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Reduced age-related terminal duct lobular unit (TDLU) involution has been linked to increased breast cancer risk and triple-negative breast cancer. Associations of TDLU involution levels with race and ethnicity remain incompletely explored. Herein, we examined the association between genetic ancestry and TDLU involution in normal breast tissue donated by 2014 healthy women in the United States. Women of African ancestry were more likely than European women to have increased TDLU counts (odds ratio [OR]trend = 1.36, 95% confidence interval [CI] = 1.07 to 1.74), acini counts per TDLU (OR = 1.47, 95% CI = 1.06 to 2.03), and median TDLU span (ORtrend = 1.44, 95% CI = 1.08 to 1.91), indicating lower involution, whereas East Asian descendants were associated with decreased TDLU counts (ORtrend = 0.52, 95% CI = 0.35 to 0.78) after controlling for potential confounders. These associations are consistent with the racial variations in incidence rates of triple-negative breast cancer in the United States and suggest opportunities for future work examining whether TDLU involution may mediate the racial differences in subtype-specific breast cancer risk.

Cell-type origin is one of the factors that determine molecular features of tumors, but resources to validate this concept are scarce because of technical difficulties in propagating major cell types of adult organs. Previous attempts to generate such resources to study breast cancer have yielded predominantly basal-type cell lines. We have created a panel of immortalized cell lines from core breast biopsies of ancestry-mapped healthy women that form ductal structures similar to normal breast in 3D cultures and expressed markers of major cell types, including the luminal-differentiated cell-enriched ERα–FOXA1–GATA3 transcription factor network. We have also created cell lines from PROCR (CD201)+/EpCAM− cells that are likely the “normal” counterpart of the claudin-low subtype of breast cancers. RNA-seq and PAM50-intrinsic subtype clustering identified these cell lines as the “normal” counterparts of luminal A, basal, and normal-like subtypes and validated via immunostaining with basal-enriched KRT14 and luminal-enriched KRT19. We further characterized these cell lines by flow cytometry for distribution patterns of stem/basal, luminal-progenitor, mature/differentiated, multipotent PROCR+ cells, and organogenesis-enriched epithelial/mesenchymal hybrid cells using CD44/CD24, CD49f/EpCAM, CD271/EpCAM, CD201/EpCAM, and ALDEFLUOR assays and E-cadherin/vimentin double staining. These cell lines showed interindividual heterogeneity in stemness/differentiation capabilities and baseline activity of signaling molecules such as NF-κB, AKT2, pERK, and BRD4. These resources can be used to test the emerging concept that genetic variations in regulatory regions contribute to widespread differences in gene expression in “normal” conditions among the general population and can delineate the impact of cell-type origin on tumor progression.Significance: In addition to providing a valuable resource for the breast cancer research community to investigate cell-type origin of different subtypes of breast cancer, this study highlights interindividual differences in normal breast, emphasizing the need to use "normal" cells from multiple sources as controls to decipher the effects of cancer-specific genomic aberrations. Cancer Res; 78(17); 5107–23. ©2018 AACR.

The effect of TGFβ1 on stem/differentiated cell associated cell surface marker expression.

Gene expression analyses of KTB cell lines and IPA analyses

PDF file, 109K, Gelsolin and Nm23-H1 overexpression in 4T1 and MDA-MB-231T cells is not associated with apoptosis.

PDF file, 92K, A cloned GFP tag on Gelsolin protein does not affect Gelsolin interaction with Nm23-H1 in 4T1 and MDA-MB-231T cells.

PDF file, 189K, Gelsolin effect on the cell motility of 4T1 and MDA-MB-231T cells is motility factor-dependent.

PDF file, 58K, Nm23-H1 binds the regulatory C-terminal domain of Gelsolin.

