Although chemopreventative agents targeting the estrogen/estrogen receptor (ER) pathway have been effective for ER+ breast cancers, prevention of hormone receptor-negative breast cancers, such as Her2/erbB-2+ breast cancers, remains a significant issue. Previous studies have demonstrated that administration of EGFR/erbB-2-targeting lapatinib to MMTV-erbB-2 transgenic mice inhibited mammary tumor development. The prevention, however, was achieved by prolonged high dose exposure. The tolerance to high dose/long-term drug administration may hinder its potential in clinical settings. Therefore, we aimed to test a novel, short-term chemopreventative strategy using lapatinib during the premalignant risk window in MMTV-erbB-2 mice.

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.

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.

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.

Metformin, the widely used anti-diabetic drug, is emerging as a promising anti-cancer agent. However, response variation among different tumors remains a significant challenge. Hence, identification of the factors that determine metformin sensitivity is of greatest significance for its clinical implementation. In this study, we showed that MDA-MB-468 cells were most sensitive among the five breast cancer cell lines tested. We found that metformin-induced inhibition of MDA-MB-468 cells was correlated with downregulation of c-Met at both protein and mRNA levels. To understand the functional significance of c-Met downregulation in metformin-mediated tumor inhibition, we established control and c-Met overexpressing sublines of MDA-MB-468 cells (468/C and 468/Met) using lentiviral expression system. We demonstrated that overexpression of c-Met significantly attenuated metformin induced inhibition of MDA-MB-468 cells. Metformin-induced inhibition of ALDH1+ cells, which are enriched with cancer stem cells, was also abrogated in 468/Met cells as compared to 468/C cells. Signal transduction analysis of the paired cell lines indicated that c-Met-induced activation of STAT3 and AKT1, and upregulation of Gab1 are related to c-Met-modulated metformin responsiveness. These findings highlight c-Met as a potential key regulator of metformin-mediated inhibition of proliferation and stemness of breast cancer cells, indicating that c-Met overexpression may be a critical factor contributing to metformin resistance. The data also suggest that combination of metformin with c-Met inhibitors could be a useful strategy to improve metformin-mediated anti-cancer efficacies in breast cancer treatment.

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.

Recently, a new strain of Cercospora sojina (Race15) has been identified, which has caused the breakdown of resistance in most soybean cultivars in China. Despite this serious yield reduction, little is known about why this strain is more virulent than others. Therefore, we sequenced the Race15 genome and compared it to the Race1 genome sequence, as its virulence is significantly lower. We then re-sequenced 30 isolates of C. sojina from different regions to identifying differential virulence genes using genome-wide association analysis (GWAS).

Cercospora armoraciae causes leaf spot disease on Armoracia rusticana. Exudation of droplets, when grown on PDA, distinguishes this fungi from other members of the genus Cercospora. The role this exudate plays in the virulence of this pathogen has not been elucidated. To explore this, we characterized the transcriptome of C. armoraciae and the proteome of exudate associated with this plant pathogen.

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.

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.

Bisphenol A (BPA) is the most common environmental endocrine disrupting chemical. Studies suggest a link between perinatal BPA exposure and increased breast cancer risk, but the underlying mechanisms remain unclear. This study aims to investigate the effects of in utero BPA exposure on mammary tumorigenesis in MMTV-erbB2 transgenic mice. Pregnant mice were subcutaneously injected with BPA (0, 50, 500 ng/kg and 250 µg/kg BW) daily between gestational days 11-19. Female offspring were examined for mammary tumorigenesis, puberty onset, mammary morphogenesis, and signaling in ER and erbB2 pathways. In utero exposure to low dose BPA (500 ng/kg) induced mammary tumorigenesis, earlier puberty onset, increased terminal end buds, and prolonged estrus phase, which was accompanied by proliferative mammary morphogenesis. CD24/49f-based FACS analysis showed that in utero exposure to 500 ng/kg BPA induced expansion of luminal and basal/myoepithelial cell subpopulations at PND 35. Molecular analysis of mammary tissues at PND 70 showed that in utero exposure to low doses of BPA induced upregulation of ERα, p-ERα, cyclin D1, and c-myc, concurrent activation of erbB2, EGFR, erbB-3, Erk1/2, and Akt, and upregulation of growth factors/ligands. Our results demonstrate that in utero exposure to low dose BPA promotes mammary tumorigenesis in MMTV-erbB2 mice through induction of ER-erbB2 crosstalk and mammary epithelial reprogramming, which advance our understanding of the mechanism associated with in utero exposure to BPA-induced breast cancer risk. The studies also support using MMTV-erbB2 mouse model for relevant studies.

