Prevention Of ER-negative Breast Cancer Research Articles

Targeting the mTOR Pathway for the Prevention of ER-Negative Breast Cancer.

Our results show that everolimus delays mammary tumor formation in multiple mouse models, suggesting that mTOR inhibitors will be useful for the prevention of ER-negative and triple-negative breast cancer in humans. See related Spotlight, p. 787.

Abstract 715: Targeting the mTOR pathway for the prevention of er-negative breast cancer

Abstract Background: Triple-Negative breast cancer (TNBC) is an aggressive cancer that lacks expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. These cancers have a poor prognosis and are treated predominantly with chemotherapy. Therefore, we are working to find preventive therapies for these potentially lethal cancers. Dysregulation of PI3K-mTOR pathway is commonly associated with TNBC and other ER-negative breast cancers. We hypothesized that targeting mTOR may prevent development of ER-negative breast cancers and in this study tested whether the mTOR inhibitor, everolimus, prevents cancer in several mouse models of ER-negative breast cancer. Methods: MMTV-erbB2 mice were purchased from the Jackson Laboratory. C3(1)/SV40TAg and BRCA1co/co;MMTV-Cre;p53+/- were generated through breeding. P53-null and p53-mutant mammary gland mice were generated by transplanting p53-null or p53-mutant mammary glands into cleared fat pads of p53-wild type mice. The mice were treated with either control or everolimus (5mg/kg), administered by oral gavage daily (5X/week). All mice develop palpable mammary tumors within 8-12 months. Mice were observed daily for tumor formation and drug toxicity. The percentage of tumor free mice was recorded, and time to tumor development was visualized using Kaplan-Meier curves and analyzed using the generalized Wilcoxon test. Results: Treatment with everolimus significantly increased median survival of all mouse models. Everolimus treatment was also able to completely prevent mammary tumor formation in 27% of C3(1)/SV40Tag mice and 44% p53-mutant mammary gland mice. In p53-null mammary gland mice, 46% of control mice had developed tumors after 420 days, compared to 7% of everolimus treated mice. Long term treatment of everolimus was associated with mild toxicity that includes slight weight loss (<10%) and skin changes (matted hair, skin erythema). Conclusions: Everolimus significantly delayed mammary tumorigenesis in all five breast cancer mouse models. Our results suggest that everolimus is a promising cancer preventive drug for ER-negative tumors, and that further studies of everolimus in combination with other targeted therapies are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients. Supported by NCI PREVENT Cancer Preclinical Drug Development Program (HHSN-2612015000-18I (PB). Citation Format: Abhijit Mazumdar, Jamal Hill, William Tahaney, Yanxia Ma, Alejandro Contreras, Shizuko Sei, Altaf Mohammed, Powel Brown. Targeting the mTOR pathway for the prevention of er-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 715.

Abstract 2610: Targeting the mTOR pathway for the prevention of ER-negative breast cancer