Nm23-H1 has been identified as a metastasis suppressor gene, but its protein interactions have yet to be understood with any mechanistic clarity. In this study, we evaluated the proteomic spectrum of interactions made by Nm23-H1 in 4T1 murine breast cancer cells derived from tissue culture, primary mammary tumors, and pulmonary metastases. By this approach, we identified the actin-severing protein Gelsolin as binding partner for Nm23-H1, verifying their interaction by coimmunoprecipitation in 4T1 cells as well as in human MCF7, MDA-MB-231T, and MDA-MB-435 breast cancer cells. In Gelsolin-transfected cells, coexpression of Nm23-H1 abrogated the actin-severing activity of Gelsolin. Conversely, actin severing by Gelsolin was abrogated by RNA interference–mediated silencing of endogenous Nm23-H1. Tumor cell motility was negatively affected in parallel with Gelsolin activity, suggesting that Nm23-H1 binding inactivated the actin-depolymerizing function of Gelsolin to inhibit cell motility. Using indirect immunoflourescence to monitor complexes formed by Gelsolin and Nm23-H1 in living cells, we observed their colocalization in a perinuclear cytoplasmic compartment that was associated with the presence of disrupted actin stress fibers. In vivo analyses revealed that Gelsolin overexpression increased the metastasis of orthotopically implanted 4T1 or tail vein–injected MDA-MB-231T cells (P = 0.001 and 0.04, respectively), along with the proportion of mice with diffuse liver metastases, an effect ablated by coexpression of Nm23-H1. We observed no variation in proliferation among lung metastases. Our findings suggest a new actin-based mechanism that can suppress tumor metastasis. Cancer Res; 73(19); 5949–62. ©2013 AACR.

PDF file, 86K.

PDF file, 33K, Nm23-H1 impairs Gelsolin- depolymerization activity of scratch-wounded cell layers.

Introduction: Breast tissue age, measured with DNA methylation-based estimates, is accelerated in healthy women compared with matched peripheral blood samples. While we have shown that the degree of acceleration in breast epigenetic age is related to hormonal factors including elevated body mass index and earlier age at menarche, no prior work has examined alterations in gene expression that accompany breast epigenetic aging. In this study, we examine gene expression patterns associated with epigenetic age acceleration in healthy breast using six epigenetic clocks. Methods: Using the AllPrep (Qiagen) DNA/RNA Kit, we extracted DNA and RNA from the same breast tissue specimens from 192 healthy women aged 19-90 years who donated breast tissue to the Susan G. Komen Tissue Bank at the Indiana University Simon Comprehensive Cancer Center. Transcriptome analysis was performed using the QuantSeq 3’MRNA-SeqFWD kit to generate RNA sequencing libraries. DNA methylation was quantified using the Illumina EPIC 850K array platform, and methylation age was estimated using beta-values from these experiments. Age acceleration is defined using the residuals of a linear regression of methylation age on chronologic age, and samples with positive residuals were characterized as exhibiting epigenetic age acceleration. Differential gene expression analysis was performed using DESeq2 bioconductor software to estimate variance-mean dependence in count data and using a negative binomial distribution model to test for differential gene expression in samples with accelerated epigenetic age in 6 epigenetic clocks: the Horvath pan-tissue clock, Hannum Age, Phenotypic Age, Grim Age, Skin and Blood clock, and the Epigenetic Pacemaker clock (EPM) , a newly developed clock that accounts for curvilinearity in methylation-based estimates of age over time. Results: Principal components analysis revealed that a large proportion of the variance in gene expression was explained by the first two components (PC1 8.6%, PC2 8.2%). Using a false discovery rate cutoff of 0.05, we found 17 (pan-tissue), 909 (Hannum), 3573 (Phenotypic), 1459 (Grim), 3081 (Skin and Blood), and 227 (EPM) genes differentially expressed with the presence of epigenetic age acceleration for each clock. Examining overlap of these genes revealed distinct groups of genes differentially expressed with acceleration in each clock, with no genes in common for all clocks, Phenotypic and Skin and Blood clocks sharing 1137 genes; Phenotypic, Grim, and Skin and Blood clocks sharing 726 genes; and few genes shared by 5 out of 6 clocks (41 genes in all clocks except pan-tissue, 5 genes in all clocks except EPM). Gene set enrichment analysis revealed activation of pathways involved in cellular respiration, oxidative phosphorylation, energy derivation, organic acid metabolic processes, and immune response, and suppression of genes involved in embryonic epithelial morphogenesis, epidermal cell differentiation, chromatin organization, and lipoprotein transport. Conclusion: We identified significant alterations in gene expression patterns that accompany breast epigenetic acceleration in healthy female breast, with distinct patterns observed for six epigenetic clocks examined. Pathways involved include epithelial morphogenesis and differentiation, potentially representing a mechanistic link between breast accelerated aging and carcinogenesis. Citation Format: Mary Elizabeth Sehl, Wenbin Guo, Colin Farrell, Natascia Marino, Jill E. Henry, Anna Maria Storniolo, Jeannette Papp, Jingyi Jessica Li, Matteo Pellegrini, Steve Horvath, Patricia A. Ganz. Differential gene expression patterns in healthy breast tissue exhibiting epigenetic age acceleration [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-11-06.

