Search

Resistance to CDK4/6 inhibitors (CDKis) is emerging as a clinical challenge. Identification of the factors contributing to CDKi resistance, with mechanistic insight, is of pivotal significance. Recent studies linked aberrant FGFR signaling to CDKi resistance. However, detailed mechanisms are less clear. Based on control and FGFR1 overexpressing luminal A cell line models, we demonstrated that FGFR1 overexpression rendered the cells resistant to palbociclib. FGFR1 overexpression abolished palbociclib-mediated cell cycle arrest, as well as the attenuated palbociclib-induced inhibition of G1/S transition regulators (pRb, E2F1, and cyclin D3) and factors that promote G2/M transition (cyclin B1, cdc2/CDK1, and cdc25). Importantly, FGFR1-induced palbociclib resistance was associated with promotion of cancer cell stemness and the upregulation of Wnt/β-catenin signaling. We found that palbociclib may function as an ER agonist in MCF-7/FGFR1 cells. Upregulation of the ER-mediated transcription in MCF-7/FGFR1 cells was associated with ERα phosphorylation and enhanced receptor tyrosine kinase signaling. The combination of palbociclib with FGFR-targeting AZD4547 resulted in remarkable synergistic effects on MCF-7/FGFR1 cells, especially for the inhibition of cancer cell stemness. Our findings of FGFR1-induced palbociclib resistance, promotion of cancer stem cells and associated molecular changes advance our mechanistic understanding of CDKi resistance, which will facilitate the development of strategies targeting CDKi resistance in breast cancer treatment.

Triple negative breast cancer (TNBC) is the breast cancer subtype that is most refractory to treatment. Thus, identification of novel molecular targets and drug treatments are needed to improve prognosis for patients. The widely used anti-diabetic drug Metformin has been reported to possess anti-cancer properties. Although it is believed that Metformin may exert its anti-cancer effects through indirect reduction of glucose and insulin levels, and directly activating the AMPK kinase and inhibiting downstream cancer-related pathways, the molecular mechanisms underlying its anti-cancer function in TNBC have yet to be fully elucidated. In this study, we investigated the role and mechanism of Metformin-mediated downregulation of oncogenic c-Met in MDA-MB-468 TNBC cells. Our results demonstrated greater sensitivity of MDA-MB-468 cells to Metformin compared to other breast cancer cell lines. Metformin-induced inhibition of MDA-MB-468 cell proliferation was correlated with the downregulation of c-Met at both protein and mRNA levels. Overexpression of c-Met in MDA-MB-468 cells using a lentiviral vector system rendered the cells resistant to Metformin, which was accompanied by the promotion of cancer stem cell stemness. Functionally, we demonstrated that c-Met induced activation of receptor tyrosine kinase (RTK) signaling pathways play a critical role in c-Met-modulated Metformin responsiveness. These novel findings highlight c-Met as a key regulator of Metformin-mediated inhibition of TNBC cell growth, suggesting that c-Met overexpression may be a critical factor contributing to metformin resistance. To this end, combination of Metformin with c-Met inhibitors could be a useful strategy to improve Metformin-mediated anti-cancer efficacies in TNBC. Citation Format: Dana M. Gant, Zhikun Ma, Amanda B. Parris, Xiaohe Yang. c-Met downregulation contributes to Metformin-induced inhibition of MDA-MB-468 triple negative breast cancer cells [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 1123.

Although ErbB2-targeted therapeutics have significantly improved ErbB2+ breast cancer patient outcomes, therapeutic resistance remains a significant challenge. Therefore, the development of novel ErbB2-targeting strategies is necessary. Importantly, ErbB2 is a sensitive client protein of heat shock protein 90 (HSP90), which regulates client protein folding, maturation, and stabilization. HSP90 inhibition provides an alternative therapeutic strategy for ErbB2-targeted degradation. In particular, ganetespib, a novel HSP90 inhibitor, is a promising agent for ErbB2+ cancers. Nevertheless, the anti-cancer efficacy and clinical application of ganetespib for ErbB2+ breast cancer is largely unknown. In our study, we examined the anti-cancer effects of ganetespib on ErbB2+ BT474 and SKBR3 breast cancer cells, and isogenic paired cancer cell lines with lentivirus-mediated ErbB2 overexpression. Ganetespib potently inhibited cell proliferation, cell cycle progression, survival, and activation/phosphorylation of ErbB2 and key downstream effectors in ErbB2+ breast cancer cells. Moreover, ganetespib decreased the total protein levels of HSP90 client proteins and reduced ErbB2 protein half-life. ErbB2-overexpressing cancer cells were also more sensitive to ganetespib-mediated growth inhibition than parental cells. Ganetespib also strikingly potentiated the inhibitory effects of lapatinib in BT474 and SKBR3 cells. Ultimately, our results support the application of ganetespib-mediated HSP90 inhibition as a promising therapeutic strategy for ErbB2+ breast cancer.

Metformin, a first line medication for type II diabetes, initially entered the spotlight as a promising anti-cancer agent due to epidemiologic reports that found reduced cancer risk and improved clinical outcomes in diabetic patients taking metformin. To uncover the anti-cancer mechanisms of metformin, preclinical studies determined that metformin impairs cellular metabolism and suppresses oncogenic signaling pathways, including receptor tyrosine kinase, PI3K/Akt, and mTOR pathways. Recently, the anti-cancer potential of metformin has gained increasing interest due to its inhibitory effects on cancer stem cells (CSCs), which are associated with tumor metastasis, drug resistance, and relapse. Studies using various cancer models, including breast, pancreatic, prostate, and colon, have demonstrated the potency of metformin in attenuating CSCs through the targeting of specific pathways involved in cell differentiation, renewal, metastasis, and metabolism. In this review, we provide a comprehensive overview of the anti-cancer actions and mechanisms of metformin, including the regulation of CSCs and related pathways. We also discuss the potential anti-cancer applications of metformin as mono- or combination therapies.

// Zhiying Guo 1 , Ming Zhao 1 , Erin W. Howard 1 , Qingxia Zhao 1 , Amanda B. Parris 1 , Zhikun Ma 1 and Xiaohe Yang 1 1 Julius L. Chambers Biomedical/Biotechnology Research Institute, Department of Biological and Biomedical Sciences, North Carolina Central University, Kannapolis, North Carolina, USA Correspondence to: Xiaohe Yang, email: xyang@nccu.edu Keywords: phenformin, breast cancer, epithelial-mesenchymal transition, insulin-like growth factor 1 receptor, MMTV-ErbB2 transgenic mice Received: November 04, 2016 Accepted: June 19, 2017 Published: July 22, 2017 ABSTRACT Reports suggest that metformin, a popular anti-diabetes drug, prevents breast cancer through various systemic effects, including insulin-like growth factor receptor (IGFR) regulation. Although the anti-cancer properties of metformin have been well-studied, reports on a more bioavailable/potent biguanide, phenformin, remain sparse. Phenformin exerts similar functional activity to metformin and has been reported to impede mammary carcinogenesis in rats. Since the effects of phenformin on specific breast cancer subtypes have not been fully explored, we used ErbB2-overexpressing breast cancer cell and animal models to test the anti-cancer potential of phenformin. We report that phenformin (25–75 μM) decreased cell proliferation and impaired cell cycle progression in SKBR3 and 78617 breast cancer cells. Reduced tumor size after phenformin treatment (30 mg/kg/day) was demonstrated in an MMTV-ErbB2 transgenic mouse syngeneic tumor model. Phenformin also blocked epithelial-mesenchymal transition, decreased the invasive phenotype, and suppressed receptor tyrosine kinase signaling, including insulin receptor substrate 1 and IGF1R, in ErbB2-overexpressing breast cancer cells and mouse mammary tumor-derived tissues. Moreover, phenformin suppressed IGF1-stimulated proliferation, receptor tyrosine kinase signaling, and epithelial-mesenchymal transition markers in vitro . Together, our study implicates phenformin-mediated IGF1/IGF1R regulation as a potential anti-cancer mechanism and supports the development of phenformin and other biguanides as breast cancer therapeutics.

// Ming Zhao 1 , Erin W. Howard 1 , Amanda B. Parris 1 , Zhiying Guo 1 , Qingxia Zhao 1, 2 , Zhikun Ma 1 , Ying Xing 2 , Bolin Liu 3 , Susan M. Edgerton 3 , Ann D. Thor 3 and Xiaohe Yang 1, 4 1 Julius L. Chambers Biomedical/Biotechnology Research Institute and Department of Biological and Biomedical Sciences, North Carolina Central University, Kannapolis, North Carolina, USA 2 Basic Medical College of Zhengzhou University, Zhengzhou, Henan, P.R. China 3 Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA 4 College of Medicine, Henan University of Sciences and Technology, Luoyang, Henan, P.R. China Correspondence to: Xiaohe Yang, email: xyang@nccu.edu Keywords: CIP2A, lapatinib resistance, ErbB2, breast cancer, PP2A Received: November 05, 2016 Accepted: July 11, 2017 Published: July 19, 2017 ABSTRACT Lapatinib, a small molecule ErbB2/EGFR inhibitor, is FDA-approved for the treatment of metastatic ErbB2-overexpressing breast cancer; however, lapatinib resistance is an emerging clinical challenge. Understanding the molecular mechanisms of lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast cancer. Our study investigated the role of CIP2A in the anti-cancer efficacy of lapatinib in ErbB2-overexpressing breast cancer cells. We found that lapatinib concurrently downregulated CIP2A and receptor tyrosine kinase signaling in ErbB2-overexpressing SKBR3 and 78617 cells; however, these effects were attenuated in lapatinib-resistant (LR) cells. CIP2A overexpression rendered SKBR3 and 78617 cells resistant to lapatinib-induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via lentiviral shRNA enhanced cell sensitivity to lapatinib-induced growth inhibition and apoptosis. Results also suggested that lapatinib downregulated CIP2A through regulation of protein stability. We further demonstrated that lapatinib-induced CIP2A downregulation can be recapitulated by LY294002, suggesting that Akt mediates CIP2A upregulation. Importantly, lapatinib induced differential CIP2A downregulation between parental BT474 and BT474/LR cell lines. Moreover, CIP2A shRNA knockdown significantly sensitized the BT474/LR cells to lapatinib. Collectively, our results demonstrate that CIP2A is a molecular target and resistance factor of lapatinib with a critical role in lapatinib-induced cellular responses, including the inhibition of the CIP2A-Akt feedback loop. Further investigation of lapatinib-mediated CIP2A regulation will advance our understanding of lapatinib-associated anti-tumor activities and drug resistance.

Heat shock protein 90 (HSP90) regulates several important cellular processes via its repertoire of 'client proteins'. These client proteins have been found to play fundamental roles in signal transduction, cell proliferation, cell cycle progression and survival, as well as other features of malignant cells, such as invasion, tumor angiogenesis and metastasis. Thus, HSP90 is an emerging target for cancer therapy. To this end, we evaluated ganetespib (STA-9090), a novel and potent HSP90 inhibitor, for its activity in gastric cancer cell lines. Ganetespib significantly inhibited the proliferation of AGS and N87 human gastric cancer cell lines and potently induced G2/M cell cycle arrest and apoptosis. Upregulation of cleaved poly(ADP-ribose) polymerase (c-PARP), c-caspase-3, c-caspase-8 and c-caspase-9 and suppression of gastric cancer‑associated HSP90 client proteins, including ErbB2, Erk, Akt, mTOR, GSK3 and Src, were observed in ganetespib-treated cells. These findings demonstrate that the ganetespib-induced mechanism of cell growth inhibition involves the activation of death receptor and mitochondrial pathways and the inhibition of receptor tyrosine kinase signaling pathways. Our study implicates ganetespib as a potential strategy for gastric cancer treatment, which warrants further preclinical and clinical investigation.

