diet was unanticipated, since chow-fed LDLr(-/-) and LDLr(-/-)/apoA-I(-/-) mice began the study with similar LDL levels and differed primarily in their HDL concentration. The 6-fold diet-induced increase in TPC observed in the LDLr(-/-) mice occurred mainly in VLDL/LDL and not in HDL. Mid-study plasma samples taken after 8 weeks of diet feeding showed that LDLr(-/-) mice had TPC concentrations approximately 60% of their 16-week level, while the LDLr(-/-)/apoA-I(-/-) mice had reached 100% of their 16-week TPC concentration after only 8 weeks of diet. Male LDLr(-/-) mice showed similar aortic cholesterol levels to male LDLr(-/-)/apoA-I(-/-) mice despite a 4-fold higher VLDL/LDL concentration in the LDLr(-/-) mice. A direct comparison of the severity of aortic atherosclerosis between female LDLr(-/-) and LDLr(-/-)/apoA-I(-/-) mice was compromised due to the loss of female LDLr(-/-)/apoA-I(-/-) mice between 10 and 14 weeks into the study. Diet-fed female and, with time, male LDLr(-/-)/apoA-I(-/-) mice suffered from severe ulcerated cutaneous xanthomatosis. This condition, combined with a complete depletion of adrenal cholesterol, manifested in fatal wasting of the affected mice. In conclusion, LDLr(-/-) and LDLr(-/-)/apoA-I(-/-) mice showed dramatic TPC differences in response to dietary fat and cholesterol challenge, while despite these differences both genotypes accumulated similar levels of aortic cholesterol.

Inflammation and skin cholesterol in LDLr−/−, apoA-I−/− mice: link between cholesterol homeostasis and self-tolerance?

Diet-fed low density lipoprotein receptor-deficient/apolipoprotein A-I-deficient (LDLr−/−, apoA-I−/−) mice accumulate a 10-fold greater mass of cholesterol in their skin despite a 1.5- to 2-fold lower plasma cholesterol concentration compared with diet-fed LDLr−/− mice. The accumulation of cholesterol predominantly in the skin has been shown to occur in a growing number of other hypercholesterolemic double knockout mouse models sharing deficits in genes regulating cellular cholesterol homeostasis. Exploring the relationship between cholesterol balance and inflammation, we have examined the time course of cholesterol accumulation in a number of extrahepatic tissues and correlated with the onset of inflammation in diet-fed LDLr−/−, apoA-I−/− mice. After 4 weeks of diet, LDLr−/−, apoA-I−/− mice showed a significant increase in skin cholesterol mass compared with LDLr−/− mice. In addition, after 4 weeks on the diet, cholesterol accumulation in the skin was also found to be associated with macrophage infiltration and accompanied by increases in tumor necrosis factor-α, cyclooxygenase-2, and langerin mRNA, which were not seen in the liver. Overall, these data suggest that as early as 4 weeks after starting the diet, the accumulation of skin cholesterol and the onset of inflammation occur concurrently. In summary, the use of hypercholesterolemic LDLr−/−, apoA-I−/− mice may provide a useful tool to investigate the role that apoA-I plays in maintaining cholesterol homeostasis and its relationship to inflammation.

Regional Distribution of Proliferating Cells and Hormone Receptors in the Mammary Gland of Surgically Postmenopausal Macaques

OBJECTIVE To define the relative proliferative response and hormone receptors status in ten sites in the mammary gland of surgically postmenopausal cynomolgus macaques given hormone replacement therapy. METHODS Surgical postmenopausal cynomolgus macaques were given either no treatment (n = 4), conjugated equine estrogens (CEE, n = 4), or combined therapy with CEE and medroxyprogesterone acetate (n = 4). The drugs were administered in the diet, at doses equivalent on a caloric basis to 0.625 mg/woman/day for CEE and 2.5 mg/woman/day for medroxyprogesterone acetate. Immunostaining of mammary sections was done for estrogen receptor, progesterone receptor, and the proliferation marker Ki-67 MIB-1 (MIB). Comparisons were made between central and peripheral gland, by quadrant, left versus right, and with respect to distance from the nipple within each quadrant. RESULT There were no significant differences in hormone receptor or MIB expression within different sites within the gland. CONCLUSIONS In the surgically postmenopausal, hormone-treated macaque, regional differences in estrogen and progesterone receptors and MIB staining are not apparent. The assumption of homogeneity throughout the gland makes aspiration cytology and multiple biopsy studies feasible in this species.

