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Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and lacks effective targeted treatment strategies. Previously, we identified 33 transcription factors highly expressed in TNBC. Here, we focused on six sex determining region Y-related HMG-box (SOX) transcription factors (SOX4, 6, 8, 9, 10, and 11) highly expressed in TNBCs. Our siRNA screening assay demonstrated that SOX9 knockdown suppressed TNBC cell growth and invasion in vitro. Thus, we hypothesized that SOX9 is an important regulator of breast cancer survival and metastasis, and demonstrated that knockout of SOX9 reduced breast tumor growth and lung metastasis in vivo. In addition, we found that loss of SOX9 induced profound apoptosis, with only a slight impairment of G1 to S progression within the cell cycle, and that SOX9 directly regulates genes controlling apoptosis. On the basis of published CHIP-seq data, we demonstrated that SOX9 binds to the promoter of apoptosis-regulating genes (tnfrsf1b, fadd, tnfrsf10a, tnfrsf10b, and ripk1), and represses their expression. SOX9 knockdown upregulates these genes, consistent with the induction of apoptosis. Analysis of available CHIP-seq data showed that SOX9 binds to the promoters of several epithelial–mesenchymal transition (EMT)- and metastasis-regulating genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of genes involved in EMT (vim, cldn1, ctnnb1, and zeb1) and that SOX9 knockdown suppresses the expression of these genes. Implications: Our studies identified the SOX9 protein as a “master regulator” of breast cancer cell survival and metastasis, and provide preclinical rationale to develop SOX9 inhibitors for the treatment of women with metastatic triple-negative breast cancer.

Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIβ (PI4KIIIβ). Molecular, biochemical, and cell biological studies show that PI4KIIIβ-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIβ-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIβ antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIβ-dependent secretion for cancer cell survival and tumor progression.

Estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and HER2-negative, or “triple negative,” breast cancer (TNBC) is a poor prognosis clinical subtype that occurs more frequently in younger women and is commonly treated with toxic chemotherapy. Effective targeted therapy for TNBC is urgently needed. Our previous studies have identified several kinases critical for TNBC growth. Since phosphatases regulate the function of kinase signaling pathways, we sought to identify critical growth-regulatory phosphatases that are expressed differentially in ER-negative, as compared to ER-positive, breast cancers. In this study, we examined the role of one of these differentially expressed phosphatases, the protein phosphatase Mg + 2/Mn + 2 dependent 1A (PPM1A) which is underexpressed in ER-negative breast cancer as compared to ER-positive breast cancers, in regulating TNBC growth. We found that PPM1A is deleted in ~40% of ER-negative breast cancers, and that induced expression of PPM1A suppresses in vitro and in vivo growth of TNBC cells. This study demonstrates that induction of PPM1A expression blocks the cell cycle and reduces CDK and Rb phosphorylation. These results suggest PPM1A is a crucial regulator of cell cycle progression in triple negative breast cancer. Our results also suggest that PPM1A loss should be explored as a predictive biomarker of CDK inhibitor sensitivity.

Background: SRY (Sex Determining Region Y)-related HMG-box (SOX) genes belong to a super-family of genes, which is characterized by a homologous sequence called the HMG-box residing on the Y-chromosome. There are 20 SOX genes present in humans and mice. We performed a siRNA screen of SOX transcription factors, and found that SOX9 was essential for breast cancer cell growth. The SOX9 protein recognizes the sequence CCTTGAG along with other members of the HMG-box class DNA-binding proteins and has been shown to be required for development, differentiation and lineage commitment. Moreover, SOX9 is expressed in adenocarcinomas, and is highly expressed in the most aggressive cancers. Our previous data shows SOX9 is highly expressed in “triple negative breast cancer” (TNBC) than in non-TNBC. Thus, we hypothesized that the SOX9 transcription factor acts as an essential molecule regulating TNBC growth and invasion. To test the hypothesis, we used SOX9-overexpressed, or SOX9-knockdown/knockout breast cancer cell models to determine whether SOX9 is necessary and/or sufficient to regulate TNBC cell proliferation, migration and invasion. Methods: We measured the cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell migration & invasion assays in ER-positive (MCF7 and ZR75-1) and ER-negative (MDA231 and MDA468) breast cancer cells. DOX-inducible SOX9-knockout cell lines were established in MDA231, MDA468, and LM2 cell lines using an inducible Cas9-CRISPR system. A SOX9 expressing lentivirus was used to overexpress SOX9, and siRNAs was used to knockdown SOXs in the different breast cancer cells. Protein and mRNA levels of SOX9 in TNBC, non-TNBC, immortalized human breast epithelial cell lines were examined by western blotting and qRT-PCR assay. Results: Knockdown of SOXs by siRNA caused decreased cell proliferation of MDA231 by ≥50% and of MDA468 by 30%-50% in siSOX4, siSOX6, siSOX9, siSOX10 and siSOX11 treatment groups (but not in siSOX8 and siSOX17 treatment groups). However, in MCF7 and T47D cell lines, treatment with siRNA to these SOX factors did not cause significant cell growth reduction. We demonstrated that SOX9 is more highly expressed in TNBC cells at both the mRNA and protein levels. Knockdown of SOX9 decreased cell migration and invasion of MDA231 to 25% and 50% respectively. The same effect also was observed in MDA468 cells, with approximately a 50% decline in migration and invasion. In SOX9-knockout MDA231, MDA468, and LM2 cells, cell proliferation, migration, and invasion were significantly reduced. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. We are now conducting in vivo studies to determine the effect of SOX9 on breast cancer cell metastasis. Conclusion: SOX9 is a critical regulator of TNBC cell proliferation, migration and invasion. These studies suggest that regulating SOX9 transcription factor and its signaling pathway will be a promising therapeutic strategy to treat TNBC and prevent metastasis. This work was supported by a Susan G. Komen Scientific Advisory Board Grant, SAB1600006 (PB), and a grant from the Breast Cancer Research Foundation 2015-2016 BCRF grant(PB), and by the Charles Cain Endowment (PB). Citation Format: Ma Y, Shepherd J, Mazumdar A, Zhao D, Bollu L, Hill J, Zhang Y, Brown P. SOX9 is a critical regulator of triple-negative breast cancer cell growth and invasion [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-04.

