Amanda B. Parris

p53 is required for metformin-induced growth inhibition, senescence and apoptosis in breast cancer cells

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.

Alcohol promotes migration and invasion of triple-negative breast cancer cells through activation of p38 MAPK and JNK.

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.

Genistein targets the cancerous inhibitor of PP2A to induce growth inhibition and apoptosis in breast cancer cells

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.

Phenformin inhibits growth and epithelial-mesenchymal transition of ErbB2-overexpressing breast cancer cells through targeting the IGF1R pathway

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.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.

Activation of cancerous inhibitor of PP2A (CIP2A) contributes to lapatinib resistance through induction of CIP2A-Akt feedback loop in ErbB2-positive breast cancer cells

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.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.

p53 pathway determines the cellular response to alcohol-induced DNA damage in MCF-7 breast cancer cells

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.

FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice

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.

Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

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.

DMBA promotes ErbB2‑mediated carcinogenesis via ErbB2 and estrogen receptor pathway activation and genomic instability

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 restriction inhibits mammary tumorigenesis in MMTV-ErbB2 transgenic mice through the suppression of ER and ErbB2 pathways and inhibition of epithelial cell stemness in premalignant mammary tissues

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 (CD24highCD49flow), putative mammary reconstituting unit subpopulation (CD24highCD49fhigh) and luminal progenitor cells (CD61highCD49fhigh). 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.