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.

There is increasing evidence that prenatal exposure to environmental factors may modify breast cancer risk later in life. This study aimed to investigate the effects of in utero exposure to low-dose alcohol on mammary development and tumor risk. Pregnant MMTV-erbB-2 mice were exposed to alcohol (6 g/kg/day) between day 13 and day 19 of gestation, and the female offspring were examined for tumor risk. Whole mount analysis indicated that in utero exposure to low-dose alcohol induced significant increases in ductal extension at 10 weeks of age. Molecular analysis showed that in utero alcohol exposure induced upregulation of ERα signaling and activation of Akt and Erk1/2 in pubertal mammary glands. However, enhanced signaling in the EGFR/erbB-2 pathway appeared to be more prominent in 10-week-old glands than did signaling in the other pathways. Interestingly, tumor development in mice with in utero exposure to low-dose alcohol was slightly delayed compared to control mice, but tumor multiplicity was increased. The results indicate that in utero exposure to low-dose alcohol induces the reprogramming of mammary development by mechanisms that include altered signaling in the estrogen receptor (ER) and erbB-2 pathways. The intriguing tumor development pattern might be related to alcohol dose and exposure conditions, and warrants further investigation.

Co-expression of several receptor tyrosine kinases (RTKs), including erbB2 and erbB3, is frequently identified in breast cancers. A member of the RTK family, the kinase-deficient erbB3 can activate downstream signaling via heterodimer formation with erbB2. We studied the expression of RTK receptors in mammary tumors from the wild-type (wt) rat c-neu transgenic model. We hypothesized that physical and functional interactions between the wt rat neu/ErbB2 transgene and mouse ErbB3-encoded proteins could occur, activating downstream signaling and promoting mammary oncogenesis.

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.

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.

Cercospora sojina is a fungal pathogen that causes frogeye leaf spot in soybean-producing regions, leading to severe yield losses worldwide. It exhibits variations in virulence due to race differentiation between strains. However, the candidate virulence-related genes are unknown because the infection process is slow, making it difficult to collect transcriptome samples.

Frogeye leaf spot (FLS) is a destructive fungal disease that affects soybean production. The most economical and effective strategy to control FLS is the use of resistant cultivars. However, the use of a limited number of resistant loci in FLS management will be countered by the emergence of new high-virulence Cercospora sojina races. Therefore, we identified quantitative trait loci (QTL) that control resistance to FLS and identified novel resistant genes using a genome-wide association study (GWAS) on 234 Chinese soybean cultivars.

While p73 overexpression has been associated with increased apoptosis in cancer tissues, p73 overexpressing tumors appear to be of high grade malignancy. Why this putative tumor suppressor is overexpressed in cancer cells and what the function of overexpressed p73 is in breast cancers are critical questions to be addressed. By investigating the effect of p53 inactivation on p73 expression, we found that both protein and mRNA levels of TAp73 were increased in MCF-7/p53siRNA cells, MCF-7/p53mt135 cells and HCT-116/p53-/- cells, as compared to wild type control, suggesting that p53 inactivation by various forms upregulates p73. We showed that p53 knockdown induced p73 was mainly regulated at the transcriptional level. However, although p53 has a putative binding site in the TAp73 promoter, deletion of this binding site did not affect p53 knockdown mediated activation of TAp73 promoter. Chromatin immuno-precipitation (ChIP) data demonstrated that loss of p53 results in enhanced occupancy of E2F-1 in the TAp73 promoter. The responsive sequence of p53 inactivation mediated p73 upregulation was mapped to the proximal promoter region of the TAp73 gene. To test the role of E2F-1 in p53 inactivation mediated regulation of p73 transcription, we found that p53 knockdown enhanced E2F-1 dependent p73 transcription, and mutations in E2F-1 binding sites in the TAp73 promoter abrogated p53 knockdown mediated activation of TAp73 promoter. Moreover, we demonstrated that p21 is a mediator of p53-E2F crosstalk in the regulation of p73 transcription. We concluded that p53 knockdown/inactivation may upregulate TAp73 expression through E2F-1 mediated transcriptional regulation. p53 inactivation mediated upregulation of p73 suggests an intrinsic rescuing mechanism in response to p53 mutation/inactivation. These findings support further analysis of the correlation between p53 status and p73 expression and its prognostic/predictive significance in human cancers.

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.