Abstract Background: Prevention of estrogen receptor (ER)-positive breast cancer prevention is now possible antiestrogen drugs; however, this treatment is ineffective against ER-negative breast cancers. ER-negative breast cancer occurs more often in younger women and is associated with a poor prognosis. Dysregulation of PI3K-mTOR pathway, specifically upregulation of mTOR and loss of phosphatase and tensin homolog (PTEN) has been commonly associated with ER-negative breast cancers and been shown to be associated with poor prognosis. We hypothesized that by targeting mTOR, will suppress the growth of ER-negative and triple negative breast cancers. To test the hypothesis, we used MMTV-erbB2, C3(1)/SV40T, and p53 null mammary gland mouse models to determine whether mTOR inhibitor everolimus is effective in preventing growth of ER-negative mammary tumors. Methods p53-null Balb/C donor mice were transplanted into both cleared inguinal mammary fat pads of Balb/C p53 wild-type mice. All mice were separated into 2 groups 1) sesame oil control and 2) everolimus (5 mg/kg). All treatments were given by oral gavage, five days a week from 3 months of age for MMTV-erbB2 and 2 months of age for SV40T mice, and in case of p53 mice, two months after transplantation. Mice were observed daily for tumor formation, toxicity and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the generalized Wilcoxon test. Results: In MMTV-erbB2 mice the mTOR inhibitor, everolimus reduced tumor incidence and was associated with an increase in median survival time from 240 days to 410 days in. At 365 days, when all mice in control group died, only half of the mice treated with everolimus had developed mammary tumors. Long term treatment of everolimus was associated with mild toxicity that includes weight loss (<10%) and skin changes (matted hair, skin erythema). Everolimus also reduced tumor incidence in C3 (1)/SV40 mice, with an increase in median survival time from 130 days to median survival which has not reached yet (>180 days). At 150 days, when all mice from the control group developed tumor only 35% mice from treatment group had developed tumors. Everolimus also significantly reduced tumor incidence in p53 null mammary gland mice. At 420 days, when approximately in 50% of the control mice developed mammary tumors compare to only 7% of the everolimus treated mice developed tumors. Conclusion: The mTOR inhibitor everolimus prevented ER-negative mammary in two mouse models (MMTV-erbB2, C3 (1)/SV40, and p53 null mammary gland mice). Based on our results Everolimus is an effective cancer preventive drug. Our results suggest that, studies with reduced everolimus dose alone or in combination with other targeted therapies such as selective estrogen receptor modulators are warranted. In the future, clinical trials of the everolimus should be considered for the prevention of breast cancer in high-risk patients. Citation Format: Abhijit Mazumdar, Yun Zhang, Jamal Hill, Powel Brown. Targeting the mTOR pathway for the prevention of ER-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2610.

Abstract 2804: The thiazolidinedione pioglitazone enhances the cancer preventive activity of the rexinoid LG100268 in Her2/neu induced breast cancer

Abstract Two major unresolved issues for breast cancer prevention are the lack of effective preventive agents for estrogen receptor (ER)-negative disease and the potential toxicity of long-term pharmacologic treatment. The ability of retinoid X receptor (RXR)-selective retinoids (rexinoids) to inhibit breast cancer formation independently of ER-status was previously demonstrated in various preclinical models. The goal of our study was to identify ideal druggable targets for the synergistic amplification of the cancer preventive effect of synthetic rexinoids and test the efficacy of a low-dose combination treatment in vivo. We identified the nuclear hormone receptors essential for rexinoid-dependent growth suppression by a high throughput siRNA screen of mammary epithelial cells. Knock-down of ELP, COUP-TF II, GR, TR2, VDR and PPARgamma partially reversed the anti-proliferative effect of the rexinoid bexarotene. Conversely, expression profiling showed that the largest fraction of genes up-regulated by the synthetic rexinoid LG100268 represented regulators of lipid metabolism and putative target genes of the RXR partner receptor PPARgamma. Combination dose-response experiments showed that the PPARgamma agonist pioglitazone was synergistic with low doses (100-300 nM) of LG100268. Long-term daily treatment of MMTV-ErbB2 mice with LG100268 (25 mg/kg) or pioglitazone (20 mg/kg), or their combination resulted in a significant delay of tumor formation, while serum triglyceride levels were not significantly elevated by the combination. Suppression of cell proliferation measured by reduced Ki67 staining was enhanced by the addition of pioglitazone, in both lesions and intact mammary epithelium, while pioglitazone also increased the frequency of cells containing cleaved caspase 3. Furthermore, pioglitazone treatment was associated with a marked induction of microvesicle formation in the adipose issue. These data suggest that the combination of LG100268 and pioglitazone, while potentially targeting the same heterodimer, can invoke distinct biological processes resulting in more effective cancer prevention than achieved by either agent alone, and may be an effective strategy for the prevention of ER-negative breast cancer. Citation Format: Iván P. Uray, Jennifer M. Rodenberg, Yun Zhang, Jamal L. Hill, Powel H. Brown. The thiazolidinedione pioglitazone enhances the cancer preventive activity of the rexinoid LG100268 in Her2/neu induced breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2804. doi:10.1158/1538-7445.AM2015-2804