Exposure to bisphenol A (BPA), an endocrine-disrupting compound, is associated with increased risk of estrogen-related diseases, including estrogen receptor-positive (ER+) breast cancer. Although bisphenol analogs, i.e. bisphenol AF (BPAF), have replaced BPA in industrial settings, increasing data indicate that these alternatives may have similar or even more potent estrogenic effects. As such, BPAF exhibits increased ER binding affinities than BPA in biochemical assays. However, preclinical studies exploring the effects of BPAF on ER+ breast cancer are missing mechanistic data. Thus, we aimed to characterize the effects of BPAF on MCF-7 and T47D ER+ breast cancer cells with mechanistic insight. We found that BPAF promoted cell growth and cell cycle progression concurrently with BPAF-induced ERα transcriptional activity and ER-RTK signaling activation. ER signaling blockage revealed that BPAF-induced cell proliferation and ER-RTK crosstalk were ER-dependent. Gene expression data demonstrated that AREG is a sensitive target of BPAF in our in vitro models. Importantly, we determined that AREG upregulation is necessary for BPAF-induced cellular responses. Ultimately, our novel finding that AREG mediates BPAF-induced ER-RTK crosstalk in ER+ breast cancer cells supports future studies to characterize the impact of BPAF on human ER+ breast cancer risk and to assess the safety profile of BPAF.

Metformin has been suggested as an anti-cancer agent. However, increasing reports show that some tumors are resistant to metformin. Identification of factors affecting metformin mediated cancer therapy is of great significance. FGFR1 is a receptor-tyrosine-kinase that is frequently overexpressed in breast cancer, which is associated with poor-prognosis. To investigate the effect of FGFR1 overexpression on metformin-induced inhibition of breast cancer cells, we demonstrated that FGFR1 overexpression rendered MCF-7 and T47D cells resistant to metformin. In particular, we found that, in addition to AKT and ERK1/2 activation, FGFR1-induced activation of IRS1 and IGF1R, key regulators connecting metabolism and cancer, was associated with metformin resistance. Targeting IRS with IRS1 KO or IRS inhibitor NT157 significantly sensitized FGFR1 overexpressing cells to metformin. Combination of NT157 with metformin induced enhanced inhibition of p-IGF1R, p-ERK1/2 and p-mTOR. Moreover, we demonstrated that IRS1 functions as a critical mediator of the crosstalk between FGFR1 and IGF1R pathways, which involves a feedback loop between IRS1 and MAPK/ERK. Our study highlights the significance of FGFR1 status and IRS1 activation in metformin-resistance, which will facilitate the development of strategies targeting FGFR overexpression-associated metformin resistance.

Although alcohol is an established breast cancer risk factor, the underlying mechanisms remain unclear. Previous studies examined the general association between alcohol consumption and breast cancer risk; however, the risk for different breast cancer subtypes has been rarely reported. Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking hormone receptors and HER2 expression, and having poor prognosis. Understanding the molecular mechanisms of TNBC etiology remains a significant challenge. In this study, we investigated cellular responses to alcohol in two TNBC cell lines, MDA-MB-231 and MDA-MB-468. Our results showed that alcohol at low concentrations (0.025-0.1% v/v) induced cell proliferation, migration, and invasion in 1% FBS-containing medium. Molecular analysis indicated that these phenotypic changes were associated with alcohol-induced reactive oxygen species production and increased p38 and JNK phosphorylation. Likewise, p38 or JNK inhibition attenuated alcohol-induced cell migration and invasion. We revealed that alcohol treatment activated/phosphorylated NF-κB regulators and increased transcription of NF-κB-targeted genes. While examining the role of acetaldehyde, the major alcohol metabolite, in alcohol-associated responses in TNBC cells, we saw that acetaldehyde induced cell migration, invasion, and increased phospho-p38, phospho-JNK, and phospho-IκBα in a pattern similar to alcohol treatment. Taken together, we established that alcohol promotes TNBC cell proliferation, migration, and invasion in vitro. The underlying mechanisms involve the induction of oxidative stress and the activation of NF-κB signaling. In particular, the activation of p38 and JNK plays a pivotal role in alcohol-induced cellular responses. These results will advance our understanding of alcohol-mediated development and promotion of TNBC. © 2016 Wiley Periodicals, Inc.

Genistein is a soy isoflavone with phytoestrogen and tyrosine kinase inhibitory properties. High intake of soy/genistein has been associated with reduced breast cancer risk. Despite the advances in genistein-mediated antitumor studies, the underlying mechanisms remain unclear. In the present study, we investigated genistein-induced regulation of the cancerous inhibitor of protein phosphatase 2A (CIP2A), a novel oncogene frequently overexpressed in breast cancer, and its functional impact on genistein-induced growth inhibition and apoptosis. We demonstrated that genistein induced downregulation of CIP2A in MCF-7-C3 and T47D breast cancer cells, which was correlated with its growth inhibition and apoptotic activities. Overexpression of CIP2A attenuated, whereas CIP2A knockdown sensitized, genistein-induced growth inhibition and apoptosis. We further showed that genistein-induced downregulation of CIP2A involved both transcriptional suppression and proteasomal degradation. In particular, genistein at higher concentrations induced concurrent downregulation of E2F1 and CIP2A. Overexpression of E2F1 attenuated genistein-induced downregulation of CIP2A mRNA, indicating the role of E2F1 in genistein-induced transcriptional suppression of CIP2A. Taken together, our results identified CIP2A as a functional target of genistein and demonstrated that modulation of E2F1-mediated transcriptional regulation of CIP2A contributes to its downregulation. These data advance our understanding of genistein-induced growth inhibition and apoptosis, and support further investigation on CIP2A as a therapeutic target of relevant anticancer agents.

Due to the growing health concern of bisphenol A (BPA), a popular endocrine-disrupting compound (EDC), the FDA has banned its use in the production of baby bottles. Bisphenol S (BPS) is a substitute of BPA and was believed to be less estrogenic/toxic, which has been widely used in the production of many household products. However, recent studies suggest that BPS exhibits proestrogenic/proliferative effects similar to BPA. Whether/how BPS exposure impacts human health at various stages, especially in children/adolescents, is emerging as a new public health concern. In this study, we investigated the in vitro and in vivo effect of BPS on breast cancer cell proliferation and signaling and mouse mammary development. Results from MTT and clonogenic assays using MCF-7 and T47D cells, two ER+ breast cancer cell lines, indicated that BPS at 0.5–5 μM promoted cell proliferation. Cell cycle analysis showed that BPS at 1 and 5 μM significantly increased the percentage of cells in S phase and decreased cells in G0/G1 phase. Luciferase reporter assays also indicated that BPS potently activated estrogen response element (ERE)-mediated transcription. Analysis of signal transduction demonstrated that PBS induced the upregulation and activation/phosphorylation of ERα, erbB3, Akt, and Erk/12. Examination of the mRNA levels of key regulators in ER signaling and growth regulation showed that BPS induced significant upregulation of AREG, MYC, IGF1R/2R, and CCND1. We also demonstrated that BPS at 1 μM attenuated tamoxifen-mediated growth inhibition of MCF-7 and T47D cells. These data demonstrate that BPS is a potent endocrine disruptor. To test the in vivo effect of in utero exposure to BPS on pubertal mammary development, MMTV-erbB2 transgenic mice were exposed to BPS via drinking water at doses of 0, 15, and 30 μM BPS/L between gestation day 10 to day 19. Mammary glands of female offspring at week 5 and week 10 of age were analyzed for morphogenesis, colony formation cell (CFC) assay, and mammary epithelial subpopulations. BPS-treated mammary tissues at 5 weeks of age displayed decreased ductal elongation and CFC numbers. At 10s week of age, mammary tissues with 30 μM BPS/L exposure displayed more complex ductal trees, CFC numbers, and increased CD24high/CD49fhigh and CD61high/CD49fmid cells, which are enriched with putative mammary stem/progenitor subpopulations. Overall, we demonstrated that BPS has a proestrogenic effect that may promote the growth of ER+ breast cancer cells. In vivo results demonstrated that in utero exposure to BPS modifies the dynamics of mammary development. The health impact of BPS exposure on mammary development warrants further investigation. Citation Format: Qingxia Zhao, Amanda B. Parris, Zhikun Ma, Xiaohe Yang. Bisphenol S induces proestrogenic effect in vitro and modifies mammary development dynamics after in utero exposure [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A27.

As de novo and acquired resistance to standard first line endocrine therapies is a growing clinical challenge for estrogen receptor-positive (ER+) breast cancer patients, understanding the mechanisms of resistance is critical to develop novel therapeutic strategies to prevent therapeutic resistance and improve patient outcomes. The widespread post-transcriptional regulatory role that microRNAs (miRNAs) can have on various oncogenic pathways has been well-documented. In particular, several miRNAs are reported to suppress ERα expression via direct binding with the 3’ UTR of ESR1 mRNA, which can confer resistance to estrogen/ERα-targeted therapies. In turn, estrogen/ERα activation can modulate miRNA expression, which may contribute to ER+ breast carcinogenesis. Given the reported oncogenic and tumor suppressor functions of miRNAs in ER+ breast cancer, the targeted regulation of specific miRNAs is emerging as a promising strategy to treat ER+ breast cancer and significantly improve patient responsiveness to endocrine therapies. In this review, we highlight the major miRNA-ER regulatory mechanisms in context with ER+ breast carcinogenesis, as well as the critical miRNAs that contribute to endocrine therapy resistance or sensitivity. Collectively, this comprehensive review of the current literature sheds light on the clinical applications and challenges associated with miRNA regulatory mechanisms and novel miRNA targets that may have translational value as potential therapeutics for the treatment of ER+ breast cancer.

Environmental factors, including 7,12-dimethylbenz [a]anthracene (DMBA) exposure, and genetic predisposition, including ErbB2 overexpression/amplification, have been demonstrated to increase breast cancer susceptibility. Although DMBA- and ErbB2-mediated breast cancers are well-studied in their respective models, key interactions between environmental and genetic factors on breast cancer risk remain unclear. Therefore, the present study aimed to investigate the effect of DMBA exposure on ErbB2-mediated mammary tumorigenesis. MMTV-ErbB2 transgenic mice exposed to DMBA (1 mg) via weekly oral gavage for 6 weeks exhibited significantly enhanced mammary tumor development, as indicated by reduced tumor latency and increased tumor multiplicity compared with control mice. Whole mount analysis of premalignant mammary tissues from 15-week-old mice revealed increased ductal elongation and proliferative index in DMBA-exposed mice. Molecular analyses of premalignant mammary tissues further indicated that DMBA exposure enhanced epidermal growth factor receptor (EGFR)/ErbB2 and estrogen receptor (ER) signaling, which was associated with increased mRNA levels of EGFR/ErbB2 family members and ER-targeted genes. Furthermore, analysis of tumor karyotypes revealed that DMBA-exposed tumors displayed more chromosomal alterations compared with control tumors, implicating DMBA-induced chromosomal instability in tumor promotion in this model. Together, the data suggested that DMBA-induced deregulation of EGFR/ErbB2-ER pathways plays a critical role in the enhanced chromosomal instability and promotion of ErbB2-mediated mammary tumorigenesis. The study highlighted gene-environment interactions that may increase risk of breast cancer, which is a critical clinical issue.