Background: Breast cancer is the most common cancer in women, and about 12% U.S. women will develop invasive breast cancer over the lifetime. Triple-negative breast cancers (TNBCs) which lack expression of the estrogen and progesterone receptors and the HER2 protein.have a poor prognosis and are typically treated with chemotherapy. Our previous studies have demonstrated that SOX9 is essential for survival and metastasis of TNBCs via suppressing apoptosis and promoting epidermal to mesenchymal transition (EMT). Hypothesis: SOX9-regulated proteins control TNBC growth, and represent promising targets for the treatment of these potentially lethal breast cancers. Material and Methods: Several breast cancer datasets were used to analyze the association between overall survival and SOX9 expression. A doxycycline-inducible Cas9-CRISPR knockout or siRNA knockdown system was used to inhibit SOX9 in cells. RNA-Seq analysis was used to identify SOX9 downstream targets in TNBC cells (MDA MBA-232 and MDA MB-468 with SOX9 knockdown by siRNA transfection) or non-TNBC cells (MCF-7 with SOX9 overexpression by transfection of SOX9-expression plasmid), with support from MD Anderson's genomic biostatistics core. After identification of SOX9 downstream genes, we treated TNBC cells with or without siRNAs of these identified genes to evaluate their effect on TNBC growth. Cell growth was measured using an automated cell counting assay. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. Significant genes are defined by using a false discovery rate (FDR) cut-off of 0.01 and log2 fold change of 2. Significant common genes are defined by using a FDR cut-off of 0.05 and log2 fold change of 1 among the cell lines. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student's t-test unless otherwise indicated. Results: TNBCs and late stage breast cancers showed higher SOX9 expression compared to non-TNBCs and early stage breast cancers. Using SOX9 inhibited cells, we demonstrated that SOX9 is critical for TNBC cell growth in vitro and in vivo. Using mRNA-seq analysis, we identified downstream genes regulated by SOX9 in breast cancer. 340 genes and 843 genes were suppressed in MDA MB-231 and MDA MB-468 respectively upon SOX9 knockdown, while 250 genes were upregulated in MCF-7 cells upon SOX9 overexpression. Between MDA MB-231 and MDA MB-468 cell line, there were 301 common genes were down-regulated upon SOX9 knockdown. 38 genes were down-regulated upon SOX9 knockdown in TNBC cell lines, and up-regulated upon SOX9 overexpression in MCF-7 cells. Among them, the top 10 genes that show largest difference between TNBCs and MCF-7 cells are MIOX, LAMB2P1, SLCO4C1, RGCC, PRTN3, LINC00173, UCN, LOC100129, VPREB3 and CTF1 genes. We then investigated the effect of these SOX9 downstream targets on cell growth in vitro. Loss of RGCC, PRTN3, and SLCO4C1 reduced cell growth in MDA MBA-231, and MDA MB-468, while having minimal effect on the growth of MCF-7 cells. Conclusion: Our results demostrate that the SOX9-regulated genes RGCC, PRTN3, and SLC04C1 are required for TNBC growth. These genes are critical regulators of TNBC growth and are potential novel targets for the treatment of these aggressive breast cancers. Grant Support: These studies were supported by a Breast Cancer Research Foundation (BCRF) grant (PB). Citation Format: Yanxia Ma, William M. Tahaney, Jing Qian, Jamal Hill, Yun Zhan, Abhijit Mazumdar, Powel H. Brown. PRTN3, RGCC and SLCO4C1 are critical SOX9-regulated genes that control TNBC growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1303.

Estrogen receptor–negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor–negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.

Background: There are few effective targeted therapies available for triple-negative breast cancers (TNBCs), the most aggressive form of breast cancer. A better understanding of the critical molecular regulators is necessary to develop effective targeted therapies for TNBCs. Our previous study focused on SOX9 transcription factor and determined that SOX9 is highly expressed in TNBCs which is required for TNBC cell survival and metastasis. Hypothesis: SOX9 controls TNBC cell growth and metastasis via directly regulating cell apoptosis and epithelial-mesenchymal transition (EMT) genes. Material and Methods: The breast cancer datasets were used to analyze the association between overall survival and SOX9 expression. Cell growth was measured using an automated cell counting assay. Cell migration and invasion were detected by transwell migration and invasion assays. A doxycycline-inducible Cas9-CRISPR knockout or siRNA knockdown system was used to inhibit SOX9 in cells. Xenograft mice model was established to evaluate the effect of SOX9 on TNBC cell growth in vivo. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. An EMT PCR array and the CHIP-seq enrichment analysis based on published SOX9-CHIP-seq datasets were performed to identify effect of SOX9 on downstream pathways. SOX9 binding peaks in gene promoter region were determined and confirmed by SOX9-CHIP-seq datasets and CHIP assay. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student’s t-test unless otherwise indicated. Results: Using available gene profiling datasets, we found that high expression of SOX9 was correlated with worse overall survival in Boersma breast dataset, but not in other datasets. Late stage breast cancers showed higher SOX9 expression compared to early stage breast cancers. Using SOX9 inhibited cells, we demonstrated that SOX9 is critical for TNBC cell survival and invasion in vitro and in vivo. To identify the molecular mechanisms of these functions, we used CHIP-seq data and CHIP assays to demonstrate that SOX9 directly binds to the promoter of several extrinsic apoptosis-inducing factors (such as fadd and tnfrsf10b) and represses their expressions. Loss of SOX9 caused up-regulation of the expression of these genes, consistent with the induction of apoptosis. We then investigated the effect of SOX9 on EMT-regulating genes. Loss of SOX9 induced down-regulation of most EMT genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of several EMT genes (such as vim and ctnnb1) and increases the expression of these genes. Conclusion: Our results demonstrate that by directly binding and regulating cell apoptosis and EMT gene expression, SOX9 controls TNBC survival and induces EMT resulting in aggressive and potentially lethal tumors. Grant Support: These studies were supported by a Breast Cancer Research Fund (BCRF) grant (PB). Citation Format: Yanxia Ma, Lakshmi Bollu, Jamal Hill, Yun Zhang, William M. Tahaney, Abhijit Mazumdar, Powel H. Brown. SOX9 controls TNBC growth and metastasis via regulating the expression of apoptosis and EMT genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2618.