Osteoprotegerin (OPG), The Endogenous Inhibitor of Receptor Activator of NF-κB Ligand (RANKL), is Dysregulated in BRCA Mutation Carriers.

Breast cancer development in BRCA1/2 mutation carriers is a net consequence of cell-autonomous and cell nonautonomous factors which may serve as excellent targets for cancer prevention. In light of our previous data we sought to investigate the consequences of the BRCA-mutation carrier state on RANKL/osteoprotegerin (OPG) signalling. We analysed serum levels of RANKL, OPG, RANKL/OPG complex, oestradiol (E2), and progesterone (P) during menstrual cycle progression in 391 BRCA1/2-mutation carriers and 782 noncarriers. These studies were complemented by analyses of RANKL and OPG in the serum and mammary tissues of female cynomolgus macaques (n = 88) and serum RANKL and OPG in postmenopausal women (n = 150). BRCA-mutation carriers had lower mean values of free serum OPG in particular in BRCA1-mutation carriers (p = 0.018) compared with controls. Among BRCA1/2 mutation carriers, lower OPG levels were associated with germline mutation locations known to confer an increased breast cancer risk (p = 0.003). P is associated with low OPG levels in serum and tissue, particularly in BRCA-mutation carriers (rho = − 0.216; p = 0.002). Serum OPG levels were inversely correlated (rho = − 0.545, p < 0.001) with mammary epithelial proliferation measured by Ki67 expression and increased (p = 0.01) in postmenopause. The P–RANKL/OPG system is dysregulated in BRCA-mutation carriers. These and previously published data provide a strong rationale for further investigation of antiprogestogens or an anti-RANKL antibody such as denosumab for breast cancer prevention.

Dietary Vitamin D Exposure Prevents Obesity-Induced Increase in Endometrial Cancer in Pten+/− Mice

The possibility that dietary vitamin D3 (VD3) exposure inhibits endometrial carcinogenesis in an animal model and modifies the enhanced risk of endometrial carcinoma associated with obesity was investigated. At 4 weeks of age, Pten+/− and wild-type mice were each divided into four treatment groups and fed AIN93G control diet, or AIN93G-based diet containing either 25,000 international units of VD3 per kilogram of diet, 58% fat to induce obesity (high fat), or high fat and 25,000 international units of VD3 per kilogram of diet. Mice were kept on these diets until they were sacrificed at week 28. Although VD3 did not affect endometrial cancer risk, it inhibited obesity-induced increase in endometrial lesions. Specifically, high-fat diet increased focal glandular hyperplasia with atypia and malignant lesions from 58% in the control diet–fed Pten+/− mice to 78% in obese mice. Dietary VD3 decreased the incidence of endometrial pathology in obese Pten+/− mice to 25% (P < 0.001). VD3 altered the endometrial expression of 25-hydroxylase, 1α-hydroxylase, and vitamin D receptor in the wild-type and Pten+/− mice. Estrogen receptor-α mRNA levels were higher (P < 0.014) and progesterone receptor protein levels in the luminal epithelium were lower (P < 0.04) in the endometrium of control diet–fed Pten+/− than wild-type mice, but the expression of these receptors was not affected by the dietary exposures. VD3 reversed the obesity-induced increase in osteopontin (P < 0.001) and significantly increased E-cadherin expression (P < 0.019) in the endometrium of obese Pten+/− mice. Our data confirm the known association between obesity and endometrial cancer risk. Dietary exposure to VD3 inhibited the carcinogenic effect of obesity on the endometrium. This protective effect was linked to a reduction in the expression of osteopontin and increase in E-cadherin. Cancer Prev Res; 3(10); 1246–58. ©2010 AACR.

Use of green fluorescent protein to measure tumor growth in an implanted bladder tumor model.