Caloric intake influences the onset of many diseases, including cancer. In particular, caloric restriction (CR) has been reported to suppress mammary tumorigenesis in various models. However, the underlying cancer preventive mechanisms have not been fully explored. To this end, we aimed to characterize the anticancer mechanisms of CR using MMTV-ErbB2 transgenic mice, a well-established spontaneous ErbB2-overexpressing mammary tumor model, by focusing on cellular and molecular changes in premalignant tissues. In MMTV-ErbB2 mice with 30% CR beginning at 8 weeks of age, mammary tumor development was dramatically inhibited, as exhibited by reduced tumor incidence and increased tumor latency. Morphogenic mammary gland analyses in 15- and 20-week-old mice indicated that CR significantly decreased mammary epithelial cell (MEC) density and proliferative index. To understand the underlying mechanisms, we analyzed the effects of CR on mammary stem/progenitor cells. Results from fluorescence-activated cell sorting analyses showed that CR modified mammary tissue hierarchy dynamics, as evidenced by decreased luminal cells (CD24(high)CD49f(low)), putative mammary reconstituting unit subpopulation (CD24(high)CD49f(high)) and luminal progenitor cells (CD61(high)CD49f(high)). Mammosphere and colony-forming cell assays demonstrated that CR significantly inhibited mammary stem cell self-renewal and progenitor cell numbers. Molecular analyses indicated that CR concurrently inhibited estrogen receptor (ER) and ErbB2 signaling. These molecular changes were accompanied by decreased mRNA levels of ER-targeted genes and epidermal growth factor receptor/ErbB2 family members and ligands, suggesting ER-ErbB2 signaling cross-talk. Collectively, our data demonstrate that CR significantly impacts ER and ErbB2 signaling, which induces profound changes in MEC reprogramming, and mammary stem/progenitor cell inhibition is a critical mechanism of CR-mediated breast cancer prevention.

The p53 tumor repressor gene is commonly mutated in human cancers. The tumor inhibitory effect of metformin on p53-mutated breast cancer cells remains unclear. Data from the present study demonstrated that p53 knockdown or mutation has a negative effect on metformin or phenformin-induced growth inhibition, senescence and apoptosis in breast cancer cells. We also found that p53 reactivating agent nutlin-3α and CP/31398 promoted metformin-induced growth inhibition, senescence and apoptosis in MCF-7 (wt p53) and MDA-MB-231 (mt p53) cells, respectively. Treatment of MCF-7 cells with metformin or phenformin induced increase in p53 protein levels and the transcription of its downstream target genes, Bax and p21, in a dose-dependent manner. Moreover, we demonstrated that AMPK-mTOR signaling played a role in metformin-induced p53 up-regulation. The present study showed that p53 is required for metformin or phenformin-induced growth inhibition, senescence and apoptosis in breast cancer cells. The combination of metformin with p53 reactivating agents, like nutlin-3α and CP/31398, is a promising strategy for improving metformin-mediated anti-cancer therapy, especially for tumors with p53 mutations.

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) regulates signaling pathways involved in cell proliferation and differentiation. Currently, the anti-tumor properties of FGFR inhibitors are being tested in preclinical and clinical studies. Nevertheless, reports on FGFR inhibitor-mediated breast cancer prevention are sparse. In this study, we investigated the anti-cancer benefits of AZD4547, an FGFR1-3 inhibitor, in ErbB2-overexpressing breast cancer models. AZD4547 (1–5 µM) demonstrated potent anti-proliferative effects, inhibition of stemness, and suppression of FGFR/RTK signaling in ErbB2-overexpressing human breast cancer cells. To study the in vivo effects of AZD4547 on mammary development, mammary epithelial cell (MEC) populations, and oncogenic signaling, MMTV-ErbB2 transgenic mice were administered AZD4547 (2–6 mg/kg/day) for 10 weeks during the ‘risk window’ for mammary tumor development. AZD4547 significantly inhibited ductal branching and MEC proliferation in vivo, which corroborated the in vitro anti-proliferative properties. AZD4547 also depleted CD24/CD49f-sorted MEC populations, as well as the CD61highCD49fhigh tumor-initiating cell-enriched population. Importantly, AZD4547 impaired stem cell-like characteristics in primary MECs and spontaneous tumor cells. Moreover, AZD4547 downregulated RTK, mTOR, and Wnt/β-catenin signaling pathways in premalignant mammary tissues. Collectively, our data provide critical preclinical evidence for AZD4547 as a potential breast cancer preventative and therapeutic agent.

Background Metformin, an FDA-approved drug for the treatment of Type II diabetes, has emerged as a promising anti-cancer agent. Other biguanide analogs, including buformin and phenformin, are suggested to have similar properties. Although buformin was shown to reduce mammary tumor burden in carcinogen models, the anti-cancer effects of buformin on different breast cancer subtypes and the underlying mechanisms remain unclear. Therefore, we aimed to investigate the effects of buformin on erbB-2-overexpressing breast cancer with in vitro and in vivo models. Methods MTT, cell cycle, clonogenic/CFC, ALDEFLUOR, tumorsphere, and Western blot analyses were used to determine the effects of buformin on cell growth, stem cell populations, stem cell-like properties, and signaling pathways in SKBR3 and BT474 erbB-2-overexpressing breast cancer cell lines. A syngeneic tumor cell transplantation model inoculating MMTV-erbB-2 mice with 78617 mouse mammary tumor cells was used to study the effects of buformin (1.2 g buformin/kg chow) on tumor growth in vivo. MMTV-erbB-2 mice were also fed buformin for 10 weeks, followed by analysis of premalignant mammary tissues for changes in morphological development, mammary epithelial cell (MEC) populations, and signaling pathways. Results Buformin significantly inhibited SKBR3 and BT474 cell growth, and in vivo activity was demonstrated by considerable growth inhibition of syngeneic tumors derived from MMTV-erbB-2 mice. In particular, buformin suppressed stem cell populations and self-renewal in vitro, which was associated with inhibited receptor tyrosine kinase (RTK) and mTOR signaling. Consistent with in vitro data, buformin suppressed mammary morphogenesis and reduced cell proliferation in MMTV-erbB-2 mice. Importantly, buformin decreased MEC populations enriched with mammary reconstitution units (MRUs) and tumor-initiating cells (TICs) from MMTV-erbB-2 mice, as supported by impaired clonogenic and mammosphere formation in primary MECs. We further demonstrated that buformin-mediated in vivo inhibition of MEC stemness is associated with suppressed activation of mTOR, RTK, ER, and β-catenin signaling pathways. Conclusions Overall, our results provide evidence for buformin as an effective anti-cancer drug that selectively targets TICs, and present a novel prevention and/or treatment strategy for patients who are genetically predisposed to erbB-2-overexpressing breast cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0498-0) contains supplementary material, which is available to authorized users.

Alcohol consumption is associated with increased breast cancer risk; however, the underlying mechanisms that contribute to mammary tumor initiation and progression are unclear. Alcohol is known to induce oxidative stress and DNA damage; likewise, p53 is a critical modulator of the DNA repair pathway and ensures genomic integrity. p53 mutations are frequently detected in breast and other tumors. The impact of alcohol on p53 is recognized, yet the role of p53 in alcohol-induced mammary carcinogenesis remains poorly defined. In our study, we measured alcohol-mediated oxidative DNA damage in MCF-7 cells using 8-OHdG and p-H2AX foci formation assays. p53 activity and target gene expression after alcohol exposure were determined using p53 luciferase reporter assay, qPCR, and Western blotting. A mechanistic study delineating the role of p53 in DNA damage response and cell cycle arrest was based on isogenic MCF-7 cells stably transfected with control (MCF-7/Con) or p53-targeting siRNA (MCF-7/sip53), and MCF-7 cells that were pretreated with Nutlin-3 (Mdm2 inhibitor) to stabilize p53. Alcohol treatment resulted in significant DNA damage in MCF-7 cells, as indicated by increased levels of 8-OHdG and p-H2AX foci number. A p53-dependent signaling cascade was stimulated by alcohol-induced DNA damage. Moderate to high concentrations of alcohol (0.1-0.8% v/v) induced p53 activation, as indicated by increased p53 phosphorylation, reporter gene activity, and p21/Bax gene expression, which led to G0/G1 cell cycle arrest. Importantly, compared to MCF-7/Con cells, alcohol-induced DNA damage was significantly enhanced, while alcohol-induced p21/Bax expression and cell cycle arrest were attenuated in MCF-7/sip53 cells. In contrast, inhibition of p53 degradation via Nutlin-3 reinforced G0/G1 cell cycle arrest in MCF-7 control cells. Our study suggests that functional p53 plays a critical role in cellular responses to alcohol-induced DNA damage, which protects the cells from DNA damage associated with breast cancer risk.

Rapamycin, a well-studied mTOR inhibitor, has been demonstrated to inhibit mammary carcinogenesis at multiple stages, including initiation, invasion, and metastasis, in preclinical animal models. Nevertheless, the cancer preventative potential and underlying mechanisms remain unclear, especially in individual breast cancer subtypes like ErbB2/Her2-positive breast cancers. ErbB2 amplification/overexpression is a particular clinical concern because it occurs in approximately one-third of human breast cancers and is associated with poor prognosis. Therefore, we used MMTV-ErbB2 transgenic mice as our model system to test the efficacy of rapamycin in the prevention of ErbB2-mediated mammary tumor development. Our initial data provided proof of concept regarding the anti-cancer effects of rapamycin in vivo. Indeed, rapamycin (1.5 mg/kg/day for 12 days) significantly reduced the volume and weight of syngeneic 78617 cell-derived mammary tumors in MMTV-ErbB2 mice, despite observed decreases in CD4+ and CD8+ immune cells. Since advanced mammary gland development can serve as an indicator of breast cancer risk, we investigated the effects of rapamycin on mammary gland development in MMTV-ErbB2 mice that were treated with low-dose rapamycin (1 mg/kg/day) between weeks 10 and 20 of age. As such, rapamycin significantly attenuated mammary morphogenesis at 20 weeks of age, as indicated by decreased branching density, ductal elongation, and proliferative index of the premalignant mammary glands. Flow cytometric analysis of isolated primary mammary epithelial cells (MECs) was performed using CD24 and CD49f markers to identify MEC populations. We found that rapamycin has a significant impact on MEC stemness based on changes in luminal (CD24highCD49flow), mammary stem cell (MaSC)-enriched (CD24highCD49fhigh), and myoepithelial/basal (CD24low/highCD49fhigh) MEC populations. We also used CD61 and CD49f markers to identify a population enriched with luminal progenitor cells (CD61highCD49fhigh) that was selectively inhibited by rapamycin. Consistent with our flow cytometric analyses, rapamycin inhibited the luminal progenitor cell-enriched population, self-renewal, and anchorage-independent cell growth of primary MECs, as demonstrated by colony-forming cell, mammosphere, and 3D culture assays, respectively. These functional stem cell assays further corroborate that rapamycin suppresses the stemness of primary MECs. Molecular analysis of MECs demonstrated that rapamycin inhibited mTOR signaling, as expected. Importantly, rapamycin also significantly suppressed the receptor tyrosine kinase/ErbB2, estrogen receptor, Wnt/β-catenin, and TGFβ/Smad3 signaling pathways prior to malignant transformation. Collectively, our study provides evidence that rapamycin has potential cancer preventative effects in the mammary glands of MMTV-ErbB2 mice during the premalignant risk window. These rapamycin-induced anti-cancer effects ultimately highlight the promising clinical significance of rapamycin for the prevention and treatment of human ErbB2-overexpressing breast cancers. Citation Format: Yang X, Zhao Q, Parris AB, Howard EW, Zhao M, Guo Z, Xing Y, Ma Z. Rapamycin inhibits the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-07-04.

