Cj Grubbs

Differences in Plasma and Nipple Aspirate Carotenoid by Lactation Status

BackgroundDietary antioxidants, such as provitamin A carotenoid, have a protective effect against breast cancer. The transport of carotenoid from the blood into the breast microenvironment may be enhanced by lactation. ObjectiveTo examine the association between plasma and nipple aspirate carotenoid levels by lactation and post-wean status. MethodsThe sample consisted of 43 women, ages 18–45, who were at least 12 months postpartum. Women who had breastfed their last infant were at least 3 months post-wean. Women collected breast fluid every other day for 17 days and had a venipuncture for total nipple aspirate and plasma carotenoid, and completed a written health assessment, ResultsThe association between plasma and nipple aspirate carotenoid levels was significant for breastfeeding women (r =.39, p =.03), but not for non-breastfeeding women (r =.31, p =.27). However, while the association between plasma and nipple aspirate carotenoid levels was significant for women at or less than 9 months post-wean (r =.65, p = .01), the effect for women after 9 months post-wean (r =.21, p =.45) was not significant. ConclusionLactation may be protective by enhancing the delivery of chemopreventive substances available in the blood to the cell level of the breast, even after breast involution has occurred post lactation.

Abstract P3-11-04: Metabolic profiles in female Sprague-Dawley rats receiving either a standard (4% fat) or Western (20% fat) diet and changes in rats bearing mammary cancers

The methylnitrosourea(MNU)-induced model of ER + mammary cancers in female Sprague-Dawley rats has been routinely used in our laboratories for screening chemopreventive agents. We recently reported that metformin was ineffective in preventing mammary cancers in this model when given to rats on a standard diet (Thompson, et al, Cancer Prev Res. 2015;8:231-9). In this study, we evaluated metformin in rats placed on either standard diet (4% fat) or a Western diet (20% fat, low calcium). As fat calories, these diets were 8% and 42%, respectively. The rats were placed on standard diet or Western diet at 43 days of age, given MNU (via the jugular vein) once at 50 days of age, and administered metformin or vehicle at 57 days of age for the remainder of the study. Serum of the rats in the various groups was obtained at 78 days of age, and at the end of the study (when mammary tumors were present). The levels of approximately 500 metabolites were compared in the serum based on data obtained by Metabolon (Research Triangle Park, NC). These studies showed that each of the four groups [(standard diet (early and late) or Western diet (early and late)] yielded four clearly distinct patterns based on an unsupervised principal component analysis. Certain of the metabolites which were differentially expressed in serum from standard vs Western diets at the early time point were alpha-10-undecanoate, 13-methylmyristic acid, 4-hydroxy-benzoate, 2-amino-heptanpate, tocopherol, and nicotinamide. Certain of these were fatty acids and lipid soluble vitamins that one would expect to be altered. Comparing serum from standard vs Western diets at the late time point confirmed many of these metabolic differences. We also compared serum from the early and late time points since the latter serums were typically from animals with a significant number of mammary cancers. We observed a number of metabolite changes including 4-hydroxy-butyrate, acetyl-carnitine, oxalate, and threonate. These cancer- related profiles will be discussed at greater lengths. Altered profiles caused by the administration of metformin and other chemopreventive agents will also be discussed. Of interest, metformin was consistently ineffective in preventing mammary cancers in rats given either standard or Western diet. Supported by NCI contract HHSN261201200021I. Citation Format: Thompson MD, Grubbs CJ, Moeinpour F, Steele VE, Miller MS, Lubet RA. Metabolic profiles in female Sprague-Dawley rats receiving either a standard (4% fat) or Western (20% fat) diet and changes in rats bearing mammary cancers. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-11-04.

Abstract P1-08-02: Gene expression changes in methylnitrosourea (MNU)-induced ER+ mammary cancers following short-term treatment of rats with SERMs (Tamoxifen and Arzoxifene)