Abstract B33: Target STAT3 signaling for the prevention of ER-negative breast cancer

Abstract Estrogen receptor (ER)-negative breast cancer counts for approximately 30-40% of breast cancer cases. Although a large portion of ER-positive breast cancer can be prevented with ER modulators and aromatase inhibitors, these drugs fail to prevent any ER-negative breast cancers or the rest, resistant ER-positive breast cancers. Particularly, there is no targeted therapy for preventing ER-negative breast cancer, due to its lack of ER expression and resulting non-responsive nature to ER modulators. Thus, prevention of ER-negative breast cancer is an unmet need and challenge, and it is urgent to identify novel molecular targets and develop effective chemopreventive agents for the prevention of all types of breast cancers. The STAT3 transcription factor is a major contributor in the development of multiple cancers since STAT3 signaling is required for the growth of stem cell-like cancer cells. In our preliminary studies, we found that STAT3 is activated in human mammary epithelial cells that overexpress tert and ras oncogenes, in the premalignant mammary glands of MMTV-erbB2 mice before mammary tumors develop, and in human breast premalignant hyperplasia lesion and ductal carcinoma in situ (DCIS). Thus, STAT3 activation is an early molecular event in breast carcinogenesis and therefore, STAT3 represents a promising, new molecular target for the prevention of breast cancer. We hypothesized that STAT3 is a driving factor in breast cancer development, and blockade of STAT3 activity will prevent ER-negative breast cancer formation. However, STAT3 is traditionally termed as “un-druggable” target. Previous strategies of inhibiting STAT3 activity did not sufficiently block STAT3 activation in cancer cells due to their limited potency, poor solubility and bioavailability. We therefore developed orally active STAT3 inhibitors for cancer prevention use. Our newly developed STAT3 inhibitors suppress STAT3 activation and ER-negative breast cancer cell proliferation at low μM to nM potency, induce apoptosis, and suppress xenograft tumor growth in vivo. The inhibitors also show low toxicity and superior aqueous solubility as well as oral bioavailability. We treated the MMTV-erbB2 transgenic mice that develop spontaneous ER-negative mammary tumors, at the age of 3 months when mammary glands were fully developed. The STAT3 inhibitor, HJC0152, was given via oral gavage for 5 days per week at a dosage of 25 mg/kg for 2-8 months. While our experiments are still ongoing, we have started to observe significant chemopreventive effect of HJC0152 on reducing oncogene-induced ER-negative mammary tumor formation in MMTV-Neu model. These studies suggest that inhibition of STAT3 activation is a promising strategy for the prevention of ER-negative breast cancer. This work was supported by Grants P50 CA097007, P30DA028821, and R21MH093844 (J.Z.) from NIH, R. A. Welch Foundation Chemistry and Biology Collaborative Grant from Gulf Coast Consortia (GCC), John Sealy Memorial Endowment Fund (J.Z.), the DFI Seed Funding Program from MD Anderson Cancer Center (Q.S.). Citation Format: Zhengduo Yang, Haijun Chen, Chunyong Ding, Christopher Wild, Jia Zhou, Qiang Shen. Target STAT3 signaling for the prevention of ER-negative breast cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr B33.