Palbociclib is the first CDK4/6-Cyclin D inhibitor approved by the FDA for ER+/Her2(erbB2)-breast cancer treatment. With the success of the drug in hormone-positive breast cancer, it is encouraging to explore more options of Palbociclib as an anticancer agent. erbB2 is a receptor tyrosine kinase (RTK) frequently overexpressed in breast cancer. The molecular hallmarks of erbB2+ breast cancer include the upregulation of cyclin D. The aim of this study was to explore the potential of Palbociclib as a preventive agent using MMTV-erbB2 transgenic mice, with a focus on the stemness of mammary epithelial cells (MECs) in the premalignant tissues. We first tested the in vivo effect of Palbociclib on tumor cells on MMTV-erbB2 mammary tumors using syngeneic tumor graft models. The animals bearing grafted tumors were treated with saline (control), low (75 mg/kg/day) and high (150 mg/kg/day) Palbociclib via oral gavage every 3 days for 29 days. We found that Palbociclib significantly inhibited tumor growth in a concentration dependent manner. To test the effect of Palbociclib on MEC stemness in premalignant mammary tissues, the animals at 9 wks of age were treated with saline, low and high (0, 75, 150 mg/kg/day) doses of Palbociclib every 3 days for 4 wks. We found the efficiency of both primary and secondary mammosphere formation of MECs from drug treated mice was significantly inhibited, suggesting the inhibition of mammary stemness in the premalignant tissues. Consistently, results from 3D cultures showed that colony numbers of the cells from drug treated mice were also lower than the control. Using CD24 and CD49f as markers, flow cytometry was performed to analyze the effect of Palbociclib on MEC subpopulations, including luminal, basal, and stromal populations. We showed that the percentage of luminal cells and putative mammary reconstitution units (MRUs) in drug treated mice was significantly inhibited. We also found that the percentage of CD61+/CD49+ cells, which are enriched with luminal progenitor cells and the origin of tumor initiation cells of this model, in Palbociclib treated tissues were also inhibited. The data suggest that Palbociclib induces MEC reprogramming in the premalignant tissues. Moreover, we found that the protein levels of pRb, E2F1, cyclin D1, c-myc, and cdc2 in cell cycle regulation, and pAKT, pERK, pmTOR, and p4EBP1 in the RTK pathway, pER, and DVL2 and LRP6 in the Wnt pathway were significantly downregulated in Palbociclib treated tissues, which was more evident in the high dose group. Taken together, our data indicate that Palbociclib inhibits MEC proliferation and stemness in the premalignant mammary tissues. Downregulation of cell cycle regulators and the signaling in ER, RTK, and Wnt pathway plays a critical role in this process. Our data support further investigation of Palbociclib in the prevention of ER+/ErbB2+ breast cancer. Citation Format: Amanda B. Parris, Yongxuan Liu, Zhikun Ma, Erin W. Howard, Xiaoshan Feng, Xiaohe Yang. Palbociclib inhibits the stemness of mammary epithelial cells in premalignant tissues of MMTV-erbB2 transgenic mice [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 5071.

Palbociclib is the first CDK4/6 inhibitor approved by the FDA for treating ER+/HER2- breast cancer. Palbociclib resistance is emerging as a clinical challenge. Identification of the factors that determine the sensitivity to Palbociclib is of pivotal significance. Fibroblast Growth Factor Receptor (FGFR) overexpression is frequently detected in breast cancer, which has been associated with poor prognosis. To investigate the impact of FGFR overexpression on Palbociclib resistance, we first established stable sublines of MCF7 and T47D cells that overexpress FGFR1, and examined their responses to Palbociclib. Data from MTT and clonogenic assays demonstrated that overexpression of FGFR1 rendered ER+ MCF7 and T47D cells resistant to Palbociclib significantly. Cell cycle analysis showed that the percentage of cells in S phase in Palbociclib treated MCF7/FGFR1 and T47D/FGFR1 cells was significantly higher than that of the control. FGFR overexpression also significantly attenuated Palbociclib-associated inhibition of cancer cell invasion. We further found that Palbociclib-induced inhibition of cancer cell stemness, as indicated by colony formation in soft agar and mammosphere assays, was significantly reduced in MCF7/FGFR1 and T47D/FGFR1 cells, as compared to control. The data indicate that FGFR1 overexpression in ER+ breast cancer cells are resistant to Palbociclib. We next examined the signaling of the key pathways in the paired cell lines treated with Palbociclib. We found that Palbociclib-induced inhibition of phospho-Rb, E2F, cyclin D3, cyclin B1, cdc-2, c-myc, and cdc-25 was significantly attenuated in MCF-7/FGFR1 and T47D/FGFR1 cells. The protein levels of p-ERK, p-AKT, and p-p38 in MCF-7/FGFR1 and T47D/FGFR1 cells were significantly higher than the control cells with/without Palbociclib treatment. The results also showed that ER alpha phosphorylation, estrogen response element (ERE)-luciferase activity and mRNA levels of ER target genes, including PS2, c-myc, E2F, FGF2 and FGFR2, in MCF-7/FGFR1 and T47D/FGFR1 cells were maintained at higher levels post Palbociclib, as compared to control. We then treated control and FGFR1 overexpressing MCF-7 and T47D cells with Palbociclib in combination with FGFR inhibitor, AZD 4547. The combined drug treatment resulted in remarkably synergistic effect on MCF-7/FGFR1 and T47D/FGFR1 cells, as indicated by MTT and clonogenic assays. Key markers in cell cycle regulation, RTK signaling and ER pathway were significantly inhibited in the cells treated with both Palbociclib and AZD 4547. Taken together, our results demonstrated that FGFR1 overexpression is a critical factor affecting Palbociclib sensitivity. The underlying mechanisms involve FGFR1 overexpression-associated RTK and ER signaling. Combination of Palbociclib with FGFR targeting agents may overcome FGFR-associated Palbociclib resistance. Citation Format: Yujie Shi, Zhikun Ma, Yudan Wu, Amanda B. Parris, Qiong Cheng, Lingfei Kong, Xiaohe Yang. FGFR1 overexpression ER positive breast cancer cells confer the cells resistant to Palbociclib via upregulation of RTK-ER signaling [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 3009.

Animal models are powerful tools for the understanding of tumor development and cancer stem cell (CSC) origin. C3-1-TAg transgenic mice are a model for human triple negative breast cancer. Although general patterns of tumor development in this model have been well characterized, the tumor initiation cells of this model have not been defined. The objective of this study was to characterize the stemness and growth properties of mammary tumor cells in context with mammary epithelial cell (MEC) subpopulations of C3-1-TAg transgenic mice to identify the origin of tumor initiation cells. We first isolated MECs from mammary tissues of C3-1-TAg mice at various ages (8, 17, and 23 weeks old) and primary tumor cells from tumors of this model. Flow cytometry analysis with CD24 and CD49f as cell markers showed that cells from mammary tissues were separated into three subpopulations, including luminal, basal, and stromal populations. We found that the percentage of luminal subpopulation increased with age. Analysis of tumor derived cells using the same markers indicated that the majority of the cells in the plots overlapped with the position of luminal epithelial cells, suggesting their mutual connections. Next, we performed FACS sorting of mammary epithelial cells from the glands based on CD24 and CD49f and obtained four subpopulations of cells, including P1(stromal), P2(luminal), P3(CD24high/CD49fhigh, top of basal population on the plot), and P4(CD24mid/CD49fhigh, lower part of basal population), followed by the characterization of each subpopulations. Colony formation cell (CFC) assay and mammosphere assay were used for evaluation of luminal progenitor cells and self-renewal potential, respectively. Data from CFC assays showed that CFC colony numbers were the highest from the P2 population, followed by a few colonies from P3 population. Mammosphere assay showed that cells from P1 population formed typical mammospheres, while as cells from P2 and P3 formed cellular clusters different from typical mammospheres. FACS sorting with cells from C3-1-TAg mammary tumors obtained P2 and P3 subpopulations. Cells from P2, not P3, subpopulation were efficient in mammosphere formation. Because cells in P2 population are luminal mammary epithelial cells and are enriched with luminal progenitor cells, integration of data above suggest that C3-1-TAg mammary tumor cells are mainly derived from luminal progenitor cells. Using CD61, which is a luminal progenitor cell marker, we found that the percentage of luminal progenitor cells (CD61+/CD49fhigh) in the mammary glands was increased with age, and most tumor derived cells were CD61+/CD49fhigh. Taken together, our data demonstrate that the tumor initiation cells in the C3-1-TAg mammary tumors were derived from luminal progenitor cells. These findings lay a foundation for further characterization of cellular basis and molecular analysis of tumorigenesis in C3-1-TAg transgenic models. Citation Format: Qiong Cheng, Amanda B. Parris, Zhikun Ma, Yujie Shi, Lingfei Kong, Xiaohe Yang. Characterization of tumor initiation cells in mammary tissues of C3-1-TAg transgenic mice [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 1168.

A translocation between chromosomes 19 and 21 [dic/t(19;21)(p13;v)] is very rare. To date, only three cases of this particular chromosomal abnormality have been reported. The translocations in these three cases were secondary changes in acute lymphoblastic leukemia (ALL) patients with the t(9;22) translocation. The gene(s) at the breakpoints of either chromosome 19p13 or 21q have not yet been identified. Here, we present a case study of a 21-year-old female with a diagnosis of precursor B cell ALL, with the t(9;22) translocation and secondary changes including a der(19)t(19;21) and an extra Philadelphia (Ph+) chromosome [der(22)t(9;22)]. Array comparative genomic hybridization (aCGH) analysis identified UHRF1 and NRIP1 as genes that were interrupted at the breakpoints of 19p13.3 and 21q21.1, and joined together as a possible fusion gene, 5′- NRIP1/UHRF1 -3′, on the derivative chromosome 19. To our knowledge, this is the first description of possible genes involved in the unbalanced translocation between chromosomes 19 and 21 in a patient with an ALL-positive for a t(9;22) translocation.

erbB-2 is amplified or overexpressed in approximately 30% of human breast cancers, and has been associated with poor prognosis and therapeutic resistance. Previous studies have suggested that erbB-2 overexpression in transgenic mice induces genomic instability; however, the patterns of genetic lesions vary with individual model systems. The development of mammary tumors in multiparous murine mammary tumor virus (MMTV)-erbB-2 transgenic mice is accelerated due to hormonal interactions which induce the overexpression of MMTV-mediated erbB-2. However, whether or not accelerated tumor development is associated with modified cytogenetic patterns remains to be determined. In this study, chromosomal changes were characterized in mammary tumor cells derived from multiparous MMTV-erbB-2 transgenic mice, and compared with tumor cells derived from control virgin mice. Immunohistochemistry and Western blotting were used to detect erbB-2 overexpression in mammary tissues. Each of the five tumors from the multiparous MMTV-erbB-2 transgenic mice was found to exhibit a marked chromosomal imbalance, compared with only one tumor with aberrant chromosomes among the five tumors from the control virgin mice. In particular, trisomy 5 and loss of the X chromosome were recurrent cytogenetic lesions in tumors from the parous mice, which is a novel pattern compared with previous studies. The elevated number of genetic lesions in tumors from parous mice, which were characterized by enhanced erbB-2 overexpression and increased receptor tyrosine kinase activation in the mammary glands, suggest a causal role for erbB-2 in the genomic instability present in these tumors. These data advance our understanding of erbB-2-mediated pathogenesis and underscore the role of cytogenetic alteration in this process.