Background: Understanding how hereditary, lifestyle and demographic risk factors influence the development of breast cancer is critical for its prevention and treatment. For this purpose, the Susan G. Komen Tissue Bank at the IU Simon Comprehensive Cancer Center (KTB) serves as the world’s primary repository for normal breast tissue and blood samples from healthy women (~5,500 tissue + blood donors and ~4,500 blood-only donors). In addition, KTB has received mammograms, H&E images and additional longitudinal health and wellness data from its donors via ongoing annual surveys. Whole genome sequencing has also recently been completed on a subset of 500 donors, including ~125 initially healthy donors who later went on to develop breast cancer. To make these disparate, multi-modal data accessible to the larger research community and add advanced precision health analytics capabilities to the KTB database, LifeOmic's Precision Health Cloud (PHC), a secure healthcare-compliant cloud platform, was chosen to host the KTB data moving forward. The PHC provides browsing and querying capabilities across all data including genomic, demographic, histology, donor surveys, and scanned images. Data from donors can be viewed individually as well as analyzed across cohorts of interest. Computation resources, such as informatics workflows and machine learning infrastructure, are available on the same secure environment to leverage the power of cloud computing without having to download large files. This is in addition to visual and custom analytics using Jupyter Notebooks accessible to collaborators. Methods: We loaded all available KTB data from ~10,000 donors into the PHC. Donor-reported data collected at the time of donation and subsequent follow-up questionnaire responses were loaded as FHIR Observations and Medications in the PHC. Additional imaging and genomic data loaded into the PHC include approximately 12,000 mammogram images, 5,000 H&E images, 5,000 ancestry-informative genotypes and 500 VCF and BAM files from whole genome sequencing. A timeline view of each donor shows data collected from each donation and follow-up surveys. All genetic variants were annotated upon ingestion into the PHC with functional effect on genes, population allele frequency, ClinVar clinical significance and in silico predictions for functional impact. Results: To demonstrate the utility of KTB data hosted in the PHC, we will show how the PHC can be used to directly analyze genomic and donor-reported data, specifically focusing on the recent whole genome sequencing data from 500 donors. Whole genome sequencing data will be used to calculate the genetic ancestry of each donor compared to their self-reported ancestry and previously predicted ancestry from a 41-SNP ancestry panel. Additionally, we will calculate genomewide breast cancer polygenic risk scores and compare these with the survey-based Tyrer-Cuzick and Gail score risk measures. We will assess how the polygenic risk score further stratifies the donors that have germline pathogenic variants in a known breast cancer gene. For the ~125 sequenced donors that went on to develop breast cancer we will also investigate correlations between family history of cancer, age of diagnosis, prior gynecological history and lifestyle differences. All generated results will remain in the PHC for access by future KTB collaborators to continue to build the utility and value of the KTB data. Conclusions: We have shown how hosting KTB data in the PHC opens new opportunities for advanced precision health research for breast cancer researchers worldwide. As more specimens and diverse data sets including genomic, longitudinal and imaging information are being tracked and deposited into KTB, this resource will continue to grow to enable diverse research needs. Citation Format: Steven M Bray, Swee Seong Wong, Baiju G Parikh, Kevin C Wood, Samuel T Rysdyk, Jill E Henry, Natascia Marino, Milan Radovich, Anna Maria V Storniolo. Advanced precision health resources in the Susan G Komen tissue bank at the IU simon comprehensive cancer center [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-14.