Abstract A62: Combination of BXL-0124, a vitamin D analog and LG100268 for the prevention of ER-negative breast cancer

Abstract Background: ER-positive Breast cancer prevention is now possible using antiestrogen drugs; however, this treatment is ineffective against estrogen receptor (ER)-negative breast cancers. Current treatments for ER-negative tumors include chemotherapy, or for those ER-negative cancers that overexpress HER2, the anti-HER2 antibody herceptin. Targeted therapy for ER-negative tumors, particularly those that do not express HER2 (triple-negative breast cancer) is urgently needed. Epidemiological evidence from several case control and cohort studies support an inverse relationship between vitamin D intake and breast cancer incidence. Vitamin D deficiency was also linked to poor outcomes in patients with early breast cancer. Using a transgenic mouse model of ER-negative breast cancer, our laboratory previously demonstrated that rexinoid LG100268 reduced mammary tumor development in these mice. We hypothesized that the combination of a rexinoid and a vitamin D analog (BXL-0124) would more effectively prevent the development of ER-negative breast cancer. To test the hypothesis, we used MMTV-erbB2 mouse tumors that do not express ER, to determine whether LG100268 and BXL-0124 combination is more effective than either treatment alone in preventing ER-negative mammary tumors. Methods: Virgin female MMTV-erbB2 mice (Jackson Lab) were housed in the institutional animal facilities and fed a purified diet (AIN-76A; Harlan Teklad). Mice were separated into 4 treatment groups: 1) sesame oil and DMSO control 2) Rexinoid (25 mg/kg) plus DMSO 3) BXL-0124 (3ug) plus sesame oil. 4) LG100268 (25mg/kg) plus BXL-0124 (3ug). All treatments were given by oral gavage, five days a week from 3 months of age. Mice were observed daily for tumor formation, toxicity and the percentage of tumor free mice were recorded. Tumor incidence and time to tumor formation was visualized using Kaplan Meier curves and analyzed using the generalized Wilcoxon test. Results: Rexinoid LG100268 reduced tumor incidence and was associated with an increase in median survival time from 242 days to 365 days. BXL-0124 also reduced tumor incidence with an increase in median survival time from 242 days to 288 days. The combination of LG 100268 and BXL-0124 had additive effect in preventing ER-negative mammary tumor development with a median survival time increase from 242 days to 422. days. We are investigating blood and tissue biomarkers currently and that results are forthcoming. Conclusion: Rexinoid LG100268 and vitamin D analog BX-0124 reduced tumor incidence in MMTV-erbB2 mice. The combination treatment of LG100268 and BXL0124 was more effective in preventing mammary tumor development compare to either agent alone. Based on our results, more combinatorial studies of LG100268 and BXL-0124 in other ER-negative models are warranted. In future clinical trials of the combination of BXL-0124 and LG100268 should be considered for the prevention of breast cancer in high-risk patients. (Supported by NIH R01 CA078480 (PB) and MDACC CCSG P30 CA 016672) Citation Format: Abhijit Mazumdar, Yun Zhang, Jamal Hill, Nanjoo Suh, Powel Brown. Combination of BXL-0124, a vitamin D analog, and LG100268 for the prevention of ER-negative breast cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A62.

Prevention of ER-negative breast cancer

Oestrogen receptor-negative and triple-negative breast cancers are types of aggressive tumours that account for approximately 30 and 15% of total breast cancers, respectively. Selective oestrogen receptor modulators and aromatase inhibitors are unable to treat and prevent these subtypes of mammary tumours. Thus, it is worth identifying new pathways, biomarkers, and agents that are effective in the treatment and prevention of these subtypes. Several classes of drugs have been studied, and many are still currently under investigation. We have attempted to conduct a state-of-the-art study on this important issue.

Abstract 2838: Brief exposure to gefitinib/Iressa during premalignant risk window provides lifelong protection from mammary tumor development in MMTV-erbB-2 transgenic mice