ErbB2/Her2 overexpression is detected in approximately 30% of human breast cancers and is associated with poor prognosis. Lapatinib, a small molecule dual inhibitor targeting ErbB2 and EGFR, is FDA-approved for the treatment of ErbB2-positive (ErbB2+) advanced or metastatic breast cancer. However, lapatinib resistance (LR) is emerging as a critical issue in clinical oncology. To understand the molecular mechanisms of LR, we first developed a lapatinib-resistant cell line from ErbB2-overexpressing BT474 breast cancer cells by prolonged exposure of parental BT474 cells to gradually increasing concentrations of lapatinib (up to 8 µM). We performed microarray analyses on parental and LR cells to identify novel factors/pathways that contribute to LR. Our microarray data indicated that the gene expression profile of the BT474/LR cells is significantly different from the parental BT474 cells. We found that DARPP32/PPP1R1B is one of the most significantly upregulated genes in the BT474/LR cells. To investigate the role of DARPP32 in LR development, we examined the effect of DARPP32 knockdown via lentiviral shRNA on BT474/LR cell proliferation and apoptosis. Results from MTT, clonogenic, cell cycle, and cleaved caspase-3 and PARP analyses indicated that DARPP32 knockdown significantly sensitized BT474/LR cells to lapatinib. We also found that DARPP32 knockdown renders MDA-MB-361 breast cancer cells, which express high endogenous levels of DARPP32, more sensitive to lapatinib. These data demonstrate that DARPP32 overexpression contributes to LR. To understand the mechanism of DARPP32 overexpression-mediated LR, we focused on the regulation of transcription factor cAMP response element-binding protein (CREB) in this process. As such, DARPP32 overexpression was generally associated with CREB upregulation. We also found that CREB-mediated transcription was increased in BT474/LR cells, and DARPP32 knockdown in BT474/LR and MDA-MB-361 cells significantly downregulated CREB protein levels and CREB-mediated transcription, as indicated by luciferase reporter assays. CREB overexpression rendered BT474 cells resistant to lapatinib and reversed DARPP32 knockdown-induced sensitization in BT474/LR cells. In contrast, CREB knockdown sensitized the cells to lapatinib. Further analysis with MG132 blockage suggests that DARPP32 may regulate CREB protein levels through the proteasomal degradation pathway. Taken together, we have identified DARPP32 overexpression as a critical mediator of acquired lapatinib resistance. The underlying mechanisms involve the deregulation of CREB protein and activity levels. These novel findings advance our understanding of the molecular mechanisms of LR and provide fundamental support for testing the DARPP32-CREB axis in clinical settings for the management of LR breast cancers, which is of significant translational value. Citation Format: Zhikun Ma, Erin W. Howard, Amanda B. Parris, Xiaohe Yang. Upregulation of DARPP32/PPP1R1B is a critical mediator of acquired lapatinib resistance in ErbB2-overexpressing breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 903.

Bisphenol AF (BPAF), a BPA analog, is widely used in the manufacturing of plastics, and has been detected in the environment and in human urine samples. Since BPAF may exhibit more potent endocrine-disrupting activities than BPA, the potential effect of BPAF exposure on breast cancer risk is a health concern. Previous reports highlight the interaction of BPAF with estrogen receptors (ERs), but the molecular pathways of BPAF-mediated cellular activities remain unclear. Therefore, we investigated BPAF-mediated cellular responses in MCF-7 and T47D ER+ breast cancer cells. Results from MTT and clonogenic assays indicated that BPAF (0.01 – 5 µM) induces cell proliferation in a concentration-dependent manner. Cell cycle analysis showed that BPAF (0.5 – 1 µM) significantly increased the percentage of cells in S phase and decreased cells in G0/G1 phase. Data from luciferase reporter assays indicated that BPAF potently activated estrogen response element (ERE)-mediated transcription, which was accompanied by the upregulation and activation/phosphorylation of ERα and ERβ. These data demonstrate that BPAF is a potent endocrine disruptor. Importantly, we also found that BPAF induces ErbB3 phosphorylation, and significantly activates downstream PI3K/Akt and MAPK pathways in both cell lines, as indicated by increased Akt, Erk1/2, and Src phosphorylation. We next examined the mRNA levels of key regulators downstream of ER and receptor tyrosine kinase (RTK) pathways, including AREG, TFF1, MYC, IGF1R/2R, NRG1, EGFR, ERRB3, ESR1/2, and CCND1, in BPAF-treated cells. We found that the most significantly upregulated gene by BPAF was AREG (Amphiregulin), an ER-targeted gene that encodes an RTK ligand protein. Concurrent ER and RTK signaling activation with AREG upregulation suggests that BPAF induces ER-RTK crosstalk through AREG-mediated signaling. To demonstrate the impact of ER signaling on BPAF-induced ER-RTK crosstalk, we blocked estrogen signaling with ICI-182,780 (Fulvestrant) in BPAF-treated cells, and found that estrogen signaling inhibition significantly suppressed BPAF-associated cell growth, ERE-mediated transcriptional activity, and signaling in ER and RTK pathways. We next examined the effect of AREG knockdown on the signaling in both RTK and ER pathways to determine the role of AREG in BPAF-induced ER-RTK crosstalk. Our data indicated that AREG knockdown not only markedly inhibited BPAF-induced proliferation and RTK signaling, but also significantly downregulated ER-mediated transcription. Overall, we demonstrated that ER-RTK crosstalk is essential for BPAF-induced oncogenic cellular responses. Importantly, we identified AREG as a critical mediator of the BPAF-induced signaling interactions. These novel findings highlight the potential clinical implications of BPAF-associated ER+ breast cancer risk and underscore the necessity to further evaluate BPAF as an endocrine disruptor. Citation Format: Qingxia Zhao, Amanda B. Parris, Erin W. Howard, Ying Xing, Xiaohe Yang. Bisphenol AF promotes ER+ breast cancer through amphiregulin-mediated ER-RTK signaling crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5046.

Increasing evidence suggests molecular interactions between erbB2 and other receptor tyrosine kinases, and estrogenic compounds and their cognate receptors. We have recently reported that downregulation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells. On the basis of these data, we hypothesized that erbB3 may play a major role connecting these two sentinel pathways. Interactions were studied using mammary/breast cancer cell lines from wild-type rat c-neu gene transgenic mice and humans. Estradiol promoted cell proliferation and activated erbB2/neu tyrosine kinase, Akt, and mitogen-activated protein kinase signaling exclusively in mammary and breast epithelial cell lines with coexpression of both erbB2 and erbB3. Estradiol action was independent of the transgene promoter (MMTV-LTR) activity, both in vitro and in vivo, as well as c-neu transgene or endogenous erbB2 gene expression. Estrogen induction of cell growth promotion, erbB2/neu activation, and downstream signaling was abrogated by blockade of estrogen receptor (ER) with the pure ER antagonist ICI 182,780 or knockdown of erbB3 expression via specific siRNA. These data suggest that activation of both ER and erbB2/erbB3 signaling is requisite for estrogen-induced mitogenesis and erbB2/neu tyrosine kinase activation.(Mol Cancer Res 2009;7(11):1882–92)

Receptor tyrosine kinase activity is essential for erbB2 (HER2/neu) promotion of breast carcinogenesis, metastasis and therapeutic resistance. erbB2 kinase can be activated by dimerization with another erbB receptor, most of which bind ligands. Of these, the erbB2/erbB3 heterodimer is the most potent oncogenic complex. erbB2 reportedly requires erbB3 to promote cellular proliferation, although this may occur without changes in erbB2 tyrosine kinase activity in some model systems. Our investigations focus on the role(s) of erbB3 in erbB2-associated kinase activity and tamoxifen resistance. Using tumor-derived cell lines from wild type rat c-neu transgenic mice and human breast cancers, we demonstrate that erbB3 plays a critical role in the activation of erbB2 tyrosine kinase activity and erbB2-associated tumorigenesis. Mechanistically, downregulation of erbB3 by specific siRNA reduces erbB2 tyrosine phosphorylation, decreases the PI-3K/Akt signaling, and inhibits mammary/breast cancer cell proliferation and colony formation. Specific erbB3 siRNA sensitizes erbB2 transfected MCF-7 cells (MCF-7/erbB2) to tamoxifen-associated inhibition of both cell growth and colony formation and enhances tamoxifen-induced apoptosis, in contrast to control siRNA transfected MCF-7/erbB2 cells which are tamoxifen-resistant. Our data indicates that erbB2/erbB3 heterodimerization is a prerequisite for erbB2 tyrosine kinase activation in mammary/breast cancer cells and that downregulation of erbB3 inhibits erbB2-associated procarcinogenic activity via inactivation of the PI-3K/Akt pathway. Furthermore, erbB3 also contributes to erbB2-mediated tamoxifen resistance and therefore may be a clinically relevant therapeutic target in addition to erbB2.

Aberrant apoptosis has been associated with the development and therapeutic resistance of cancer. Recent studies suggest that caspase deficiency/downregulation is frequently detected in different cancers. We have previously shown that caspase-3 reconstitution significantly sensitized MCF-7 cells to doxorubicin and etoposide. In contrast to the well established role of caspase-3 as an effector caspase, the focus of this study is to delineate caspase-3 induced feedback activation of the apical caspases-2, -8, -9 and -10A in doxorubicin and TNF-alpha induced apoptosis. Using cell-free systems we show that caspases-9 and 2 are the most sensitive, caspase-8 is less sensitive and caspase-10A is the least sensitive to caspase-3 mediated-cleavage. When apoptosis is induced by doxorubicin or TNF-alpha in an intact cell model, cleavage of caspases-8 and -9, but not caspase-2, was markedly enhanced by caspase-3. Caspase-3 mediated-feedback and activation of caspase-8 and -9 in MCF-7/C3 cells is further supported by an increase in the cleavage of caspase-8 and 9 substrates and cytochrome c release. These data indicate that, in addition to its function as an effector caspase, caspase-3 plays an important role in maximizing the activation of apical caspases and crosstalk between the two major apoptotic pathways. The significant impact of caspase-3 on both effector and apical caspases suggests that modulation of caspase-3 activity would be a useful approach to overcome drug resistance in clinical oncology.

During herpes simplex virus 1 (HSV-1) infection, apoptosis is initiated by immediate early gene transcription and is later modulated by proteins synthesized in infected cells. We have previously shown that procaspase 3 levels are reduced during HSV-1 replication. We now demonstrate that a replication-defective HSV-1 recombinant virus which is incapable of packaging viral DNA into capsids activated caspase 3 but retained the ability to prevent the apoptotic process from killing the infected cells. This implies that HSV-1-dependent apoptosis is not merely a response to abortive infection. Maximum accumulation of the active form of caspase 3 accompanied complete HSV-1-dependent apoptosis. Additionally, caspase 7 was found to be activated during HSV-1-dependent apoptosis. Infected MCF-7 cells which ectopically express caspase 3 underwent more efficient apoptosis than their caspase 3-null parental counterparts, confirming that caspase 3 contributes to HSV-1-dependent apoptosis. However, caspase 3 reconstitution did not make the MCF-7 cells as sensitive as HEp-2 cells to HSV-1-dependent apoptosis, suggesting that other cellular factors may be involved in conferring resistance to this process. These results indicate that caspase 3 activation is a consequence of HSV-1 infection and have important implications in our understanding of the interactions of the virus with host cells.

Amplification and overexpression of the erbB-2 (HER-2/ neu) proto-oncogene and exposure to the cell cycle mitogenic hormone estrogen (E2) have been associated with mammary tumorigenesis. Phytoestrogens found in soy act as selective estrogen receptor modulators and may also modify mammary carcinogenesis. We have used the wt-erbB-2 transgenic mouse model to study the effects of estrogen and dietary phytoestrogens on erbB-2-associated mammary tumorigenesis. Transgenic mice were treated with short-term E2 or placebo pellets during the early reproductive period and fed a casein or soy diet for life. Mammary tumors from the different treatment groups were used for the derivation of novel cell lines. Comparative genomic hybridization (CGH), flow cytometry, assays of cell proliferation and soft agar cloning were performed to study genomic instability and in vitro characteristics. CGH data were compared with corresponding parental tumors. Mammary tumors exhibited significantly fewer genetic changes than cell lines by CGH. Cell lines from soy-fed animals (that developed tumors with a longer latency) demonstrated the greatest frequency of chromosomal gain and loss. The E2-treated, casein-fed animals (that developed tumors with the shortest latency) had the fewest genetic changes in derived lines by CGH. Nonetheless, E2-associated tumors in vivo and lines in vitro had the most aggressive phenotypes. In addition, over 40% of all derived cell lines, and both tumors from the placebo-treated casein-fed mice, exhibited loss of chromosome 4 by CGH. In aggregate, our data suggest that estrogenic signaling influences mammary tumor development in this transgenic mouse model bearing the rat wt-erbB-2 gene. Once induced, tumors and derived lines demonstrate persistent phenotypic characteristics, including tumor aggression and shortened latency in E2-treated mice. Loss of chromosome 4 was commonly identified in derived lines and may have facilitated immortalization or passage in culture.