Estrogen receptor (ER)-negative cancers have a poor prognosis, and few targeted therapies are available for their treatment. Our previous analyses have identified potential kinase targets critical for the growth of ER-negative, progesterone receptor (PR)-negative and HER2-negative, or “triple-negative” breast cancer (TNBC). Because phosphatases regulate the function of kinase signaling pathways, in this study, we investigated whether phosphatases are also differentially expressed in ER-negative compared to those in ER-positive breast cancers. We compared RNA expression in 98 human breast cancers (56 ER-positive and 42 ER-negative) to identify phosphatases differentially expressed in ER-negative compared to those in ER-positive breast cancers. We then examined the effects of one selected phosphatase, dual specificity phosphatase 4 (DUSP4), on proliferation, cell growth, migration and invasion, and on signaling pathways using protein microarray analyses of 172 proteins, including phosphoproteins. We identified 48 phosphatase genes are significantly differentially expressed in ER-negative compared to those in ER-positive breast tumors. We discovered that 31 phosphatases were more highly expressed, while 11 were underexpressed specifically in ER-negative breast cancers. The DUSP4 gene is underexpressed in ER-negative breast cancer and is deleted in approximately 50 % of breast cancers. Induced DUSP4 expression suppresses both in vitro and in vivo growths of breast cancer cells. Our studies show that induced DUSP4 expression blocks the cell cycle at the G1/S checkpoint; inhibits ERK1/2, p38, JNK1, RB, and NFkB p65 phosphorylation; and inhibits invasiveness of TNBC cells. These results suggest that that DUSP4 is a critical regulator of the growth and invasion of triple-negative breast cancer cells. Electronic supplementary material The online version of this article (doi:10.1007/s10549-016-3892-y) contains supplementary material, which is available to authorized users.

Triple-negative breast cancer (TNBC) has the worst prognosis of all breast cancers, and women diagnosed with TNBC currently lack targeted treatment options. To identify novel targets for TNBC, we evaluated phosphatase expression in breast tumors and characterized their contributions to in vitro and in vivo growth of TNBC. Using Affymetrix microarray analysis of 102 breast cancers, we identified 146 phosphatases that were significantly differentially expressed in TNBC compared with estrogen receptor (ER)-positive tumors. Of these, 19 phosphatases were upregulated (0.66-fold; FDR = 0.05) in TNBC compared with ER-positive breast cancers. We knocked down 17 overexpressed phosphatases in four triple-negative and four ER-positive breast cancer lines using specific siRNAs and found that depletion of six of these phosphatases significantly reduced growth and anchorage-independent growth of TNBC cells to a greater extent than ER-positive cell lines. Further analysis of the phosphatase PTP4A3 (also known as PRL-3) demonstrated its requirement for G1–S cell-cycle progression in all breast cancer cells, but PTP4A3 regulated apoptosis selectively in TNBC cells. In addition, PTP4A3 inhibition reduced the growth of TNBC tumors in vivo. Moreover, in silico analysis revealed the PTP4A3 gene to be amplified in 29% of basal-like breast cancers, and high expression of PTP4A3 could serve as an independent prognostic indicator for worse overall survival. Collectively, these studies define the importance of phosphatase overexpression in TNBC and lay the foundation for the development of new targeted therapies directed against phosphatases or their respective signaling pathways for TNBC patients. Cancer Res; 76(7); 1942–53. ©2016 AACR.

The chemopreventive effects of three agents, rexinoid bexarotene, tyrosine kinase inhibitor gefitinib, and celecoxib, were tested on mammary tumor development arising in p53-null mammary epithelium. The rexinoid bexarotene was the most efficacious inhibitor as it reduced mammary tumor development by 75% in virgin mice and significantly delayed mean tumor development by 98 days in hormone-stimulated mice. The tyrosine kinase inhibitor gefitinib reduced mammary tumor incidence by 50% in virgin mice but did not significantly delay mean tumor latency in hormone-stimulated mice. Celecoxib did not reduce tumor incidence or mean tumor latency in either of the two models. The high doses of the rexinoid and the tyrosine kinase inhibitor did not affect the progression of tumors arising from the premalignant mammary outgrowth line, PN8a. A comparison of these agents with tamoxifen shows the superiority of tamoxifen in preventing tumor development in p53-null mammary cells. Similarly, a comparison of the results of the p53 model with other transgenic models in their response to the chemopreventive agents showed that mammary tumors arising from different oncogenic events will respond differently to the different agents.

Genetically engineered mouse cancer models are among the most useful tools for testing the in vivo effectiveness of the various chemopreventive approaches. The p53-null mouse model of mammary carcinogenesis was previously characterized by us at the cellular, molecular, and pathologic levels. In a companion article, Medina et al. analyzed the efficacy of bexarotene, gefitinib, and celecoxib as chemopreventive agents in the same model. Here we report the global gene expression effects on mammary epithelium of such compounds, analyzing the data in light of their effectiveness as chemopreventive agents. SAGE was used to profile the transcriptome of p53-null mammary epithelium obtained from mice treated with each compound versus controls. This information was also compared with SAGE data from p53-null mouse mammary tumors. Gene expression changes induced by the chemopreventive treatments revealed a common core of 87 affected genes across treatments (P < 0.05). The effective compounds, bexarotene and gefitinib, may exert their chemopreventive activity, at least in part, by affecting a set of 34 genes related to specific cellular pathways. The gene expression signature revealed various genes previously described to be associated with breast cancer, such as the activator protein-1 complex member Fos-like antigen 2 (Fosl2), early growth response 1 (Egr1), gelsolin (Gsn), and tumor protein translationally controlled 1 (Tpt1), among others. The concerted modulation of many of these transcripts before malignant transformation seems to be conducive to predominantly decrease cell proliferation. This study has revealed candidate key pathways that can be experimentally tested in the same model system and may constitute novel targets for future translational research.