SERMs have proven to be highly effective in both therapy and prevention of ER + breast cancers. Tamoxifen has been the most commonly used SERM, but arzoxifene (another high- affinity competitive SERM agonist) showed strong activity in early clinical trials against ER + breast cancer; although further development was discontinued. Both SERMs have shown a dose dependent effect on prevention and therapy of rat mammary tumors in the ER + MNU model of cancer. Of interest, the doses required for prevention was significantly lower than the doses required for therapy. In the present study, rats bearing mammary cancers induced by MNU were treated with tamoxifen (0.66, 3.3, 20 and 100 ppm in diet) or arzoxifene (3.0 ppm in diet) for 5 days. Global gene expression analysis showed that more than 100 genes were down-regulated and more than 100 genes were up-regulated (p 1.5) in cancers treated with tamoxifen doses > 3 ppm; and that many of these gene changes were dose dependent. The genes modulated by tamoxifen and arzoxifene were enriched in the cell cycle pathway that were related to chromosome condensation in prometaphase [including Aurora-A, Aurora-B, Bub1B, non-SMC condensing I complex, subunit H (BRRN1), Condensin, CAP-G, CAP-G/G2, CAP-H/H2, CAP-D2/D3, CAP-E, TOP2, Cyclin A, Cyclin B, CDK1, Histone H1 and inter-centromere protein (INCENP]. Employing a different set of tamoxifen treated samples, we were able to confirm that many of the same genes were modulated employing a quantitative RT-PCR assay. Finally, we will compare certain of the gene changes obtained in the animal model with gene changes observed in human neoadjuvant trials. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-08-02.

P3-10-03: Bardoxolone (5MeCDDO) Inhibits Cancer Initiation but Promotes Progression in Rodent Models of Breast Cancer. What Does It Mean for the Antioxidant Response Element (ARE) as a Primary Prevention Target?

The preventive efficacies of the triterprenoid 5MeCDDO were examined in three preclinical models of breast cancer. We initially evaluated 5MeCDDO in an ER+ mammary model in which female Sprague-Dawley rats were administered MNU, i.v., at 50 days of age. 5MeCDDO (27 ppm) was administered in the diet beginning 5 days after MNU, and continuing for the duration of the study. Doses >50 ppm were toxic. 5MeCDDO failed to decrease tumor latency or multiplicity and, in fact, increased the size of the cancers which did develop. This concentration of 5MeCDDO greatly increased liver to body weight ratios. We also examined the preventive efficacy of 5MeCDDO (54 and 27 mg/kg diet) in a bitransgenic model (MMTV-Neu/p53KO) of ER − mammary cancer. In this model, animals develop cancers which overexpress Neu and fail to have a mutation in the transmembrane domain of Neu. Similarly to results in the MNU model, 5 MeCDDO did not alter either tumor latency or multiplicity. The effect of 5MeCDDO was further evaluated in a third model in which ER+ tumors were induced by the procarcinogen dimethylbenzanthracene (DMBA). DMBA was administered by gavage to female Sprague-Dawley rats at 50 days of age. In this model (which examines the ability of an agent to alter the activation of the carcinogen) the preventive agent was administered beginning 7 days prior to DMBA and was continued until 7 days post DMBA. 5,6 Benzoflavone (500 ppm), a positive control, decreased tumor multiplicity >90%, while 5MeCDDO (27 or 2.7 ppm) decreased multiplicity 65 and 35%, respectively. The efficacy observed in this model is in agreement with the ability of the agent to stimulate the ARE, and to induce a variety of ARE related genes (e.g., GST PI, quinone reductase, etc). Thus, as expected, high induction of the ARE was associated with a decrease in DMBA-induced cancer initiation, but not a decrease in the progression stage of mammary cancer development in three cancer models. This latter finding brings into question whether merely measuring induction of the ARE (e.g., quinone reductase) is sufficient to imply the general preventive efficacy of a given agent or mixture. Supported by NCI contract number HHSN261200433001C. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-10-03.

P3-10-01: Alternative Dosing Regimens with the EGFr Inhibitors (Gefitinib and Lapatinib) in Mammary Cancer Models: Prevention and Therapeutic Efficacy.