Rapamycin is a potent inhibitor of the mTOR pathway and has demonstrated anti-cancer properties in various cancer models. Although rapamycin was reported to inhibit mammary carcinogenesis at multiple stages, including initiation, invasion, and metastasis, the underlying mechanisms remain unclear, especially in individual breast cancer subtypes. Specifically, ErbB2/Her2 amplification occurs in approximately 25% of invasive human breast cancers, and is characterized by Akt-mTOR pathway deregulation. Recent reports also suggest that cancer stem cells (CSCs) play a critical role in tumor development and metastasis. Therefore, we investigated the efficacy of rapamycin in the prevention of mammary tumor development in MMTV-ErbB2 transgenic mice by focusing on its effects on CSCs/tumor-initiating cells (TICs). In this study, MMTV-ErbB2 mice were inoculated with syngeneic 78617 cells, which are derived from MMTV-ErbB2 mammary tumors, followed by rapamycin treatment (1.5 mg/kg/day) for 12 days. We found that rapamycin elicited strong anti-tumorigenic effects on the syngeneic tumors, as indicated by substantially reduced tumor volume and weight. To determine the effect of rapamycin on mammary epithelial cells (MECs) in premalignant tissues, MMTV-ErbB2 mice were treated with low-dose rapamycin (1 mg/kg/day) between weeks 10 and 20 of age. Rapamycin significantly inhibited mammary morphogenesis at 20 weeks of age as compared to the control mice. As such, the branching density, ductal elongation, and proliferative index of the mammary glands from the rapamycin-treated mice were markedly decreased. Flow cytometric analysis of primary MECs demonstrated that rapamycin selectively inhibited the CD61highCD49fhigh subpopulation, which are enriched with luminal progenitor cells and TICs. 3D culture of FACS-sorted MEC subpopulations indicated that CD61highCD49fhigh cells are the major subpopulation that gives rise to 3D colonies. We further demonstrated that rapamycin significantly suppressed colony-forming cell (CFC) number and primary/secondary sphere formation of primary MECs and spontaneous MMTV-ErbB2 tumor cells, indicating rapamycin-induced inhibition of mammary progenitor cells and CSC self-renewal, respectively. Furthermore, molecular analysis of MECs demonstrated that rapamycin inhibited signaling associated with ER, ErbB2, and mTOR. In particular, rapamycin strikingly inhibited Wnt/β-catenin and TGFβ/Smad3 signaling. Taken together, our results demonstrate that rapamycin-mediated prevention of MMTV-ErbB2 mammary tumor development involves selective targeting of mammary progenitor cells and potential TICs in the premalignant mammary tissues through regulation of MEC stemness and multiple signaling pathways, which contributes to its potent anti-tumor effects on ErbB2-overexpressing breast cancer. Citation Format: Qingxia Zhao, Amanda B. Parris, Erin W. Howard, Ming Zhao, Zhiying Guo, Ying Xing, Zhikun Ma, Xiaohe Yang. Rapamycin suppresses ErbB2-overexpressing mammary tumors through selective inhibition of luminal progenitor cells and tumor-initiating cells [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 2223. doi:10.1158/1538-7445.AM2017-2223

The fibroblast growth factor receptor (FGFR) family (FGFR1-4) of receptor tyrosine kinases (RTKs) regulates signaling pathways involved in cell proliferation and differentiation. In particular, FGFR1 and FGFR2, which are found in the terminal end buds of developing mammary ducts, play a role in mammary development and glandular morphogenesis involving the regulation of mammary stem cells (MaSCs) in mice. As such, a number of FGFR inhibitors are being tested in preclinical studies and clinical trials for anti-tumor properties. Nevertheless, reports on FGFR inhibitor-mediated breast cancer prevention are sparse. In this study, we aimed to investigate the anti-cancer benefits of AZD4547, a small molecule inhibitor of FGFR1-3, on ErbB2-overexpressing breast cancer models. We particularly focus on the effects of AZD4547 on MaSCs and tumor-initiating cells (TICs) in the premalignant tissues of MMTV-ErbB2 transgenic mice. We first demonstrated the anti-proliferative effects of AZD4547 (1-5 µM) on human ErbB2-overexpressing breast cancer cell lines. We further showed that AZD4547 confers potent inhibition of the stemness of these breast cancer cells, as indicated by significant depletion of ALDH+ cells and impaired tumorsphere formation. To study the in vivo effects of AZD4547 on the stemness of mammary epithelial cells (MECs), MMTV-ErbB2 transgenic mice were administered AZD4547 (2-6 mg/kg/day) for 10 weeks (weeks 8-18 of age) during the ‘risk window’ for mammary tumor development. Histopathological analysis indicated that AZD4547 significantly inhibited ductal branching and MEC proliferation. To examine the effect of AZD4547 on MEC subpopulations and tissue hierarchy dynamics in the premalignant mammary tissues of this model, we performed flow cytometry analyses on the primary MECs using CD24/CD49f and CD61/CD49f cell surface markers. The results showed that AZD4547 treatment substantially reduced MaSC-derived luminal and myoepithelial cell populations. AZD4547 also selectively suppressed the CD61highCD49fhigh cell population, which is enriched with luminal progenitor cells that give rise to TICs during MMTV-ErbB2 mammary tumor development. Mammosphere and colony-forming cell (CFC) assays on primary MECs demonstrated that the stemness of these cells was also blocked by AZD4547 prior to malignant transformation. Consistently, AZD4547 inhibited the anchorage-independent growth of cells from spontaneous tumors. Moreover, we demonstrated that AZD4547 downregulated multiple pathways, including the inactivation of FGFR, EGFR, and Wnt/β-catenin signaling. Collectively, the morphogenic, MaSC/TIC, and signaling regulation associated with AZD4547 treatment provides critical evidence for AZD4547 as a breast cancer preventative and therapeutic agent, which ultimately reveals clues for more effective eradication of refractory mammary tumors. Citation Format: Qingxia Zhao, Amanda B. Parris, Erin W. Howard, Ming Zhao, Ying Xing, Zhikun Ma, Xiaohe Yang. FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice [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 1908. doi:10.1158/1538-7445.AM2017-1908

Metformin is an oral biguanide used for type II diabetes. Epidemiologic studies suggest a link between metformin use and reduced risk of breast and other types of cancers. ErbB2-expressing breast cancer is a subgroup of tumors with poor prognosis. Previous studies demonstrated that metformin is a potent inhibitor of ErbB2–overexpressing breast cancer cells; metformin treatment extends the life span and impedes mammary tumor development in ErbB2 transgenic mice in vivo. However, the mechanisms of metformin associated antitumor activity, especially in prevention models, remain unclear. We report here for the first time that systemic administration of metformin selectively inhibits CD61high/CD49fhigh subpopulation, a group of tumor-initiating cells (TIC) of mouse mammary tumor virus (MMTV)-ErbB2 mammary tumors, in preneoplastic mammary glands. Metformin also inhibited CD61high/CD49fhigh subpopulation in MMTV-ErbB2 tumor-derived cells, which was correlated with their compromised tumor initiation/development in a syngeneic tumor graft model. Molecular analysis indicated that metformin induced downregulation of ErbB2 and EGFR expression and inhibited the phosphorylation of ErbB family members, insulin-like growth factor-1R, AKT, mTOR, and STAT3 in vivo. In vitro data indicate that low doses of metformin inhibited the self-renewal/proliferation of cancer stem cells (CSC)/TICs in ErbB2–overexpressing breast cancer cells. We further demonstrated that the expression and activation of ErbB2 were preferentially increased in CSC/TIC-enriched tumorsphere cells, which promoted their self-renewal/proliferation and rendered them more sensitive to metformin. Our results, especially the in vivo data, provide fundamental support for developing metformin-mediated preventive strategies targeting ErbB2–associated carcinogenesis. Cancer Prev Res; 7(2); 199–210. ©2013 AACR.

Increasing evidence indicates that environmental exposure plays a critical role in breast cancer etiology. Bisphenol A (BPA), the building block of polycarbonate plastics with estrogenic activity, is one of the most common chemicals exposed in daily life. Recent studies suggest a link between BPA exposure and increased breast cancer risk. However, specific conditions and the underlying mechanisms of BPA exposure associated breast cancer risk remain unclear. This study aimed to investigate the effects of in utero exposure to BPA on mammary tumor development in MMTV-erbB-2 transgenic mice, which exemplifies a scenario of gene-environmental interaction. Since erbB-2 is amplified/overexpressed in approximately 30% of breast cancer, which has been associated with poor prognosis and therapeutic resistance, use of this clinically relevant mammary tumor model is of high translational value. In this study, pregnant MMTV-erbB-2 mice were subcutaneously injected with 0, 50 ng, 500 ng and 250 μg/Kg body weight BPA daily between day 13 and day 19 of gestation. We found that in utero exposed mice in the low dose (50 and 500 ng) groups, but not the high dose group (250 μg), displayed earlier vaginal opening and prolonged estrous phase, suggesting a dose dependent pro-estrogenic effect of in utero exposure to BPA. Whole mount analysis showed that mammary glands of mice with in utero exposure to low dose BPA had a longer ductal extension at 6 weeks and increased lateral branching/alveolar structures at 10 weeks, which was more significant in the 500 ng group but less evident in the high dose group. Molecular analysis of mammary tissues at 10 weeks indicated that in utero exposure to BPA increased phosphorylation/activation of erbB-2, EGFR, erbB-3, Erk1/2 and Akt, and expression of erbB-3 and EGFR. Signaling of estrogen receptor (ER) pathway was upregulated concomitantly, as reflected by increased expression and phosphorylation of ERα and upregulation of cyclin D1 and c-myc. More importantly, mice in the 500 ng group but not in the high dose group, developed tumor earlier than the mice in the control group, suggesting that dose is a critical factor affecting the outcomes. These data demonstrated that in utero exposure to BPA may promote mammary tumor development in erbB-2 transgenic mice in a dose dependent manner. Concomitant activation of the erbB-2 and ER pathways in the premalignant tissues suggests that induction of ER-erbB-2 crosstalk may play a critical role in mammary tumorigenesis promoted by in utero exposure to BPA, which underscores the significance of gene-environment interaction in early exposure to BPA associated breast cancer risk. Citation Format: Zhikun Ma, Amanda Parris, Xiaohe Yang. In utero exposure to bisphenol A induces reprogramming of mammary development and tumor risk in MMTV-erbB-2 transgenic mice. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B31.

Resveratrol is a natural polyphenolic compound with cancer chemopreventive activity. However, our understanding of the molecular mechanism responsible for resveratrol-induced apoptosis is still very limited. Here, we used MCF-7 and MDA-MB231 breast cancer cells as a model to demonstrate that resveratrol induced the expression of ASPP1, a new member of the ASPP (apoptosis stimulation protein of p53) family, which plays an important role in the regulation of apoptosis. Moreover, resveratrol enhanced apoptosis of MCF-7/ASPP1 cells, accompanied by higher expression of bax and p21. In contrast, siRNA-mediated knockdown of ASPP1 inhibited apoptosis in MB231 cells. Furthermore, we found that higher levels of ASPP1 were associated with adenovirus-mediated overexpression of E2F1 while siRNA-mediated E2F1 knockdown led to down-regulation of ASPP1. In conclusion, our results demonstrate that overexpression of ASPP1 rendered MCF-7 and MDA-MB231 breast cancer cells more sensitive to resveratrol-mediated apoptosis via the E2F pathway, thus suggesting that ASPP1 may represent a novel therapeutic target for resveratrol in human breast cancer.