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.

The oncogene erbB2 is overexpressed in 20% to 30% human breast cancers and is most commonly overexpressed in estrogen receptor (ER)–negative breast cancers. Transgenic mice expressing erbB2 develop ER-negative mammary tumors, mimicking human breast carcinogenesis. Previously, we have shown that activator protein 1 (AP-1) regulates proliferation of ER-negative breast cancer cells. We hypothesized that blockade of AP-1 in mouse mammary epithelial cells will suppress ER-negative tumorigenesis induced by erbB2. Trigenic erbB2 mice were generated by crossing a bigenic pUHD-Tam67/MMTV-rtTA mouse to a MMTV-erbB2 mouse. The resulting trigenic mice develop tumors and express a doxycycline-inducible c-Jun dominant negative mutant (Tam67) in the mammary glands. In vivo AP-1 blockade by Tam67 expression started delayed mammary tumor formation in MMTV-erbB2 mice by more than 11 weeks. By 52 weeks of age, 100% (18 of 18) of the untreated animals had developed mammary tumors, whereas 56% (9 of 16) of the doxycycline-treated trigenic mice developed tumors. In addition, the tumors that arose in the AP-1–blocked erbB2 mice failed to express Tam67. Twenty-five percent of the doxycycline-treated MMTV-erbB2 mice survived more than 72 weeks of age without developing mammary tumors. Examination of normal-appearing mammary glands from these mice showed that AP-1 blockade by Tam67 also significantly prevents the development of premalignant lesions in these glands. The expression of erbB2 either in normal mammary tissue or in mammary tumors was not altered. Our results show that blocking the AP-1 signaling in mammary cells suppresses erbB2-induced transformation, and show that the AP-1 transcription factor is a critical transducer of erbB2. These results provide a scientific rationale to develop targeted drugs that inhibit AP-1 to prevent the development of ER-negative breast cancer.

Retinoids, vitamin A analogues that bind to retinoic acid receptor (RAR) or retinoid X receptor (RXR), play important roles in regulating cell proliferation, apoptosis, and differentiation. Recently, RXR-selective ligands, also referred to as rexinoids, have been investigated as potential chemopreventive agents for breast cancer. Our previous studies demonstrated that the rexinoid bexarotene significantly prevented ER-negative mammary tumourigenesis with less toxicity than naturally occurring retinoids in animal models. To determine whether bexarotene prevents cancer at the early stages during the multistage process of mammary carcinogenesis, we treated MMTV-erbB2 mice with bexarotene for 2 or 4 months. The development of preinvasive mammary lesions such as hyperplasias and carcinoma-in-situ was significantly inhibited. This inhibition was associated with reduced proliferation, but no induction of apoptosis. We also examined the regulation of a number of rexinoid-modulated genes including critical growth and cell cycle regulating genes using breast cell lines and mammary gland samples from mice treated with rexinoids. We showed that two of these genes (DHRS3 and DEC2) were modulated by bexarotene both in vitro and in vivo. Identification of these rexinoid-modulated genes will help us understand the mechanism by which rexinoid prevents cancer. Such rexinoid-regulated genes also represent potential biomarkers to assess the response of rexinoid treatment in clinical trials.

Purpose: To test whether a novel rexinoid, LG100268, prevents the development of preinvasive and invasive estrogen receptor–negative mammary tumorigenesis in MMTV-erbB2 mice. Experimental Design: For invasive breast cancer prevention, MMTV-erbB2 mice were treated with daily gastric gavage of vehicle, LG100268 (10 mg/kg), or LG100268 (100 mg/kg) for long term starting at 3 months of age. For preinvasive lesion study, mice were treated with daily gastric gavage of vehicle or LG100268 (100 mg/kg) for 4 months. Results: Long-term treatment with LG100268 significantly prevented invasive mammary tumor development. Median time (age) to tumor development was delayed from 217 days in vehicle group to 357 days in low-dose group. In high-dose group, only 2 of 20 mice developed tumors after 430 days of treatment. Short-term treatment of LG100268 significantly prevented the development of preinvasive mammary lesions including hyperplasia and ductal carcinoma in situ. The cancer prevention effect was associated with reduced expression of Ki67 and cyclin D1 in mammary glands by >80%. Conclusion: Rexinoid LG100268 is an effective chemopreventive agent in preventing the development of both malignant and premalignant mammary lesions in MMTV-erbB2 mice.

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

A mitogenic agent in corncob bedding and fresh corn products disrupts sexual behavior and estrous cyclicity in rats. The mitogenic activity resides in an isomeric mixture of linoleic acid derivatives with a tetrahydrofuran ring and two hydroxyl groups (THF-diols) that include 9, (12)-oxy-10,13-dihydroxystearic acid and 10, (13)-oxy-9,12-dihydroxystearic acid. Synthetic THF-diols stimulated breast cancer cell proliferation in vitro and disrupted the estrous cycle in female rats at oral doses of ∼0.30 mg/kg body weight/day. Exposure to THF-diols may disrupt endocrine function in experimental animals at doses ∼200 times lower than classical phytoestrogens, promote proliferation of breast or prostate cancer, and adversely affect human health.

