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Background Rice sheath blight, caused by Rhizoctonia solani Kühn (teleomorph: Thanatephorus cucumeris), is one of the most severe diseases in rice (Oryza sativa L.) worldwide. Studies on resistance genes and resistance mechanisms of rice sheath blight have mainly focused on indica rice. Rice sheath blight is a growing threat to rice production with the increasing planting area of japonica rice in Northeast China, and it is therefore essential to explore the mechanism of sheath blight resistance in this rice subspecies. Results In this study, RNA-seq technology was used to analyse the gene expression changes of leaf sheath at 12, 24, 36, 48, and 72 h after inoculation of the resistant cultivar ‘Shennong 9819’ and susceptible cultivar ‘Koshihikari’ with R. solani. In the early stage of R. solani infection of rice leaf sheaths, the number of differentially expressed genes (DEGs) in the inoculated leaf sheaths of resistant and susceptible cultivars showed different regularity. After inoculation, the number of DEGs in the resistant cultivar fluctuated, while the number of DEGs in the susceptible cultivar increased first and then decreased. In addition, the number of DEGs in the susceptible cultivar was always higher than that in the resistant cultivar. After inoculation with R. solani, the overall transcriptome changes corresponding to multiple biological processes, molecular functions, and cell components were observed in both resistant and susceptible cultivars. These included metabolic process, stimulus response, biological regulation, catalytic activity, binding and membrane, and they were differentially regulated. The phenylalanine metabolic pathway; tropane, piperidine, and pyridine alkaloid biosynthesis pathways; and plant hormone signal transduction were significantly enriched in the early stage of inoculation of the resistant cultivar Shennong 9819, but not in the susceptible cultivar Koshihikari. This indicates that the response of the resistant cultivar Shennong 9819 to pathogen stress was faster than that of the susceptible cultivar. The expression of plant defense response marker PR1b gene, transcription factor OsWRKY30 and OsPAL1 and OsPAL6 genes that induce plant resistance were upregulated in the resistant cultivar. These data suggest that in the early stage of rice infection by R. solani, there is a pathogen-induced defence system in resistant rice cultivars, involving the expression of PR genes, key transcription factors, PAL genes, and the enrichment of defence-related pathways. Conclusion The transcriptome data revealed the molecular and biochemical differences between resistant and susceptible cultivars of rice after inoculation with R. solani, indicating that resistant cultivars have an immune response mechanism in the early stage of pathogen infection. Disease resistance is related to the overexpression of PR genes, key transcriptome factors, and PAL genes, which are potential targets for crop improvement.

Background: Frogeye leaf spot (FLS) is a destructive fungal disease in soybean production. The most economical and effective strategy to control FLS is to use resistant cultivars. However, the use of a limited number of resistant loci in FLS management will be countered by the emergence of new, and more virulent, Cercospora sojina races. Thus, 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.Results: A total of 30,890 single nucleotide polymorphism (SNP) markers were used to estimate linkage disequilibrium (LD) and population structure. The GWAS results identified six loci (pGlyma20g31630 encoding pyruvate dehydrogenase (PDH), Glyma05g28980 encoding mitogen-activated protein kinase 7 (MPK7), and Glyma20g31510, Glyma20g31520 encoding the calcium-dependent protein kinase 4(CDPK4) in the haplotype blocks were found to be associated with FLS resistance.Conclusions: The proteins encoded by these four genes directly, or indirectly participate in the biological pathway of salicylic acid (SA) and jasmonic acid (JA). These two plant hormones can induce the expression of disease resistance related genes and are essential for plant systemic acquired resistance (SAR). Therefore, the novel loci and candidate genes provide genomic location information for further advances in FLS resistance gene cloning and molecular breeding.