Introduction: Risk factors for breast cancer include advancing age, lifetime estrogen exposure, and breast density. DNA methylation-based estimates of age are elevated in breast tissue of healthy women compared with paired blood samples, and the degree of age acceleration is associated with lifetime estrogen exposure. However, no prior work has examined cell compositional changes associated with breast epigenetic aging. In this study we estimated the abundance of different cell types in healthy breast, computed using gene expression data, and investigate cell composition changes that accompany advancing chronologic age and breast epigenetic age acceleration. Methods: DNA/RNA were extracted (AllPrep, Qiagen) from breast tissue specimens from 192 healthy women aged 19-90 years who donated breast tissue to the Susan G. Komen Tissue Bank at the Indiana University Simon Comprehensive Cancer Center. Transcriptome analysis was performed using the QuantSeq 3’mRNA-SeqFWD kit to generate RNA sequencing libraries. DNA methylation age was estimated using beta-values from Illumina EPIC 850K array platform. Age acceleration is defined using the residual of a linear regression of methylation age on chronologic age. Cell deconvolution was performed using CIBERSORTx to estimate the abundance of adipocytes, luminal epithelial cells, basal myoepithelial cells, vascular endothelial cells, lymphatic endothelial cells, immune cells (dendritic cells & macrophages), pericytes & smooth muscle cells, and fibroblasts. We examined cell composition changes with chronologic age, and with age-adjusted measures of acceleration in 6 epigenetic clocks: the Horvath Pan-tissue, Hannum, Phenotypic, Grim, Skin & Blood, and Epigenetic Pacemaker clocks, as well as the DNA methylation-based estimate of telomere length, DNAmTL. Results: Advancing chronologic age was associated with an increase in the imputed proportion of adipocytes (R=0.40, p< 0.0001), vascular endothelial cells (R=0.23, p=0.0033), and immune cells (dendritic cells & macrophages) (R=0.29, p=0.00016), and a decrease in the proportion of luminal epithelial cells (R=-0.43, p< 0.0001), and basal myoepithelial cells (R=-0.27, p=0.00047). Epigenetic age acceleration was significantly associated with increases in proportions of luminal epithelial cells (p< 0.0001 for age-adjusted Hannum, Phenotypic, Grim, and Skin & Blood clocks, p< 0.05 for Pan-tissue) and basal myoepithelial cells (p< 0.0001 for Phenotypic and Skin & Blood clocks), and decreased proportions of adipocytes and vascular endothelial cells (p< 0.0001 for Hannum, Phenotypic, Grim, and Skin & Blood clocks, p< 0.05 for Pan-tissue). Conclusion: Using gene expression data in healthy female breast tissue, we identified significant changes in cell-type-specific abundance that accompany advancing chronologic age, with an increase in adipocytes and immune cells, and a decline in luminal epithelial cells and basal myoepithelial cells. By contrast, epigenetic age acceleration in breast tissue is associated with decreasing proportions of adipocytes and vascular endothelial cells and a rise in basal myoepithelial cells and luminal epithelial cells. Our findings suggest distinct patterns of cellular composition changes that accompany normal aging compared with accelerated aging in breast tissue. Citation Format: Mary E. Sehl, Wenbin Guo, Collin Farrell, Natascia Marino, Jill Henry, Anna Maria Storniolo, Jeanette Papp, Jingyi Li, Steve Horvath, Matteo Pellegrini, Patricia A. Ganz. Cell composition changes in healthy breast tissue is associated with advancing age and epigenetic age acceleration [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-03-09.