Background: Estrogen receptor (ER) -negative breast cancer is a clinical subtype that is overrepresented among younger women and is associated with a poor prognosis. Current treatments for ER-negative tumors include cytotoxic chemotherapy, or for those overexpress HER2, the anti-HER2 antibody. Targeted therapy for triple-negative breast cancers is urgently needed. In this project, we investigated phosphatases that are differentially expressed in ER-negative as compared to ER-positive breast cancers. We hypothesized that: (1) specific phosphatases govern the growth of ER-negative cancers, (2) Induced expression of specific phosphatases that are under expressed in ER-negative cancers will suppress the growth of ER-negative breast cancers. Methods: Using 102 human breast tumors (57 ER-positive & 45 ER-negative) from the neo-adjuvant studies from the Baylor Breast Center tumor bank, we isolated RNA and performed Affymetrix microarray studies. Statistical analysis was done with dChip software, and phosphatases over (>1.5 fold; FDR Results: We identified 20 over-expressed and 29 under-expressed phosphatases in ER-negative breast cancers. We selected the under expressed phosphatase PPM1A, for further study. Multivariate cox regression analysis shows PPM1A is an independent predictor for breast cancer survival. Induced expression of PPM1A in ER-negative cells inhibited anchorage-dependent and independent growth but had no effect on ER-positive cell. PPM1A expression also inhibited MDA-MB 231 cells growth in vivo. Induced expression of PPM1A blocked cell cycle progression at G1 phase. PPM1A interacts with CDK6 and inhibits phosphorylation of CDK and MDM2. Conclusions: We identified a set of over- and under- expressed phosphatases in ER-negative breast cancers as compared to ER-positive cancers. Overexpression of PPM1A in ER-negative breast cancer cells inhibits growth. By identifying the molecules that regulate breast cancer cells growth we are identifying potential new targets for the treatment of these aggressive ER-negative breast cancers. Supported by Komen Promise grant KG081694 & Komen SAB grant Citation Format: Abhijit Mazumdar, Jamal Hill, Yun Zhang, Lakshmi Reddy Bollu, Anna Tsimelzon, Jenny Chang, Gordon Mills, Powel Brown. Induced expression of PPM1A in ER-negative breast cancer cells inhibits growth by suppressing CDK phosphorylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5525. doi:10.1158/1538-7445.AM2017-5525

Triple-negative breast cancers (TNBC) are aggressive with no effective targeted therapies. A combined database analysis identified 32 inflammation-related genes differentially expressed in TNBCs and 10 proved critical for anchorage-independent growth. In TNBC cells, an LPA-LPAR2-EZH2 NF-κB signaling cascade was essential for expression of interleukin (IL)-6, IL-8, and CXCL1. Concurrent inhibition of IL-6 and IL-8 expression dramatically inhibited colony formation and cell survival in vitro and stanched tumor engraftment and growth in vivo. A Cox multivariable analysis of patient specimens revealed that IL-6 and IL-8 expression predicted patient survival times. Together these findings offer a rationale for dual inhibition of IL-6/IL-8 signaling as a therapeutic strategy to improve outcomes for patients with TNBCs. Cancer Res; 73(11); 3470–80. ©2013 AACR.

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 (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.

In pursuit of effective therapeutic agents for the estrogen receptor (ER)-negative breast cancer, we previously showed that bexarotene reduced mammary tumor development by 75% in ErbB2 mice. To further improve the effectiveness of breast cancer prevention, we have now investigated the effects of a combinatorial therapy consisting of two cancer preventive drugs. On the basis of the hypothesis, rexinoid LG100268 plus tamoxifen would more effectively prevent the development of both ER-positive and ER-negative breast cancer. We treated p53-null mammary gland mice with tamoxifen and LG100268, individually and in combination. By 60 weeks of age, vehicle-treated mice developed tumors in 52% of transplanted mammary glands, whereas mice treated with tamoxifen and LG100268 developed tumors in only 13% of transplanted mammary glands. To further define the mechanistic effects of this combinatorial treatment, we investigated the effects of tamoxifen and LG100268 on mammary tissue biomarkers. In mammary tissue harvested before tumor development, the proliferation markers Ki67 and cyclin D1 were significantly reduced in mice treated with the combination therapy. In addition, the rexinoid target genes ABCA1 and ABCG1 were induced in both the rexinoid and combination treatment groups, whereas expression remained constant in tamoxifen group. These results show that tamoxifen-LG100268 combinatorial treatment is more effective in preventing mammary tumors than either agent alone. In addition, these studies have identified relevant tissue biomarkers that can be used to show the effect of these agents on mammary tissue. These results support the development of clinical trials of antiestrogen and rexinoid combinatorial therapy for the prevention of patients with high-risk breast cancer. Cancer Prev Res; 5(10); 1195–202. ©2012 AACR.

Background: Anti-estrogen drugs such as selective estrogen receptor modulators and aromatase inhibitors effectively prevent ER-positive breast cancer, but do not prevent triple negative breast cancer (TNBC) which represents approximately 20% of all breast cancers. TNBCs are characterized by a high proliferation rate and a dysregulated cell cycle. Drugs that specifically activate RXR without activating RAR nuclear receptors (“rexinoids”) are being studied as potential cancer preventive drugs. One of the most promising rexinoids is the compound, LG100268. LG100268 is highly effective in preventing ER-negative/HER2-positive mammary tumors in MMTV-ErbB2 mice by inhibiting cyclin D-dependent pathways, but less preventive in models of TNBC (p53-null mice and C3(1)-SV40 Tag mice). Therefore, we hypothesized that inhibiting multiple cell cycle regulatory points by combining kinase inhibitors and LG100268 is an effective and safe strategy to prevent triple negative breast cancer. Methods: To identify kinases that inhibit cell growth we carried out a ‘whole kinome’ (636 kinases) high-throughput siRNA screen in combination with LG100268 (1µM) in immortalized, human mammary epithelial cells overexpressing telomerase (HME-tert). After 4 days of growth following LG100268 treatment and suppression of the respective kinase cells were stained with DAPI and cell number was determined by automated microscopy and image analysis. Combination “hits” were selected based on a p-value Results: The analysis of the kinome screen showed that the combination of LG100268 with each of 27 kinases had a significant growth suppressive effect compared to siRNA control and vehicle alone. Knockdown of the respective kinases was independently confirmed by qRT PCR. Growth assays of HME-tert cells after siRNA knockdown of kinases in combination with LG100268 demonstrated that in particular CDK1, 2 and 5 enhanced the chemopreventive effect of LG100268. Using a CDK inhibitor dinaciclib in combination with LG100268 showed that the combination additively suppressed mammary epithelial growth in vitro. In vivo in SV40 Tag mice, LG100268 or dinaciclib alone significantly reduced the area (mm2) of hyperplastic lesions (defined as any hyperplastic ductal foci comprised of more than three layers of epithelial cells) compared to the control group. The combination therapy did not result in a significant reduction of hyperplastic area compared to dinaciclib alone. Long-term treatment with LG100268 did not delay TNBC formation compared to vehicle (both MTTF = 144 days). However, long-term treatment with dinaciclib alone delayed mammary tumorigenesis with a median time to tumor formation (MTTF) of 167 days compared to a MTTF of 144 days of age in the control group (p < 0.001). The combination therapy did not further affect the tumor formation compared to dinaciclib alone (MTTF = 162 days). Conclusions: These studies demonstrate that dinaciclib alone or the combination therapy of LG100268 and dinaciclib causes inhibition of premalignant cell growth and delayed ER-negative mammary tumorigenesis in SV40 Tag mice. While an additive effect of dinaciclib and LG100268 was observed in vitro, the in vivo combination therapy was not significantly more effective than dinaciclib treatment alone. Future studies should investigate different combinations of dosages of dinaciclib and LG100268 in vivo. These studies were supported by an NCI/NIH R25T grant (R25CA057730, BL) and Susan G. Komen for the Cure Promise grant (KG081694) Citation Format: Beate Christiane Litzenburger, Ivan Peter Uray, Jamal Hill, Jun Zhang, Abhijit Mazumdar, Powel H. Brown. Targeting multiple cell cycle regulatory points for the prevention of triple-negative breast cancer. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PR03.