Rice false smut (RFS) disease, which is caused by Ustilaginoidea virens, has been widespread all over the world in recent years, causing irreversible losses. Under artificial culture conditions, exudates will appear on colonies of U. virens during the growth of the hyphae. Exudation of droplets is a common feature in many fungi, but the functions of exudates are undetermined. As the executors of life functions, proteins can intuitively reflect the functions of exudates. Shotgun proteomics were used in this study. A total of 650 proteins were identified in the exudate of U. virens, and the raw data were made available via ProteomeXchange with the identifier PXD019861. There were 57 subcategories and 167 pathways annotated with Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, respectively. Through protein–protein interaction (PPI) network analysis, it was found that 20 proteins participated in the biosynthesis of secondary metabolites. Two separate PPI analyses were performed for carbon metabolism and microbial metabolism in diverse environments. After comparing and annotating the functions of proteins of the exudate, it was speculated that the exudate was involved in the construction and remodeling of the fungal cell wall. Pathogenicity, sporulation, and antioxidant effects might all be affected by the exudate.

Background 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. Methods Virulence of three strains of C. armoraciae was evaluated in greenhouse assays. De novo sequencing was applied to assemble transcriptome from these strains. Nano-HPLC-MS/MS analysis was used to identify proteins in the pathogen exudate. Identified proteins were functionally classified and annotated using GO, KEGG, and COG/KOG bioinformatics analysis methods. Results When treated with the exudate of C. armoraciae strain SCa-01, leaves of A. rusticana showed yellowing and necrosis of the leaves and similar symptoms to plants inoculated with this fungi. A total of 14,937 unigenes were assembled from C. armoraciae, and 576 proteins comprising 1,538 peptides, 1,524 unique peptide, were identified from the exudate. GO annotation classified 411 proteins (71%) into 27 functional categories, namely, 12, seven and eight biological process, cellular component, and molecular function subcategories, respectively. KEGG analysis assigned 314 proteins to 84 signaling/metabolic pathways, and 450 proteins were annotated against the COG/KOG database. Discussion Transcriptome and GO analysis of C. armoraciae found most proteins in the exudate. GO analysis suggested that a considerable proportion of proteins were involved in cellular process and metabolic process, which suggests exudates maintain the metabolic balance of this fungi. Some proteins annotated to the phenylalanine metabolism, which suggests that the exudates may enhance the virulence of this pathogen. Some proteins annotated to the phenylalanine metabolism, which suggests that the exudates may enhance the pathogenicity of the pathogen. Also some proteins were annotated to the peroxisome metabolic pathway and the fatty acid biosynthesis pathways. These pathways may confer antifungal, antioxidant and antimicrobial activity on the exudates.

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.

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.

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.

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 &micro, g/kg BW) daily between gestational days 11&ndash, 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 ER&alpha, p-ER&alpha, 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.

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.

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.

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.

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)

Fe nanowires/chitosan nano-composites were prepared and their capacity for adsorbing bovine serum albumin (BSA) was examined. The results show that when pH of buffer solution was set at 6.0, the maximum adsorption was 148.40 mg g −1 . By crosslinking α-human chorionic gonadotropin (α-HCG), biological probes with Fe nanowires/chitosan/antibody were prepared. immunochromatographic strips were manufactured, and different concentration HCG antigens were tested. The results indicate that the sensitivity is 10 IU l −1 . Pregnancy can be detected by these immunochromatographic strips 2–3 days earlier than by the colloidal gold test strips in the market.

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.

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.

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.

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.

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner. However, the mechanism by which this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pathway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However at higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavage of PARP (i.e., an approximately 60-kDa cleavage fragment) was observed. Atypical PARP cleavage directly correlated with apoptosis in MCF-7 cells and was inhibited by the global cysteine protease inhibitors iodoacetamide and N-ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, granzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant cleavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain. beta-Lap exposure also stimulated the cleavage of lamin B, a putative caspase 6 substrate. Reexpression of procaspase-3 into caspase-3-null MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-independent activation of a noncaspase proteolytic pathway.

Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the requirements for these interactions remain unclarified. We examined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is removed and cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7.

The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (kcat/Km) of 8.7 x 10(5) and 2.8 x 10(5) M-1 s-1, respectively. These rates are of sufficient magnitude to indicate direct processing in vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors.

A new form of intercellular signaling is described for lymphocyte granule-mediated apoptosis. Christopher Froelich, Vishva Dixit and Xiaohe Yang propose that perforin delivers granzyme B into target cells by a mechanism analogous to receptor-dependent endocytosis of pathogens. Once in the cytosol, granzyme B prefers to initiate apoptosis by activating the apical protease caspase 10.