The EGFR inhibitors are effective in treatment of lung and pancreatic cancers (erlotinib, gefitinib) and Neu overexpressing breast cancer (lapatinib) in humans; as well as preventing multiple cancers in animal models. However, the development of toxicities (Iressa, skin rashes; Lapatinib, diarrhea) limit their potential use in prevention; and perhaps even in an adjuvant setting. We examined whether alternative dosing regimens which might reduce toxicity would still achieve preventive and therapeutic efficacy. Female Sprague-Dawley rats were administered a single IV dose of methylnitrosourea (MNU) at 50 days of age. In a prevention study, MNU treated rats administered Gefitinib daily (10 mg/kg BW/day, 7x/week) or Gefitinib (70 mg/kg BW, 1x/week) beginning 5 days after MNU resulted in 94 and 75% reductions, respectively, in cancer multiplicity. Simultaneous measurements of tumor load (number of tumors x tumor weight) showed that both regimens resulted in greater than a 90% decrease. In the therapeutic study (initiating treatment when animals developed a small palpable cancer), both regimens were again highly effective. A prevention study was also performed with Lapatinib (75 mg/kg BW/day, 7x/week or 525 mg/kg BW, 1x/week). While the daily dose reduced cancer multiplicity 90%, the weekly dose caused a 70% reduction. Finally, we examined the effects of daily or weekly dosing with Iressa (100 mg/kg BW/day, 5x/week or 500 or 250 mg/kg BW, 1x/week) in an ER − mouse model (using MMTV-Neu; p53 +/− mice). This study showed that while daily dosing with Iressa decreased tumor multiplicity roughly 80%, weekly dosing at either dose caused roughly a 50% decrease. These data show that even a significant alteration in the dosing of Gefitinib (EGFR 1) or Lapatinib (EGFR 2/1) still resulted in a large reduction in mammary cancers. The important clinical question will be whether this altered dosing will diminish the toxicities associated with these agents. We cannot determine this in animal models since, at the effective doses employed, the typical toxicities observed in humans are not observed. Supported by NCI contract number HHSN261200433001C. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-10-01.

P3-11-07: Mammary Gland Biopsy To Examine Surrogate Endpoint Biomarkers of Preventive Agent Efficacy.

Phase II breast cancer prevention trials examine agent activity by sampling and imaging the breast through core needle biopsy, fine needle aspiration, or imaging (e.g., mammography). The biomarker endpoints in normal or at risk breast tissue employed with biopsies or fine needle aspirates (e.g., Ki67 or apoptosis) have often not been formally validated relative to a tumor endpoint. In this study, we have attempted to examine these endpoints in a preclinical model for which we have final tumor data. In the methylnitrosourea (MNU)-induced rat mammary cancer model, estrogen receptor positive cancers develop that are histologically similar to a large majority of human tumors. Here we present a protocol for correlating surrogate endpoint biomarkers and agent efficacy in the same animal. Biopsy samples are taken of the normal mammary gland after MNU treatment, and a week later the animals are started on the preventive agent. After 2 weeks, a second biopsy is taken and the animals are followed for tumor development. To test the protocol, we examined surrogate endpoints of tamoxifen efficacy at human equivalent dosages (5mg/d and 20mg/d). Both doses of tamoxifen prevented the development of tumors in the MNU-treated rats. The pre/post biopsy analysis revealed a statistically significant reduction in Ki-67 consistent with the clinical trial data. Additional biomarkers of apoptosis and the chromosome condensation pathway will be presented. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-11-07.

Abstract P2-07-03: Tumors Resistant to Iressa Treatment Show Increased Aberrant Expression of CREB (Ser133)

Background: Activation of tyrosine kinase receptors, including EGFR, HER2, HER3 and HER4, plays a key role in the prognosis of mammary cancer. EGFR is a validated therapeutic target; but unfortunately, only a small percentage of patients with EGFR-overexpressing tumors respond to therapy, and resistance develops even in responsive patients. Iressa is a small molecule tyrosine kinase inhibitor that suppresses the activation of EGFR. Completely understanding the molecular mechanisms and protein targets involved in the effects of Iressa can help determine why efficacy varies. This requires the simultaneous identification of specific molecular markers in the complex network of signaling pathways that are secondarily modulated by Iressa in mammary cancer. Methods and Materials: In this study, female Sprague-Dawley rats (50 days old) were given methylnitrosourea (MNU) by IV injection through the jugular vein (75 mg/kg BW). The rats were palpated for mammary cancers 2 times per week. When a palpable mammary cancer of approximately 200-250 mm2 was present, the rat was administered Iressa (6 mg/kg BW/day) by gavage for 2 days. The tumor was then biopsied. The rats were treated with Iressa at the same dose for an additional 40 days and tumors were harvested. At sacrifice, the mammary cancer was rapidly removed and frozen for protein array analysis. The tumors were sorted into two groups based on sensitivity to treatment with Iressa and analyzed by a phospho-protein Proteome Profiler™ Array (RD70(24 Suppl):Abstract nr P2-07-03.