The AP-1 transcription factor is activated by multiple growth factors that are critical regulators of breast cell proliferation. We previously demonstrated that AP-1 blockade inhibits breast cancer cell growth in vitro. Yet a specific role of AP-1 in normal mammary gland development has not been studied. Using a bi-transgenic mouse expressing an inducible AP-1 inhibitor (Tam67), we found that the AP-1 factor regulates postnatal proliferation of mammary epithelial cells. Mammary epithelial proliferation was significantly reduced after AP-1 blockade in adult, prepubertal, pubertal, and hormone-stimulated mammary glands. In pubertal mice, mammary cell proliferation was greatly reduced, and the cells that did proliferate failed to express Tam67. We also observed structural changes such as suppressed branching and budding, reduced gland tree size, and less fat pad occupancy in developing mammary glands after AP-1 blockade. We further demonstrated that Tam67 suppressed the expression of AP-1-dependent genes (TIMP-1, vimentin, Fra-1, and fibronectin) and the AP-1-dependent growth regulatory genes (cyclin D1 and c-myc) in AP-1-blocked mammary glands. We therefore conclude that AP-1 factor is a pivotal regulator of postnatal mammary gland growth and development.

Tamoxifen reduces the relative risk of breast cancer developing from specific premalignant lesions. Many breast cancers that arise after tamoxifen treatment are estrogen receptor-α (ER-α)–negative, although premalignant lesions such as atypical ductal hyperplasia are highly ER-α–positive. The p53 null mouse mammary epithelial transplant model is characterized by ER-α–positive premalignant lesions that give rise to both ER-α–positive and ER-α–negative tumors. Given this progression from ER-α–positive to ER-α–negative lesions, we tested the ability of tamoxifen to block or delay mammary tumorigenesis in several versions of this model. In groups 1 and 2, p53 null normal mammary epithelial transplants were maintained in virgin mice. In groups 3 to 5, the p53 null and mammary transplants were maintained in mice continuously exposed to high levels of progesterone. In groups 6 and 7, transplants of the premalignant outgrowth line PN8a were maintained in virgin mice. Tamoxifen blocked estrogen signaling in these mice as evidenced by decreases in progesterone-induced lateral branching and epithelial proliferation in the mammary epithelium. Tamoxifen did not alter the elevated levels of progesterone in the blood while significantly reducing the circulating level of prolactin. Tamoxifen reduced tumor incidence in p53 null normal mammary epithelial transplants maintained in virgin mice from 55% to 5% and in progesterone-stimulated mice from 81% to 21%. The majority of the resultant tumors were ER-α–negative. Tamoxifen also significantly delayed tumorigenesis in the ER-α–positive high premalignant line PN8a from 100% to 75%. These results show that tamoxifen delays the emergence of ER-α–negative tumors if given early in premalignant progression.

Although antiestrogens have been effective in preventing estrogen receptor (ER)-positive breast cancer, chemopreventive agents are still needed to prevent ER-negative breast cancer. Tyrosine kinase inhibitors are promising agents for the treatment and prevention of human cancers. ZD1839 (gefitinib or Iressa) is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that blocks signal transduction pathways in epithelial cells. We examined whether ZD1839 blocks signal transduction and prevents the development of ER-negative breast cancer.The ability of ZD1839 to block signal transduction in normal, immortalized, and malignant breast cells was assessed by western blotting with specific antibodies to detect phosphorylation of cytoplasmic signaling molecules. The effect of ZD1839 on growth of these breast cells was assessed with anchorage-dependent and anchorage-independent growth assays. Its effect on ER-negative mammary tumorigenesis was assessed in MMTV-erbB2 transgenic mice. All statistical tests were two-sided.ZD1839 suppressed the phosphorylation of EGFR and mitogen-activated protein kinase in normal and malignant breast cells. ZD1839 treatment statistically significantly suppressed mammary tumorigenesis in MMTV-erbB2 transgenic mice; median time to tumor development was approximately 230 days in vehicle-treated mice and more than 310 days in mice treated with ZD1839 at 100 mg/kg (P.001). ZD1839 reduced proliferation of normal breast cells by 20.3% (95% confidence interval [CI] = -13.7% to 44.2%) and of tumor cells by 42.0% (95% CI = 20.2% to 58.2%). ZD1839 also increased expression of the cell cycle regulator p27 in normal mammary tissue by 48.7% (95% CI = 27.0% to 74.2%) and in tumor tissue by 50.3% (95% CI = 35.8% to 66.7%).This study appears to provide the preclinical rationale for the development of these EGFR tyrosine kinase inhibitors for the prevention of human breast cancer.