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.

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.

We have shown that erbB2 altered breast cancer cells are less sensitive to the anti-proliferative effects of metformin than triple negative cells, and have described the differences of molecular mechanisms of metformin action by tumor subtypes. We hypothesized that metformin may be more effective against trastuzumab-resistant erbB2-overexpressing breast cancer cells because it targets the critical signaling pathways that are altered with resistance. BT474, SKBR3 and derived trastuzumab-resistant sublines BT474-HR20 (HR20) and SKBR3-pool2 (pool2) were used to test this hypothesis. Metformin treatment resulted in significantly more inhibition of proliferation and clonogenicity in resistant sublines. It decreased erbB2/insulin-like growth factor-1 receptor (IGF-1R) complexes (present only in the resistant sublines) without altering erbB2 expression, and reduced the expression and activity of erbB3 and IGF-1R in the trastuzumab-resistant but not parental cells. Trastuzumab-resistant sublines were resistant to rapamycin induced changes in mTOR activity and cell growth. In contrast, both BT474 and HR20 cells were highly sensitive to inhibitors of Src (Dasatinib) and PI-3K (LY294002). The pool2 cells showed higher sensitivity than SKBR3 cells to LY294002, but not Dasatinib. On the basis of these data, metformin appears to be significantly more effective against trastuzumab-resistant as compared to sensitive breast cancer cells. Metformin disrupts erbB2/IGF-1R complexes, erbB3 and IGF-1R expression and activity, as well as Src kinase and/or PI-3K/Akt signaling. This action appears to be independent of mTOR signaling. Our findings provide a rationale to study the effects of metformin on patients with erbB2 positive tumors treated with trastuzumab, with or without resistance.

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.

The relationship between the expression of mitochondrial voltage-dependent anion channels (VDACs) and the protective effects ofMyrica rubraSieb. Et Zucc fruit extract (MCE) against carbon tetrachloride (CCl4)-induced liver damage was investigated. Pretreatment with 50 mg kg−1, 150 mg kg−1or 450 mg kg−1MCE significantly blocked the CCl4-induced increase in both serum aspartate aminotransferase (sAST) and serum alanine aminotransferase (sALT) levels in mice (P< .05 or .01 versus CCl4group). Ultrastructural observations of decreased nuclear condensation, ameliorated mitochondrial fragmentation of the cristae and less lipid deposition by an electron microscope confirmed the hepatoprotection. The mitochondrial membrane potential dropped from −191.94 ± 8.84 mV to −132.06 ± 12.26 mV (P< .01) after the mice had been treated with CCl4. MCE attenuated CCl4-induced mitochondrial membrane potential dissipation in a dose-dependent manner. At a dose of 150 or 450 mg kg−1of MCE, the mitochondrial membrane potentials were restored (P< .05). Pretreatment with MCE also prevented the elevation of intra-mitochondrial free calcium as observed in the liver of the CCl4-insulted mice (P< .01 versus CCl4group). In addition, MCE treatment (50–450 mg kg−1) significantly increased both transcription and translation of VDAC inhibited by CCl4. The above data suggest that MCE mitigates the damage to liver mitochondria induced by CCl4, possibly through the regulation of mitochondrial VDAC, one of the most important proteins in the mitochondrial outer membrane.

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.

Poly (sodium 4-styrenesulfonate) (PSS) was grafted onto carbon black surface by ambient surface-initiated atom radical transfer polymerization. And the carbon paste electrodes modified by CB-g-PSS and MWNTs were completed. CB-g-PSS and MWNTs had no effect on direct electron transfer based on the carbon paste electrode, but increased the redox speed of Fe [(CN)(6)](3-) / Fe[(CN)(6)](4-) and led to stronger current response. The experimental results indicate that the sensitivity of modified by CB-g-PSS is 2.1 times of no modified, and MWNTs is 4 times. Measurable linear range is 1.1 mmol/L approximately 33.3 mmol/L. For sensors modified by CB-g-PSS, testing of different concentration glucose: RSD7%; sensors modified by MWNTs, RSD7.5% (glucose15 mmol/L), RSD10% (glucose15 mmol/L) respectively.

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.

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.

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

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.