Effects of methyl derivatives of the rexinoid UAB30 on methylnitrosourea (MNU) induced mammary cancers and on various indicators of toxicity.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #1108 UAB30 is a RXR selective retinoid that has shown excellent activity in preventing chemically-induced mammary cancers with minimal toxicity (specifically not increasing serum triglycerides), and is currently undergoing a Phase I clinical trial. Several methyl analogs were synthesized in an attempt to obtain a more active preventive agent and to better understand the mechanisms of activity/toxicity of this class of agents. The retinoids included 4-methyl-UAB30, 5-methyl-UAB30, 6-methyl-UAB30, 7-methyl-UAB30, and 8-methyl-UAB30. The compounds were evaluated in the MNU-induced mammary cancer model using female Sprague-Dawley rats. MNU (75 mg/kg BW) was administered at 50 days of age and the animals observed for 120 days afterward for the development of ER+ mammary cancers. The retinoids were given at a dose of 200 mg/kg diet, except 7-methyl-UAB30 (given at 100 mg/kg diet). 4-Methyl-UAB30 and 7-methyl-UAB30 were highly active; reducing mammary cancer multiplicity by 74 and 61%, respectively. The retinoids 5-methyl-UAB30, 6-methyl-UAB30, and 8-methyl-UAB30 did not have chemopreventive activity in this model. The 8-methyl-UAB30 derivative actually caused a 108% increase in growth of the mammary cancers. As we have previously shown (Carcinogenesis 27, 1232-1239, 2006), serum triglycerides correlated with cancer preventive activity; i.e., high levels were observed with active retinoids. 4-methyl-UAB30 caused an initial large increase in body weight gain of the rats. Of interest, serum levels of 6-methyl-UAB30 and 7-methyl-UAB30 were high, while the other agents were low or could not be detected. All methyl derivatives caused varying decreases in liver retinyl palmitate levels. Structure-activity relationships are also being evaluated using crystallography of RXR/UAB-rexinoid complexes as a guide. Structure-based and dynamic-based approaches are used to facilitate the design of new rexinoids that fit into the LBD of RXRs. For example, 4-methyl-UAB30 had a 5-fold greater binding affinity to hRXR alpha LBD than 9-cis-retinoic-acid. These studies emphasize that minor modifications of a retinoid molecule can drastically change its absorption, metabolism, toxicity, binding affinities to receptors, and activity in preventing mammary cancers. (Supported by NCI Breast SPORE CA089019 and contract HHSN261200433001C). Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1108.

Lapatinib Suppresses RTK-Mediated Signaling through Multiple Signaling Pathways.

Background: Activation of tyrosine kinase receptors, including EGFR, HER2, HER3 and HER4, plays a key role in the prognosis of mammary cancer. EGFR and HER2 are validated therapeutic targets; but unfortunately, only a small percentage of patients with EGFR or HER2-overexpressing tumors respond to therapy, and resistance develops even in responsive patients. Lapatinib is a small molecule dual tyrosine kinase inhibitor that suppresses the activation of EGFR and HER2. Completely understanding the molecular mechanisms and protein targets involved in the effects of Lapatinib and other RTK inhibitors can help determine why efficacy varies. This requires the simultaneous identification of specific molecular markers in the complex network of signaling pathways that are modulated by Lapatinib in mammary cancer.Methods and Materials: In this study, female Sprague-Dawley rats (50 days old) were given methylnitrosourea (MNU) by IV injection through the jugular vein (75 mg/kg BW). The rats were palpated for mammary cancers 2 times per week. When a palpable mammary cancer of approximately 200-250 mm2 was present, the rat was administered Lapatinib (150 mg/kg BW/day) by gavage for 7 days. This dose of lapatinib was effective in a prevention study (causing a greater than 80% inhibition of mammary cancers). At sacrifice, the mammary cancer was rapidly removed and fixed for immunohistochemistry or frozen for protein array analysis. Fixed samples were analyzed for phosphorylated EGFR (Tyr1177) and HER2 expression by incubating with specific primary antibodies to detect pEGFR (Tyr1177) or HER2 and secondary antibodies conjugated to a fluorescence dye. Samples were observed by confocal microscopy. Frozen tissues were subjected to Proteome Profiler™ Arrays (RD69(24 Suppl):Abstract nr 1130.

Progress in Developing Effective Chemoprevention Agents for Cervical Neoplasia

Chemoprevention refers to the use of micronutrients or pharmaceutical agents to prevent or delay the development of cancer in healthy populations. Agents employed in chemopreventive trials should prevent or reverse the initiation, promotion, or progression processes involved with carcinogenesis. Chemoprevention strategies would therefore be best employed in patients with normal or premalignant lesions who are at high risk of developing a malignancy.