Background: Estrogen receptor (ER) -negative breast cancer is a clinical subtype that is overrepresented among younger women and is associated with a poor prognosis. Current treatments for ER-negative tumors include cytotoxic chemotherapy, or for those overexpress HER2, the anti-HER2 antibody. Targeted therapy for triple-negative breast cancers is urgently needed. In this project, we investigated phosphatases that are differentially expressed in ER-negative as compared to ER-positive breast cancers. We hypothesized that: (1) specific phosphatases govern the growth of ER-negative cancers, (2) Induced expression of specific phosphatases that are under expressed in ER-negative cancers will suppress the growth of ER-negative breast cancers. Methods: Using 102 human breast tumors (57 ER-positive & 45 ER-negative) from the neo-adjuvant studies from the Baylor Breast Center tumor bank, we isolated RNA and performed Affymetrix microarray studies. cDNA was synthesized and Biotin-labeled and hybridized onto an Affymetrix HGU133A GeneChipTM. Statistical analysis was done with dChip software, and phosphatases over (>1.5 fold; FDR Results: We identified 20 over-expressed, and 29 under-expressed phosphatases in ER-negative breast cancers. We selected the under expressed phosphatase PPM1A, for further study. Multivariate cox regression analysis shows PPM1A is an independent predictor for breast cancer survival. Induced expression of PPM1A in ER-negative cells inhibited anchorage-dependent and independent growth but had no effect on ER-positive cell. PPM1A expression also inhibited MDA-MB 231 cells growth in vivo. Induced expression of PPM1A blocked cell cycle progression at G1 phase. Conclusions: We identified a set of over- and under- expressed phosphatases in ER-negative breast cancers as compared to ER-positive cancers. Overexpression of PPM1A in ER-negative breast cancer cells inhibits growth. By identifying the molecules that regulate breast cancer cells growth we are identifying potential new targets for the treatment of these aggressive ER-negative breast cancers. Supported by Komen Promise grant KG081694 & SAB13-00006 Citation Format: Abhijit Mazumdar, Graham Poage, Jamal Hill, Yun Zhang, Anna Tsimelzon, Jenny Chang, Gordon Mills, Powel Brown. Induced expression of PPM1A in ER-negative breast cancer cells inhibits growth by suppressing cell cycle. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2014-5279

Nuclear receptors are key proteins that regulate cell proliferation, death, and differentiation. In breast cells, estrogen receptors (ERs) are critical for mammary gland development, differentiation, and tumorigenesis, and selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene, and aromatase inhibitors can prevent the development of ER-positive breast cancer. Other nuclear transcription factors are critical regulators of proliferation and differentiation including retinoic acid receptors (RARs), vitamin D receptors (VDR), and peroxisome proliferator-activator receptors (PPARs). Each of these ligand activated nuclear hormone receptors bind the retinoid X receptors (RXRs) to regulate gene expression. Ligands of these nuclear hormone receptors have been shown to prevent many different types of cancer, including breast cancer. I will present results in preclinical and early clinical studies testing retinoids, rexinoids, Vitamin D analogs, and PPAR ligands for the prevention of breast cancer. Our results and the results of others have shown that ligands that bind and activate the rexinoid receptor are highly effective in preventing breast cancer in animals, particularly ER-negative breast cancer. These preclinical results supported the testing of the RXR ligand bexarotene in women at high risk of breast cancer. This early study demonstrated a biological effect of bexarotene on breast tissue in women. We and other have conducted preclinical studies testing combinations of nuclear hormone receptor ligands (RXR ligand + anti-estrogen SERMS)(RXR ligand + Vitamin D analog) and (RXR ligand + PPAR agonist). These results demonstrate that nuclear hormone receptor ligands can be used to prevent breast cancer and that rexinoids and other ligands provide an opportunity to prevent all forms of breast cancer. Supported by RO1CA078480-11 Citation Format: Abhijit Mazumdar, Ivan Uray, Beate Litzenburger, Yun Zhang, Jamal Hill, Nanjoo Suh, Powel Brown. Targeting nuclear hormone receptors for breast cancer prevention. [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 ED05-01.

Background: Estrogen receptor (ER) -negative breast cancers are poor prognosis tumors that occur more commonly in young women. Current treatments for ER-negative tumors include cytotoxic chemotherapy, or for those ER-negative breast cancers that overexpress HER2, the anti-HER2 antibody herceptin. Similar therapy for ER-negative, Her2 negative tumors, particularly for (triple-negative breast cancers) is urgently needed. In this project, we investigated whether phosphatases that are differentially expressed in ER-negative as compared to ER-positive breast cancers. We hypothesized that: (1) the specific phosphatases that govern the growth of ER-negative cancers are different from those of ER-positive cancers, (2) Induced expression of some of those phosphatases that are lowly expressed in ER-negative breast cancers will suppress the growth and invasion of ER-negative breast cancers. Methods: Using 102 human breast tumors (57 ER-positive & 45 ER-negative) from the neo-adjuvant studies from the Baylor Breast Center tumor bank, we isolated RNA and performed Affymetrix microarray studies. cDNA was synthesized and Biotin-labeled and hybridized onto an Affymetrix HGU133A GeneChipTM. Statistical analysis was done with dChip software, and phosphatases more highly (>1.5 fold; FDR Results: We identified 20 highly-expressed (>1.5 fold; FDR=0.05), and 29 lowly-expressed ( Conclusions: We identified a set of highly and lowly expressed phosphatases in ER-negative breast cancers as compared to ER-positive cancers. Overexpression of DUSP4 in ER-negative breast cancer cells inhibits growth and invasion of these cells. By identifying the molecules that regulate breast cancer cells growth we are identifying potential new targets for the treatment of these aggressive ER-negative breast cancers. Supported by Komen Promise grant KG081694 (PHB). Citation Format: Abhijit Mazumdar, Petra Den Hollander, Graham Poage, Jamal Hill, Yun Zhang, Anna Tsimelzon, Susan Hilsenbeck, Jenny Chang, Gordon Mills, Powel H. Brown. High expression of DUSP4 in ER-negative breast cancer cells suppresses growth and invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 856. doi:10.1158/1538-7445.AM2013-856