PURPOSE Bladder cancer is a common malignancy in which local recurrence and distant failure contribute to poor patient outcome. The search for improved therapies remains a high priority in this disease. For most experimental therapies animal tumor models represent an important step between in vitro testing and clinical trials. A useful animal model of bladder cancer involves the orthotopic implantation of bladder tumor cells in sygeneic animals. This model offers the opportunity to test the efficacy of therapies administered systemically or intravesically. However, quantitation of tumor growth has been difficult. MATERIALS AND METHODS To allow the quantitative assessment of tumor mass in this model and differentiate tumor cells from normal bladder epithelium at early stages of tumor growth we transfected cells of the MBT2 murine bladder cancer cell line with a vector encoding enhanced green fluorescent protein and devised an enzyme-linked immunosorbent assay that enables the detection of green fluorescent protein in cells at the pg. level. RESULTS Using this assay tumor growth can be detected 7 days after implantation and by 14 days levels of green fluorescent protein are more than 500-fold greater than in controls. CONCLUSIONS Combined with the enzyme-linked immunosorbent assay use of this MBT2 green fluorescent protein transfectant allows tumor cell growth to be monitored with sensitivity and reproducibility. It reduces the time required to measure effects on tumor growth to 2 weeks, while preserving the advantages of this orthotopic tumor model.

Hormonal regulation of the normal breast

The breast is a target organ for reproductive hormones but basic knowledge on hormonal effects is very poor. Available data indicate that the breast is regulated in a specific manner which is distinct from the endometrium and other target organs. It seems clear that the breast undergoes cyclic changes during the menstrual cycle and that in vivo there is a direct stimulatory action of progestogens on the breast. In surgically postmenopausal female macaques continuous combined estrogen/progestogen therapy was found to induce greater proliferation than estrogen alone.

Abstract A013: DMP1β, an alternative splice isoform of tumor suppressor hDMP1 locus, has oncogenic properties in breast cancer

Despite recent advances in therapeutic approaches and early detection, breast cancer remains significant health care burden in many developing countries. In fact, it has been suggested that up to ~30% of breast cancer cases are overdiagnosed as a result of early detection mammography screening. This finding poses a hypothesis that better patient stratification using prognostic/predictive indicators, especially for the patients with early diagnosis, would provide significant improvement in clinical management and reduce health care cost. Our recent work identified Dmp1 as a critical tumor suppressor in breast cancer. Dmp1 is a transcription factor that induces cell cycle arrest and senescence by activating the p14ARF-p53 pathway. Overexpression of Her2/neu activates the Dmp1 promoter via PI3K-Akt-NFκB pathway leading to increase of p53 target genes. Loss of Dmp1 accelerates mammary tumor development in MMTV-neu mouse model without difference between Dmp1+/- and Dmp1-/- genotypes. Human DMP1 locus on 7q21 is hemizygously deleted in ~42% of breast carcinomas, which is mutually exclusive of INK4a/ARF or p53 inactivation. In the cases with hemizygous DMP1 deletion, the other DMP1 allele remains wild-type without mutation or promoter hypermethylation, which suggests that DMP1 is haploinsufficient tumor suppressor. The h DMP1 locus encodes three distinct transcripts via alternative splicing of pre-mRNA at Exon 10. The bona fide tumor suppressor protein is named DMP1α, while the two other transcripts without known biological function were named DMP1β and DMP1γ. The qPCR analysis of 46 matched breast cancer samples revealed that 30% of tumor samples have splicing alteration of DMP1 to increase DMP1β isoform. Importantly, the patients with high DMP1β/α ratio in tumor samples had shorter relapse-free survival compared to those patients without splicing alteration. Furthermore, DMP1β/α ratio was increased in MMTV-neu mouse mammary tumors. Immunohistochemistry of 50 breast tumor samples showed that DMP1β protein is overexpressed which was also associated with shorter relapse-free survival. Expression of DMP1β in non-tumorigenic breast epithelial cell line, MCF10A, significantly increased the rate of cell proliferation and size of the mammospheres in Matrigel©. Knockdown of the endogenous DMP1β inhibits proliferation of breast cancer cell lines. To ascertain role of DMP1β in development of breast cancer in vivo, we developed a MMTV-DMP1β mouse model. The mammary glands from MMTV-DMP1β female mice show dysplastic morphological changes in the epithelium with multifocal tumors at 18 months of age. The tumor tissue and surrounding mammary glands were hyperproliferative as they expressed high levels of Ki67 and Cyclin D1. Overall, we provide evidence that alternative splicing to increase DMP1β expression leads to proliferation of human cells and mouse mammary gland and offers poor prognosis for breast cancer patients. Citation Format: Dejan Maglic, Robert Kendig, Mark Cline, Guangchao Sui. DMP1β, an alternative splice isoform of tumor suppressor h DMP1 locus, has oncogenic properties in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A013.