Ra Lubet

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.

P5-06-11: Quercetin-3-Methyl Ether Inhibits Lapatinib-Sensitive and Lapatinib-Resistant Breast Cancer Cell Growth by Inducing G2/M Arrest and Apoptosis.

Background: Lapatinib, an oral, small-molecule, reversible inhibitor of both EGFR and HER2, is highly active in HER2 positive breast cancer as a single agent and in combination with other therapeutics. However, resistance against lapatinib is an unresolved problem in clinical oncology. Recently, interest in the use of natural compounds to prevent or treat cancers has gained increasing interest because of presumed low toxicity. Quercetin-3-methyl ether, a naturally occurring compound present in various plants, has potent anticancer activity. Material and Methods: Quercetin-3-methyl ether was obtained from Analyticon Discovery (Potsdam, Germany). Lapatinib-resistant SK-Br-3 (SK-Br-3-Lap R) cells were isolated in the laboratory of Cell Biology and Biotherapy at the Istituto Nazionale dei Tumori, Naples, Italy. Parental SK-Br-3 and SK-Br-3-Lap R cells were cultured in monolayers at 37 °C in a 5% CO2 incubator in 10% FBS/McCoy supplemented with penicillin/streptomycin (100 units/ml; Invitrogen). SK-Br-3 Lap R cells were routinely maintained in 1 μM lapatinib. Anchorage-dependent and -independent growth and cell cycle were assessed in the presence and absence of quercetin-3-methyl ether. Results: Here, we found that quercetin-3-methyl ether caused in a significant growth inhibition of lapatinib-sensitive and -resistant breast cancer cells. Western blot data showed that quercetin-3-methyl ether had no effect on Akt or MAPKs signaling in resistant cells. However, quercetin-3-methyl ether caused a pronounced G2/M block mainly through the Chk1-Cdc25c-cyclin B1/Cdk1 pathway in lapatinib-sensitive and -resistant cells. In contrast, lapatinib produced an accumulation of cells in the G1 phase mediated through cyclin D1, but only in lapatinib-sensitive cells. Moreover, quercetin-3-methyl ether induced significant apoptosis, accompanied with an increase the levels of in cleaved caspase 3, caspase 7 and poly(ADP-ribose) polymerase (PARP) in both cell lines. Conclusion: Overall, these results suggested that quercetin-3-methyl ether might be a novel and promising therapeutic agent in lapatinib-sensitive or -resistant breast cancer patients. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-06-11.

Abstract P1-08-04: Doses of Selective Estrogen Receptor Modulations (SERMS) Required for Prevention and Therapy of Chemically-Induced Mammary Cancers

Selective Estrogen Receptor Modulators (SERMs) interfere with the activation of the estrogen receptor. SERMs have been shown to be effective for both treatment and prevention of breast cancer. Here we examine the ability of tamoxifen and arzoxifene, a more easily absorbed form of raloxifene, to prevent mammary tumors at a significantly lower dosage than those used for treatment. For prevention studies, female Sprague-Dawley rats (50 days of age) were injected with a single i.v. dose of MNU resulting in the development of multiple ER-positive mammary tumors beginning 7 weeks after carcinogen treatment. Rats were administered the preventive agent beginning five days after carcinogen administration. When the agents were tested at does varying between 0.15-3.3 ppm in the diet, both agents caused a dose dependent increase in preventive activity. The highest doses of tamoxifen (3.3 ppm) and arzoxifene (3.0 ppm) both decreased tumor multiplicity greater than 90%. Both of these doses are less than one-fifth the human equivalent dose (HED) based on standard FDA scaling factors. Both doses also resulted in substantial decreases in mammary gland development. These dosages were then used in a therapy setting to compare the efficacy. For these studies, MNU treated rats were allowed to develop their first palpable mammary tumor (70-150 mm 2 ) before initiating treatment. Tumor growth or regression was followed by caliper measurements. Neither of the preventive doses of these SERMs exhibited therapeutic activity although substantially higher doses of tamoxifen (33 or 100 ppm) were relatively effective. These results are in contrast to our prior findings with other classes of agents (RXR agonists, EGFR inhibitors, and aromatase inhibitors) in which highly effective preventive doses were as effective as therapeutic doses in this model. These results demonstrate the unique dose differential regarding prevention and therapy with SERMs. Microarray analysis of tumors treated for 5 days with tamoxifen at doses of 3.3ppm or 100ppm reveals overlapping significant gene changes of similar magnitude in comparison to control treated tumors although there were significant gene changes seen at the therapeutic dose which were not observed at the lower dose. These studies raise two questions: 1) whether this class of agents might be used at significantly lower doses in a pure prevention setting as contrasted with a therapeutic setting where they were initially defined; 2) are there gene expression changes which might be used to help identify this differential dosing for prevention vs. therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-08-04.

