Michael F. Verderame

Supplementary Table 1 from The Impact of Fish Oil on the Chemopreventive Efficacy of Tamoxifen against Development of N-Methyl-N-Nitrosourea–Induced Rat Mammary Carcinogenesis

Supplementary Figure 1 from The Impact of Fish Oil on the Chemopreventive Efficacy of Tamoxifen against Development of N-Methyl-N-Nitrosourea–Induced Rat Mammary Carcinogenesis

The training of PhD students and early-career scientists is largely an apprenticeship in which the trainee associates with an expert to become an independent scientist. But when is a PhD student ready to graduate, a postdoctoral scholar ready for an independent position, or an early-career scientist ready for advanced responsibilities? Research training by apprenticeship does not uniformly include a framework to assess if the trainee is equipped with the complex knowledge, skills and attitudes required to be a successful scientist in the 21^st century. To address this problem, we propose competency-based assessment throughout the continuum of training to evaluate more objectively the development of PhD students and early-career scientists.

The opioid growth factor (OGF) and its receptor, OGFr, play a regulatory role in cell proliferation, and maintain homeostasis through a tonically active negative feedback mechanism. To directly evaluate the repercussion of increased OGFr expression and consequent gain-of-function in epithelium, bovine keratin 5 promoter elements were used to direct the expression of OGFr to skin in a tetracycline-regulated manner. Three founder lines overexpressing OGFr (OGFrTG/K5-tTA) were established. Evidence for increased OGFr in the epithelium included a three-fold increase in OGFr binding activity, as well as significant increases in OGFr protein, as monitored by semiquantitative immunohistochemistry. DNA synthesis in target epithelium, including cornea, tongue, and skin of transgenic mice was decreased 41% to 80% from wild-type littermates; the liver, a nonepithelial organ, was not altered. Decreased DNA synthesis in corneal epithelium induced by transgenic expression of OGFr was further reduced by treatment with exogenous OGF but reversed by exposure to the opioid antagonist, naloxone. The number of cell layers in both epidermis and cornea of OGFrTG/K5-tTA animals was reduced nearly 45% from wild-type mice. Full-thickness wounds in mice overexpressing OGFr healed 37% to 75% slower than wild-type littermates. These data demonstrate for the first time that stable genetic amplification of OGFr downregulates homeostatic cell proliferation, as well as pathophysiological processes with respect to wound repair. These mice also can serve as a valuable model to dissect the mechanism of OGF-OGFr action and may be important in understanding the etiology, pathogenesis, and treatment of epithelium-related diseases.

In these experiments, we tested the hypothesis that inhibition of the estrogen receptor (ER) with Tamoxifen and activation of PPARγ with fish oil (FO) rich in omega-3 (n-3; known PPAR agonists) inhibit the development of hormone-independent breast cancer in view of the known crosstalk between the ER and PPARγ pathways. We selected the polyoma middle T transgenic mouse model, since in this system the development of ER− tumors is preceded by ER positive preneoplastic lesions. Tamoxifen admixed with a 20% corn oil (CO) modified AIN-76A diet delayed mammary carcinogenesis and inhibited tumor multiplicity, volume, and weight in a dose-dependent (1, 10, and 100 ppm) fashion. Administration of increasing concentrations of FO in the diet (5%, 10%, and 17%) did not affect any of the tumor parameters. Combined administration of different doses of Tamoxifen and FO delayed carcinogenesis and suppressed tumor multiplicity and volume to the same extent as Tamoxifen alone. Mice fed 10% FO exhibited the expected increase in n-3/n-6 ratio in plasma and tumor based on diet analysis. Further increase in the n-3/n-6 ratio was not observed in mice fed the 17% FO diet. FO reduced tissue levels of arachidonic acid and its metabolite PGF-2α. Our results support the role of ER expression by preneoplastic lesions in the development of hormone-independent tumors and consequently the importance of including ER targeting in combination with mechanistically based novel chemopreventive agents.

Our laboratories are interested in testing the value of combining omega-3 fatty acids and antiestrogens as a safe and effective chemopreventive strategy for breast cancer. We have shown that a fish oil rich diet (FO) (17% FO + 3% corn oil [CO]) enhances the chemopreventive action of Tamoxifen (Tam) in a prepubertal model of N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis (A. Manni, et al., Cancer Prev Res 3:322, 2010). To gain insight into mechanisms of prevention and the importance of different ratios of n-3:n-6 fatty acids in inhibiting carcinogenesis, we tested the effects of two FO rich diets (17% FO + 3% CO and 10% FO + 10% CO) individually and in combination with Tam on the histologic progression of preneoplastic lesions, indices of proliferation and apoptosis, as well as expression of multiple mammary gland specific and systemic biomarkers of oxidative stress in carcinogen treated rats. Following MNU administration at 21 days of age, separate groups of rats within each experimental arm were sacrificed 24 hours, 21 days, 28 days and 35 days prior to the development of palpable tumors. Increasing the amount of FO in the diet progressively increased the n-3:n-6 ratio in the plasma from 0.13 (20% CO) to 0.38 (10% FO) and 0.96 (17% FO). Although the FO rich diets significantly reduced Ki67 expression in hyperplastic lesions in a dose-dependent fashion (17% FO > 10% FO), neither the diets nor Tam decreased the development of preneoplastic lesions indicating that the inhibitory effect on carcinogenesis results from blocking the transition from hyperplasia to adenocarcinoma, consistent with our recent published study. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers (plasma protein carbonyls and blood protein bound GSH) or mammary specific biomarkers of oxidative stress, based on oxidative damage to DNA measured as 8-hydroxy-2’-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric-acid reactive substances (TBARS). On the other hand, tissue levels of 8-isoprostanes, prostaglandin-like compounds formed by non-enzymatic oxidation of arachidonic acid in the cell membrane, were markedly reduced by both FO rich diets at all time points (pCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5571. doi:10.1158/1538-7445.AM2011-5571