Trastuzumab (or Herceptin), as the first erbB2-targeted therapy, has been successfully used to treat breast cancer patients with erbB2-overexpressing tumors. However, resistances to trastuzumab frequently occur, and novel strategies/agents are urgently needed to abrogate the resistant phenotype. Our current study explores the potential of SNDX-275, a class I HDAC inhibitor, to overcome trastuzumab resistance and investigates the combinational effects of SNDX-275 and trastuzumab on both sensitive and resistant breast cancer cells. Cell proliferation assays showed that SNDX-275 significantly enhanced trastuzumab-induced growth inhibition in trastuzumab-sensitive, erbB2-overexpressing breast cancer cells. Importantly, SNDX-275 at its therapeutic range re-sensitized trastuzumab-resistant cells to trastuzumab-mediated growth inhibition. SNDX-275 in combination with trastuzumab resulted in a dramatic reduction of erbB3 and its phosphorylation (P-erbB3), and inhibition of Akt signaling. Apoptotic-ELISA and western blot analyses confirmed that the combinations of SNDX-275 and trastuzumab as compared to SNDX-275 alone significantly enhanced DNA fragmentation and induced more PARP cleavage and caspase-3 activation in both trastuzumab-sensitive and -resistant breast cancer cells. Furthermore, co-immunoprecipitation assays revealed that SNDX-275 mainly attenuated the interactions of erbB2 and erbB3 receptors, but had no significant effect on erbB2/IGF-1R or erbB3/IGF-1R associations in the trastuzumab-resistant breast cancer cells. These data indicated that SNDX-275 enhanced trastuzumab efficacy against erbB2-overexpressing breast cancer cells, and exhibited potential to overcome trastuzumab resistance via disrupting erbB2/erbB3 interactions and inactivating PI-3K/Akt signaling. SNDX-275 may be included in erbB2-targeted regimen as a novel strategy to treat breast cancer patients whose tumors overexpress erbB2.

Molecular regulation of p73, a p53 family member, remains unclear. Here we report that p73 expression is significantly regulated by cell densities. In particular, we found that p73alpha and p73beta are differentially regulated. While p73beta protein levels were inversely correlated with cell densities, p73alpha protein levels behaved oppositely. We further showed that density-dependent changes of p73alpha follow the same patterns as E2F-1 and TAp73 mRNA levels, suggesting transcriptional regulation. Our data also suggest that high levels of p73beta at lower densities may be due to increased protein stability. However, AIP-4/Itch appeared not to be involved in downregulation of p73beta at high densities. Moreover, we also found that subcellular location of p73 isoforms changes with the culture density increases. While high level of p73beta at low density was mainly presented in the nucleus, low levels of this protein at high densities were mainly in the cytosol. Taken together, these findings reveal a novel mechanism that differentially regulates p73 isoforms and underscores the role of cell-cell interaction in p73 regulation, which may advance our understanding of p73 expression and function in human cancers.

We generated replication-defective adenovirus Ad-p53AIP1 and studied its anti-tumor efficacy both in vitro and in vivo. We demonstrated that Ad-p53AIP1 infection elicited high levels of p53AIP1 expression in cancer cells. We also found that Ad-p53AIP1 expression induced marked apoptosis and cell cycle arrest in HepG2 cells. Moreover, Ad-p53AIP1 infection significantly inhibited the tumorigenesis of 4T1 mouse mammary cancer cells in vivo. In particular, we discovered that p53AIP1 overexpression up-regulated the protein levels of p53 in HepG2 cells, which was accompanied by down-regulation of MDM2 mRNA and protein, suggesting an interaction between MDM2 and p53 in p53AIP1-induced apoptosis and cell cycle arrest. Our data demonstrated the feasibility of Ad-p53AIP1-mediated cancer gene therapy. p53AIP1-induced up-regulation of p53 protein through MDM2 suggests that p53AIP1 gene therapy may be more advantageous in tumors expressing high levels of oncoprotein MDM2 or having a mutation in MDM2 inhibitor p16INK4.

Inflammatory response plays an important role in the pathogenesis of secondary brain injury after traumatic brain injury (TBI). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that plays a crucial role in cytoprotection against inflammation. The present study investigated the role of Nrf2 in the cerebral upregulation of NF-κB activity, proinflammatory cytokine, and ICAM-1 after TBI. Wild-type Nrf2 (+/+) and Nrf2 (−/−)-deficient mice were subjected to a moderately severe weight-drop impact head injury. Electrophoretic mobility shift assays (EMSAs) were performed to analyze the activation of nuclear factor kappa B (NF-κB). Enzyme-linked immunosorbent assays were performed to quantify the production of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6 (IL-6). Immunohistochemistry staining experiments were performed to detect the expression of intercellular adhesion molecule-1 (ICAM-1). Nrf2 (−/−) mice were shown to have more NF-κB activation, inflammatory cytokines TNF-α, IL-1βand IL-6 production, and ICAM-1 expression in brain after TBI compared with their wild-type Nrf2 (+/+) counterparts. The results suggest that Nrf2 plays an important protective role in limiting the cerebral upregulation of NF-κB activity, proinflammatory cytokine, and ICAM-1 after TBI.

Genistein is a soy isoflavone with anti-tumor properties. Genistein-induced apoptosis involves Bcl-2 down-regulation. However, overexpression of Bcl-2 in breast cancer has been associated with better prognosis and response to hormonal therapy. To examine genistein's effect on breast cancer cells with different Bcl-2 levels, we established control (MCF-7/PV) and Bcl-2 overexpressing MCF-7 (MCF-7/Bcl-2) cell lines and characterized genistein regulated apoptosis and cell cycle progression in these cells. Our results demonstrate that overexpression of Bcl-2 rendered MCF-7 cells more sensitive, rather than resistant, to genistein. We found that genistein induces enhanced cytochrome c release and mitochondrial membrane depolarization in MCF-7/Bcl-2 cells, as compared to control. We also found that genistein increases Bcl-2 levels and Bcl-2/Bax ratio in the mitochondrial fractions of MCF-7/Bcl-2 cells, suggesting that disturbed Bcl-2/Bax distribution may cause cytochrome c release and apoptosis in these cells. Cell cycle analysis indicated that genistein induces G0/G1 arrest in MCF-7/PV cells but increases in G2/M arrest in MCF-7/Bcl-2 cells. This was accompanied by modified responses of several cell cycle regulators, such as p21 and cyclin Bl. Taken together, our results indicate that genistein-Bcl-2 interaction switches Bcl-2 from an anti-apoptotic protein into a proapoptotic protein, which involves disturbed Bcl-2/Bax distribution in mitochondria, increased cytochrome c release and modified cell cycle regulation.

Caspases play an essential role in the initiation/regulation of apoptosis. Aberrant apoptotic regulation has been associated with carcinogenesis and therapeutic resistance. To explore the possible involvement of altered caspase expression in breast cancer, we have systematically examined the expression of both protein and mRNA levels of 7 caspases in a panel of 18 breast cancer cell lines. We found that variation of caspase expression can occur at both protein and RNA levels. Down-regulation of these caspases, especially caspase-8 and -10, was frequently observed. Functional screening of these selected cell lines using TNF-alpha, doxorubicin and radiation induced cell injury showed that a lack of functional caspase-8 resulted in resistance to TNF-alpha-induced apoptosis. Array style examination of caspase expression profiles in breast cancer cell lines yields massive information that is valuable in establishing cell line models to study the role of caspase down-regulation/deficiency in breast cancer development and therapeutic resistance.

Deregulation of erbB-2 and EGFR is frequently detected in breast cancer, which has been associated with poor prognosis. Lapatinib, an orally active small molecule acting as a dual inhibitor of erbB-2 and EGFR, has been used for the treatment of patients with advanced or metastatic erbB-2+ breast cancer. Lapatinib resistance, however, is emerging as a clinical challenge. Understanding the molecular mechanisms of Lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance in breast cancer treatment. Cancerous inhibitor of PP2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast and other types of cancers. Through interacting with protein phosphatase 2A (PP2A), CIP2A regulates c-Myc and factors involved in cell proliferation and survival. This study aimed to investigate the role of CIP2A in Lapatinib-mediated inhibition of erbB-2 overexpressing breast cancer cells. Our data showed that Lapatinib downregulated CIP2A in erbB-2 overexpressing SK-BR-3 and 78617 cells, which was correlated with concurrent inactivation of erbB-2, EGFR, Akt, Erk1/2 and mTOR. Overexpression of CIP2A rendered the cells resistant to Lapatinib induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via siRNA sensitized the cells to Lapatinib-mediated effects. We also demonstrated that Lapatinib-induced downregulation of CIP2A can be abolished by LY294002, suggesting that CIP2A downregulation was regulated by Akt. Analysis with cycloheximide (CHX) and MG132 indicated that Lapatinib modulates CIP2A protein stability and promotes its degradation through prosteasome pathway. More importantly, we found that Lapatinib induced differential downregulation of CIP2A between the parental and Lapatinib-resistant (LR) BT-474 cells. Decrease in CIP2A downregulation in BT-474-LR cells suggests its association with Lapatinib resistance. Knockdown of CIP2A via lentiviral siRNA system significantly sensitized Lapatinib induced growth inhibition and apoptosis. Taken together, our results demonstrate that CIP2A is a molecular target of Lapatinib, which plays a critical role in Lapatinib-induced cellular responses. Lapatinib-induced downregulation of CIP2A involves the inhibition of the Akt-CIP2A feedback loop. Correlation between Lapatinib resistance and decreased CIP2A downregulation, as well as CIP2A knockdown-mediated sensitization of BT-474-LR cells indicate that CIP2A is a resistance factor of Lapatinib. Further investigation on Lapatinib-mediated regulation of CIP2A will advance our understanding of Lapatinib-associated antitumor activities and drug resistance. Citation Format: Ming Zhao, Amanda Blackwelder, Harry Lee, Xiaohe Yang. Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib. [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 3557. doi:10.1158/1538-7445.AM2015-3557

Notch signaling is believed to promote cell survival in general. However, the mechanism is not clearly understood. Here, we show that cells expressing intracellular domain of human Notch1 (NIC-1) are chemoresistant in a wild-type p53-dependent manner. NIC-1 inhibited p53 by inhibiting its activating phosphorylations at Ser15, Ser20, and Ser392 as well as nuclear localization. In addition, we found that inhibition of p53 by NIC-1 mainly occurs through mammalian target of rapamycin (mTOR) using phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) pathway as the mTOR inhibitor, rapamycin treatment abrogated NIC-1 inhibition of p53 and reversed the chemoresistance. Consistent with this, rapamycin failed to reverse NIC-1-induced chemoresistance in cells expressing rapamycin-resistant mTOR. Further, ectopic expression of eukaryotic initiation factor 4E (eIF4E), a translational regulator that acts downstream of mTOR, inhibited p53-induced apoptosis and conferred protection against p53-mediated cytotoxicity to similar extent as that of NIC-1 overexpression but was not reversed by rapamycin, which indicates that eIF4E is the major target of mTOR in Notch1-mediated survival signaling. Finally, we show that MCF7 (breast cancer) and MOLT4 (T-cell acute lymphoblastic leukemia) cells having aberrant Notch1 signaling are chemoresistant, which can be reversed by both PI3K and mTOR inhibitors. These results establish that Notch1 signaling confers chemoresistance by inhibiting p53 pathway through mTOR-dependent PI3K-Akt/PKB pathway and imply that p53 status perhaps is an important determinant in combination therapeutic strategies, which use mTOR inhibitors and chemotherapy. (Cancer Res 2006; 66(9): 4715-24)