Abstract PD03-07: Evaluation of the Anti-Diabetic Drug Metformin in the Methylnitrosourea (MNU)-Induced Model of ER+ Mammary Cancer and the MMTV-Neu/p53KO Model of ER - Mammary Cancer

Metformin, an anti-diabetic drug, activates AMP-activated protein kinase (AMPK), and has generated a great deal of interest as a potential preventive agent for a variety of cancers, including breast. This interest is based both on epidemiologic studies showing that diabetics treated with metformin have a lower incidence of a variety of cancers, and on mechanistic considerations arguing that activation of AMPK is likely to be relevant to cancer treatment and prevention. We are examining the preventive efficacy of metformin in ER+ and ER - rodent mammary cancer models. Treatment of Sprague-Dawley female rats at 50 days of age with a single dose of MNU (75 mg/kg BW) resulted in the development of multiple ER + mammary cancers beginning approximately 7 weeks after MNU treatment. This model has been highly responsive to a wide variety of agents that affect ER+ tumors in humans (SERMs, aromatase inhibitors, pregnancy, ovariectomy). Metformin was administered by gavage (7x/week) at either 150 or 50 mg/kg BW/day beginning 7 days after MNU dosing, and continuing for the duration of the study. Body weights were not altered by these dose levels and other signs of toxicity were not observed. Metformin failed to decrease mammary tumor incidence and, in fact, increased tumor multiplicity in this model by approximately 35%. In addition, we are evaluating the ability of metformin to inhibit mammary cancer in MMTV-Neu/p53KO mice (which spontaneously develop ER - cancers). In this study, metformin is being administered in the diet at a dose level of 1500 mg/kg diet. Initial supplementation of the diet with this agent began when the mice were 60 days of age, and will continue for 10 months. Preliminary results examined at 6 months into the study imply no efficacy of metformin in this model as well (average tumors/mouse: controls, 0.9; metformin, 1.6). Thus, these initial studies fail to observe any chemopreventive efficacy of metformin in two commonly employed models of ER + or ER - mammary cancer in rodents. Supported by NCI contract number HHSN261200433001C. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD03-07.

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.

Preventive Effects of Lapatinib, a Combined EGFR 1 and EGFR 2 Inhibitor, on Methylnitrosourea-Induced ER+ and MMTV-Neu/P53 KO ER- Mammary Cancers.

Lapatinib is a small molecule TK inhibitor that blocks both EGFR 1 and EGFR 2 (Neu) in the nanomolar range. While the potential usefulness of this agent in Neu over-expressing tumors appears obvious, its potential efficacy in ER + tumors that do not over-express Neu seems more problematic. The efficacy of lapatinib was tested in two animal models of mammary cancer; the MNU induced rat model that has ER + mammary cancers and the MMTV-Neu/P53 KO transgenic mouse model that has Neu over-expressing ER - cancers. In the ER + mammary model, the carcinogen (75 mg/kg BW) was administered to female Sprague-Dawley rats at 50 days of age via the jugular vein. These animals subsequently developed multiple well-differentiated mammary adenocarcinomas. Lapatinib treatment (75 or 25 mg/kg BW/day), 7x/week by gavage, was initiated five days after the carcinogen and continued until the end of the study (148 days post MNU). The high and low doses of lapatinib reduced mammary cancer multiplicity by 81% and 31%, respectively. Lapatinib at the higher dose was also an effective therapeutic agent in this model. In the MMTV-Neu/P53 KO model (over-expressed Neu under control of MMTV and heterozygous KO of P53), it was observed that lapatinib administered at a dose of 75 mg/Kg BW/day beginning when an animal was 8 weeks of age reduced cancer multiplicity by 60 %. When the short term effects of lapatinib was examined in both models, it was found that levels of phosphorylated EGFR 1 and 2, ERK and AKT, as well as the proliferation index (Ki 67), were reduced in the cancers in both models. The potential use of an agent such as lapatinib in preventing ER + tumors is of particular interest since tamoxifen and to a lesser degree aromatase inhibitors are relatively ineffective in the prevention of ER + tumors which over- express Neu. Supported by NCI contract HHSN261200433001C. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1043.

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.