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Breast cancer is a heterogeneous disease that can be subdivided into distinct types based upon the molecular classification of oncogenic driver mutations. While most breast cancers are driven by estrogen-mediated signaling (ER+) or HER2 expression (HER2+), the cellular signals that mediate transformation and growth of ER-HER2- (or basal-type) breast cancers are incompletely understood. To identify novel targets for the treatment of this aggressive and treatment-refractory disease, we investigated inflammatory signaling in these cancers, as inflammatory pathways have proven critical for oncogenic transformation of multiple cancers. In our primary analysis, we analyzed ER- and ER+ primary patient tumors (N=106, Baylor College of Medicine Tumor Bank) to determine inflammatory genes (in the KEGG Cytokine-Cytokine Receptor set, N=226) that are differentially expressed (FDR

Background: Recent research in breast biology supports the cancer stem cell hypothesis which asserts that malignancies arise in tissue stem cells through dysregulation of self-renewal. The cancer stem cell hypothesis has important implications for early detection, prevention and treatment of breast cancer. If breast cancers arise from the transformation of normal stem cells, it may be possible to target stem cells for the prevention of cancer. We have previously shown that rexinoids prevent the development of ER-negative breast cancer in transgenic mouse models. Previous studies have demonstrated a role of retinoid signaling in the regulation of normal breast stem cell self-renewal and differentiation. Bexarotene and LG100268 are rexinoids that selectively bind RXR receptors. Based on the effects of these molecularly targeted agents, we hypothesize that rexinoids prevent breast cancer development by suppressing the growth and transformation of the breast stem cell population. Methods: We performed in vivo and in vitro studies to address our hypothesis. Cell proliferation assays were used to determine whether rexinoids can inhibit the growth of normal, pre-malignant and malignant breast cell lines (HMECs, MCF10A, DCIS.COM, SUM225, HCC1937 and HCC38 cell lines). To determine whether these agents affect stem cells, we performed mammosphere assays and FACS analysis after treatment with vehicle or rexiniods. We next conducted in vivo studies using MMTV-Wnt transgenic mice treated with LG100268 (50mg/kg) or vehicle, and studied the stem cell properties of mammary epithelial cells from these mice. The stem cell population in mammary glands was quantified by staining of CD24, CD49f and using FACS analysis and mammosphere assays. Results: Our results showed that the rexionids LG100268 and bexarotene inhibit the growth of breast cell lines only at a high concentrations (the IC50 (concentration that reaches a 50% growth inhibiton) in most of the tested cell lines exceeded 10uM). Results of in vitro mammosphere assays showed that 100nM LG100268 reduced mammosphere formation efficiency by more than 40% in all breast cancer cell lines. Results from our in vivo experiments showed that LG100268 reduced the mammary epithelial stem cell population by 57% as defined by the CD24+/CD49f++ population identified by FACS and by 47% as measured by in vitro mammosphere assays. Conclusion: These results from in vitro and in vivo studies demonstrate that the rexinoid LG100268 is a potent mammary stem cell inhibitor. This study shows that rexinoids suppress stem cell expansion and suggest that mammary stem cell inhibitors will be useful breast cancer preventive agents. This work is supported by a grant from the Breast Cancer Research Foundation (to PHB) and from an NIH/NCI RO1 grant (CA078480) (to PHB). Citation Information: Cancer Prev Res 2011;4(10 Suppl):A45.

Breast cancers are now subdivided into distinct tumor types based upon molecular classification of tumor gene expression. While most breast cancers are driven by estrogen-mediated signaling (estrogen receptor-positive, (ER+) or Luminal-type breast cancers) or by addiction to the HER2 oncogene (HER2+ breast cancer), the essential cellular signals that control the growth of ER-negative and HER2-negative (or basal-type) breast cancers (approximately 15% of all breast cancers), are incompletely understood. Patients with these ER− and HER2− breast cancers have poor outcomes and few available treatment options other than non-specific chemotherapy. To identify novel targets for the treatment and prevention of basal breast cancers, we investigated the activity of inflammatory genes in these cancers, previously linked to the progression of several other types of cancer. To identify critical inflammatory genes in basal breast cancer, we performed univariate t-tests (FDR1.5 or < 0.7 in at least two of the datasets analyzed). Further analysis of inflammatory gene expression in breast cancer cell lines revealed that 24 of these genes were highly expressed in basal-like cell lines at the RNA and protein level, as compared to luminal-like ER+ breast cancer lines (showing fold-differences of >1.5 or Citation Information: Cancer Prev Res 2011;4(10 Suppl):A42.

Background: The rexinoid bexarotene (LGD1069, Targretin) is a highly selective retinoid × receptor (RXR) agonist that inhibits the growth of pre-malignant and malignant breast cells. Bexarotene was shown to suppress the development of breast cancer in transgenic mice models without side effects. The chemopreventive effects of bexarotene are due to transcriptional modulation of cell proliferation, differentiation and apoptosis. Our goal in the present study was to obtain a profile of the genes modulated by bexarotene on mammary gland from three transgenic mouse mammary cancer models in an effort to elucidate its molecular mechanism of action and for the identification of biomarkers of effectiveness. Methods: Serial analysis of gene expression (SAGE) was employed to profile the transcriptome of p53-null, MMTV-ErbB2, and C3(1)-SV40 mammary cells obtained from mice treated with bexarotene and their corresponding controls. Results: This resulted in a dataset of approximately 360,000 transcript tags representing over 20,000 mRNAs from a total of 6 different SAGE libraries. Analysis of gene expression changes induced by bexarotene in mammary gland revealed that 89 genes were dysregulated among the three transgenic mouse mammary models. From these, 9 genes were common to the three models studied. Conclusion: Analysis of the indicated core of transcripts and protein-protein interactions of this commonly modulated genes indicate two functional modules significantly affected by rexinoid bexarotene related to protein biosynthesis and bioenergetics signatures, in addition to the targeting of cancer-causing genes related with cell proliferation, differentiation and apoptosis., Centro de Investigaciones Inmunológicas Básicas y Aplicadas