The mammary gland during in utero stage is in a largely undifferentiated state, which is particularly vulnerable to environmental factors. Increasing evidence indicates that intrauterine hormonal and nutritional alterations may significantly modify breast cancer risk later in life. A well-known case is the “DES Daughter” story. However, the underlying mechanisms are largely unknown and more prenatal risk factors remain to be identified. Therefore, development of sensitive and reliable model systems to identify those factors with mechanistic insight is of great significance in breast cancer prevention. Previously, most studies on in utero exposure-modulated breast cancer risk used carcinogenic models. While this system is valuable in providing proof of concept, due to challenges in elucidating molecular pathways induced by in utero exposures and by carcinogens, molecular mechanistic studies in these models are compromised. In this study, we aimed to establish an animal model for studying in utero exposure to environmental hormones-induced mammary tumor risk using MMTV-erbB-2 transgenic mice. erbB-2 is an oncogene that is amplified in ~25-30% of human breast cancer. The MMTV-erbB-2 mice have a defined genetic background and tumor development patterns, which is of advantage in mechanistic studies. When pregnant MMTV-erbB-2 mice were injected with 20 ng/mouse/day of Estradiol (E2) between day 13 and day 19 of gestation, mammary tumor development of the offspring was significantly accelerated, as compared to control. The average tumor latencies of the control and the E2 group were 36 and 32 weeks respectively. In utero exposure to E2 also induced earlier vaginal opening and prolonged length of estrous stages. Whole mount analysis indicated that in utero exposure to E2 increased mammary epithelial density at 6 and 10 weeks of age. Importantly, we also found that in utero exposure to E2 induced a concurrent upregulation of signaling in estrogen receptor α (ER α) and receptor tyrosine kinase pathways in the mammary tissues, as indicated by increased expression of ER target genes (c-myc, cyclin D1, EGFR, and Bcl-2) and phosphorylation of EGFR, erbB-2, Akt1 and Erk1/2. Use of gefitinib also, a small molecular inhibitor of EGFR, abrogated the upregulation of both pathways in mammary tissues with in utero exposure to E2, suggesting a role of EGFR in in utero exposure to E2 induced crosstalk between the two pathways. Our data indicates that in utero exposure to E2 induces a systematic pro-estrogenic effect and enhanced signaling and crosstalk between ER and EGFR/erbB-2 pathways. Deregulation of the two critical pathways in the premalignant tissues is associated with mammary tumor risk later in life. This will lead to further mechanistic studies, such as in utero exposure to hormones, induced epigenetic regulation, and reprogramming. This model system may represent a model for studying gene-environment interaction in breast carcinogenesis and can be used to study the effects of in utero exposure to other environmental and dietary factors modulated breast cancer risk. Citation Format: Zhikun Ma, Amanda Blackwelder, Xiaohe Yang. In utero exposure to estrogen promotes mammary tumor risk in MMTV-erbB-2 transgenic mice through induction of ER-erbB-2 crosstalk. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A09.

Background and Objectives We propose that a growth factor receptor tyrosine kinase (RTK) inhibitor, such as tyrphostin A47, could serve as an adjunct to estrogen replacement therapy (ERT) for postmenopausal breast cancer survivors. Tyrphostins have been shown to block estrogen (E2)-induced proliferation in the human breast cancer MCF-7 cell line. Therefore, the effects of A47 on signal transduction, cell cycle progression, and apoptosis in E2-mediated breast cancer cell growth in vitro were investigated. Methods Cell growth was determined by MTT proliferation assay, cell cycle analysis assessed by flow cytometry, and RTK activation by Western blot. Apoptosis assays included nuclear staining, TdT-mediated dUTP-X nick end labeling, and caspase 3 activation. Results We find A47 selectively inhibits epidermal growth factor (EGF) and basic fibroblast growth factor but not insulin growth factor-1 proliferation. Although A47 inhibits EGF-induced phosphorylation of the EGF receptor in A431 cells, it does not consistently block MAP kinase phosphorylation. Conclusions Taken together, A47 blocks E2/EGF-induced activation of EGFR and therefore interferes with the proximal EGFR signaling pathway. A47 also arrests the cells at the G1-S transition of the cell cycle and induces cell death by apoptosis. Thus, a growth factor RTK may be useful in blocking hormone-dependent tumor growth in an elevated E2 environment. J. Surg. Oncol. 2002;79:17–29. © 2002 Wiley-Liss, Inc.

Metformin is an oral biguanide used for type II diabetes. Epidemiologic studies suggest a link between metformin use and reduced risk of breast and other types of cancers. erbB-2 expressing breast cancer is a subgroup of tumors with poor prognosis. Previous studies demonstrated that metformin is a potent inhibitor of erbB-2 overexpressing breast cancer cells; metformin treatment extends the life span and impedes mammary tumor development in ErbB-2 transgenic mice in vivo. However, the mechanisms of metformin associated anti-tumor activity, especially in prevention models, remain unclear. In this study, we investigated the mechanisms of metformin associated prevention/inhibition of ErbB-2 mediated breast cancer development by focusing on the potential effect of metformin on tumor initiating cells (TICs)/cancer stem cells (CSCs) in the MMTV-erbB-2 transgenic mouse model and its context with existing in vitro models. We report here for the first time that systemic administration of metformin (250 mg/kg/day, ip, between 8- and 18-week of age) selectively inhibits CD61high/CD49fhigh subpopulation, a group of TICs of MMTV-ErbB-2 mammary tumors, in preneoplastic mammary glands. Metformin also inhibited CD61high/CD49fhigh subpopulation in MMTV-erbB-2 tumor-derived cells, which was correlated with their compromised tumor initiation/development in a syngeneic tumor graft model. Molecular analysis indicated that metformin induced downregulation of erbB-2 and EGFR expression and inhibited the phosphorylation of erbB family members, IGF1R, Akt, mTOR and Stat3 in vivo. In vitro data indicate that low doses (1 mM) of metformin inhibited the self-renewal/proliferation of CSCs/TICs in ErbB-2-overexpressing breast cancer cells. We further demonstrated that the expression and activation of ErbB-2 were preferentially increased in CSC/TIC-enriched tumorsphere cells, which promoted their self-renewal/proliferation and rendered them more sensitive to metformin. Our results, especially the in vivo data, provide fundamental support for developing metformin-mediated preventive strategies targeting erbB-2-associated carcinogenesis. Citation Format: Pei Zhu, Meghan Davis, Amanda Blackwelder, Nora Bachman, Bolin Liu, Susan Edgerton, Leonard L. Williams, Ann D. Thor, Xiaohe Yang. Metformin selectively targets tumor initiating cells in erbb-2 overexpressing breast cancer models. [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 2967. doi:10.1158/1538-7445.AM2014-2967

ErbB-2 is a receptor tyrosine kinase (RTK) that is amplified/overexpressed in 20-30% of all invasive breast malignancies; it has been associated with poor prognosis and therapeutic resistance in breast cancer patients. 7,12-dimethylbenz anthracene (DMBA) is a polycyclic aromatic hydrocarbon known for its carcinogenic effects. The objective of this study was to investigate the effect of DMBA exposure on erbB-2 mediated carcinogenesis. This represents a combination of genetic predisposition and environmental assault that synergistically promote carcinogenesis. In this study, female MMTV-erbB-2 transgenic mice at 6-weeks of age were treated weekly by oral gavage with either 1.0 mg DMBA in peanut oil or vehicle alone for 6 weeks. In the control group, palpable tumors appeared at week 25, with an average latency of 36.0 weeks. All mice in the DMBA group developed mammary tumors between week 16 and 26 with an average latency of 20 weeks. By the endpoint (first tumor ∼1.5cm3), the average number of palpable tumors per mouse were 1.15 and 2 for control and DMBA groups, respectively. Lung metastasis in the DMBA group was significantly higher than control mice (40% DMBA vs. 5% control). These data show that DMBA exposure significantly promoted erbB-2 mediated carcinogenesis. Whole mount analysis showed that at 14 weeks of age there was marked expansion of the ducts, whereas lateral branching of mammary glands from DMBA mice appeared to be inhibited, suggesting that DMBA stimulates glandular growth while inhibiting differentiation in this model. Western blot analysis of mammary tissues at 14 weeks of age showed that ERα, p-ERα, cyclin D1, Bcl-2, c-myc, EGFR, erbB-2 and erbB-3 protein levels were significantly increased in DMBA treated mice, which was accompanied by increased phosphorylation/activation of erbB-2, EGFR, ERK and Akt1. These data suggest that DMBA induced activation of both ER and receptor tyrosine kinase (RTK) pathways. Interestingly, tumor histology between tumors from control and DMBA treated mice was similar. However, analysis of tumor karotypes from control and DMBA mice showed that tumors from DMBA mice displayed more chromosomal alterations; an addition to chromosome 4 and trisomy 5 in the control mouse, trisomy 2, trisomy 3 and a deletion to chromosome 4 were detected from tumors of the DMBA mice, suggesting that DMBA induced chromosomal instability contributed to tumor promotion in this model. Taken together, our data suggest that DMBA promoted erbB-2 mediated carcinogenesis. In addition to its well known mutational effect, DMBA induced deregulation of ER and erbB-2 pathways plays a critical role in this process. Citation Format: Zhikun Ma, Stanley Kosanke, Morgan Carrington, Xiaohe Yang. DMBA promotes erbB-2 mediated carcinogenesis through activation of estrogen receptor and receptor tyrosine kinase pathways. [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 5345. doi:10.1158/1538-7445.AM2014-5345

Growing evidence indicates that incongruities in the inflammatory and immune response pathways contribute to tumorigenesis. We seek to explore the underlying biological mechanisms that both promote and inhibit breast cancer (BrCa) tumorigenesis via innate immune pathways involving the Toll-like receptors (TLRs) and their associated agonists. Women of African descent (AA) are an understudied group at high risk for developing aggressive BrCa. Therefore, we previously examined the occurrence of single nucleotide polymorphisms (SNPs) in selected genes along TLR pathways among a small cohort of AA women with breast cancer and identified a SNP in the central regulatory kinase IRAK4 that was associated with increased breast cancer risk. In silico predictions suggest that the IRAK4 minor variant rs4251545 codes for an Ala428Thr missense mutation that introduces a potential phosphorylation site in the extreme carboxy terminus (XCT) of the IRAK-4 kinase domain. The current study tests the hypothesis that functional IRAK4 promotes BrCa cell growth and migration using in vitro (cultured human BrCa cell) and in vivo (zebrafish BrCa xenograft) models. Materials and methods: ER-positive MCF7 and triple-negative MDA-MB-231 (wild-type IRAK4) and MDA-MB-468 (IRAK4 w/rs4251545) human BrCa cell lines were compared. Cultured cells were treated for 24 hours with or without IRAK1/4 inhibitor. Cell migration and cell growth among BrCa lines were compared using wound healing and crystal violet assays, respectively. BrCa cell xenografts of zebrafish (ZF) embryos were examined for metastatic potential and the physiologic effects of morpholino inhibition of ZF IRAK4 among embryos with and without BrCa xenograft burdens were compared. In addition, zebrafish/tumor xenograft model was used to address the in vivo metastatic property of BrCa cells and the effect of targeting zebrafish irak4 using morpholino (MO) knockdown approach in the tumor-host environment on the metastatic behavior of MDA-MB-468 cells. Results: Treatment with IRAK1/4 inhibitor delayed BrCa migration and growth in all BrCa cells lines. Relative in vivo migration rates/ (or metastatic property) of MDA-MB-231 and MDA-MB-468 BrCa xenografts in ZF resemble in vitro rates, in which MDA-MB-231s migrate metastasized more rapidly/ (or aggressively). MO inhibition of ZF zebrafish IRAK4 irak4 gene expression in the host environment MDA-MB-468 xenografted fish inhibits MDA-MB-468 metastasis in the zebrafish/tumor xenograft. Conclusion: Inhibition of IRAK4 reduces BrCa growth and migration in vitro and in vivo in zebrafish. The development of drugs that target IRAK4 and other regulators of the innate immune pathway may help modulate BrCa tumor progression. Citation Format: K. Sean Kimbro, Karine Ferri-Lagneau, Jamil Haider, Susan Yeyeodu, Xiaohe Yang, TinChung Leung. IRAK4 and breast cancer cell migration in culture and zebrafish xenografts. [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 4983. doi:10.1158/1538-7445.AM2014-4983