Abstract Although chemopreventive agents targeting estrogen/estrogen receptor (ER) pathway have been effective for ER+ breast cancer, hormonal resistance associated with increased receptor tyrosine kinase activities, such as erbB-2 and/or EGFR, remains a significant issue in beast cancer prevention. Previous studies demonstrated that administration of gefitinib, an orally active EGFR tyrosine kinase inhibitor, to MMTV-erbB-2 transgenic mice inhibited mammary tumor development in these mice, suggesting that gefitinib is a promising agent for the prevention of ER-negative breast cancer. However, the prevention was achieved by high dose (100 mg/kg) and prolonged drug exposure (from 12 to 50 weeks). The tolerance to high dose/long-term of drug administration for the prevention regimens raised concerns in clinical application. In this study, we tested the efficacy of brief exposure to gefitinib during the high risk window in the prevention of mammary tumor development in the MMTV-erbB-2 transgenic mice and studied the underlying mechanisms. We found that brief exposure of gefitinib to these mice at a dosage of 100 mg/kg/day from 16 to 23 weeks (total 8 wks) resulted in significant chemopreventive effects. In contrast to no tumor-free animals in the control group by 52 weeks, mice in gefitinib treated group remained 65% tumor-free at the same age, which was comparable to the results from a previous report that mice with lifelong gefitinib were 75% tumor-free by 45 weeks. We further demonstrated that delayed tumor development in the treated mice was preceded with decreased mammary epithelial density. Molecular analysis indicated that gefitinib inhibited phosphorylation and expression of EGFR, erbB-3 and endogenous erbB-2 in premalignant mammary tissues. Phosphorylation of Akt1 and Erk1/2 was also significantly downregulated. Importantly, although tumors developed from this model were ER-, mammary tissues in the premalignant stage were ERα+; and gefitinib treatment drastically inhibited the phosphorylation and expression of ERα and the transcription of ER target genes, including EGF, EGFR, ESR1, Bcl-2, cyclin D1 and c-myc. Moreover, BrdU incorporation analysis demonstrates that cell proliferation in the mammary glands in gefitinib treated mice was significantly inhibited. Taken together, these data demonstrate that brief treatment with EGFR/erbB-2 targeting agents at the appropriate risk window may provide lifelong protection from mammary tumors, and inhibition of ER-erbB-2 crosstalk may play a critical role in this long lasting preventive process. Given the concerns associated with high dose/long-term gefitinib treatment, our findings are of great significance in directing clinical application of EGFR/erbB-2 targeting agents as chemopreventive agents. Citation 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 2838. doi:10.1158/1538-7445.AM2011-2838

Effect of Lapatinib on the Development of Estrogen Receptor–Negative Mammary Tumors in Mice

Lapatinib, a selective orally available inhibitor of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases, is a promising agent for the treatment of breast cancer. We examined the effect of lapatinib on the development of mammary tumors in MMTV-erbB2 transgenic mice, which express wild-type ErbB2 under the control of the mouse mammary tumor virus promoter and spontaneously develop estrogen receptor (ER)-negative and ErbB2-positive mammary tumors by 14 months of age. Mice were treated from age 3 months to age 15 months with vehicle (n = 17) or lapatinib (30 or 75 mg/kg body weight; n = 16 mice per group) by oral gavage twice daily (6 d/wk). All statistical tests were two-sided. By 328 days after the start of treatment, all 17 (100%) of the vehicle-treated mice vs five (31%) of the 16 mice treated with high-dose lapatinib developed mammary tumors (P < .001). Among MMTV-erbB2 mice treated for 5 months (n = 20 mice per group), those treated with lapatinib had fewer premalignant lesions and noninvasive cancers in their mammary glands than those treated with vehicle (P = .02). Lapatinib also effectively blocked epidermal growth factor-induced signaling through the EGFR and ErbB2 receptors, suppressed cyclin D1 and epiregulin mRNA expression, and stimulated p27 mRNA expression in human mammary epithelial cells and in mammary epithelial cells from mice treated for 5 months with high-dose lapatinib. Thus, cyclin D1, epiregulin, and p27 may represent useful biomarkers of lapatinib response in patients. These data suggest that lapatinib is a promising agent for the prevention of ER-negative breast cancer.

S22. Prevention of ER-negative breast cancer

S22. Prevention of ER-negative breast cancer