Establishing commensal dysbiosis, defined as an inflammatory gut microbiome with low biodiversity, prior to breast tumor initiation, enhances early dissemination of hormone-receptor positive (HR+) mammary tumor cells. Here, we sought to define mammary tissue mediators of dysbiosis-induced tumor dissemination. We found that commensal dysbiosis increased both the frequency and profibrogenicity of mast cells in the mammary tissue, a phenotypic change that persisted after tumor implantation. Fibroblast activation and tissue remodeling associate with enhanced breast tumor metastasis. We employed pharmacological and adoptive transfer approaches to demonstrate that mammary tissue mast cells from dysbiotic animals enhances dissemination of HR+ tumor cells. Collagen levels in mammary tissues from HR+ breast cancer patients correlated with mast cell abundance, suggesting clinical relevance of mast cell-mediated fibroblast activation. Together, these data demonstrate that a gut-mast cell axis exists that induces fibroblast activation and orchestrates early dissemination of HR+ breast tumors.SignificanceOur study defines the mechanism by which an inflammatory gut microbiome facilitates HR+ breast tumor cell dissemination. We establish that gut commensal dysbiosis triggers mammary tissue mast cells to facilitate early metastatic dissemination. These findings highlight a novel gut microbiome-innate immune cell axis involved in negative breast cancer outcomes.

BackgroundGenome-wide association studies have identified several breast cancer susceptibility loci. However, biomarkers for risk assessment are still missing. Here, we investigated cancer-related molecular changes detected in tissues from women at high risk for breast cancer prior to disease manifestation. Disease-free breast tissue cores donated by healthy women (N=146, median age=39 years) were processed for both methylome (MethylCap) and transcriptome (Illumina’s HiSeq4000) sequencing. Analysis of tissue microarray and primary breast epithelial cells was used to confirm gene expression dysregulation.ResultsTranscriptomic analysis identified 69 differentially expressed genes between women at either high and those at average risk of breast cancer (Tyrer-Cuzick model) at FDRppr≤0.5). Finally, among the participants, 35 women donated breast biopsies at two time points, and age-related molecular alterations enhanced in high-risk subjects were identified.ConclusionsNormal breast tissue from women at high risk of breast cancer bears molecular aberrations that may contribute to breast cancer susceptibility. This study is the first molecular characterization of the true normal breast tissues and provides an opportunity to investigate molecular markers of breast cancer risk, which may lead to new preventive approaches.

Background Genome-wide association studies have identified several breast cancer susceptibility loci. However, biomarkers for risk assessment are still missing. Here, we investigated cancer-related molecular changes detected in tissues from women at high risk for breast cancer prior to disease manifestation. Disease-free breast tissue cores donated by healthy women (N = 146, median age = 39 years) were processed for both methylome (MethylCap) and transcriptome (Illumina’s HiSeq4000) sequencing. Analysis of tissue microarray and primary breast epithelial cells was used to confirm gene expression dysregulation. Results Transcriptomic analysis identified 69 differentially expressed genes between women at high and those at average risk of breast cancer (Tyrer-Cuzick model) at FDR p p r ≤ 0.5). Finally, among the participants, 35 women donated breast biopsies at two time points, and age-related molecular alterations enhanced in high-risk subjects were identified. Conclusions Normal breast tissue from women at high risk of breast cancer bears molecular aberrations that may contribute to breast cancer susceptibility. This study is the first molecular characterization of the true normal breast tissues, and provides an opportunity to investigate molecular markers of breast cancer risk, which may lead to new preventive approaches.

Background: Despite advances in breast cancer diagnosis and treatment, there is a critical need to identify the determinants of breast cancer susceptibility in order to define new strategies to target cancer initiation and progression. The Susan G. Komen Tissue Bank at Indiana University (KTB), the only repository worldwide of non-malignant breast samples, can address key scientific questions by examining molecular changes, including epigenetic (DNA methylation) and transcriptomic alterations that occur in breasts of cancer-free women who are considered at high risk for developing breast cancer. Methods: Disease-free breast tissue cores donated by 80 high-risk (Tyrer-Cuzick lifetime risk ≥20%) and 80 average-risk women were processed for whole methylome (Diagenode MeCap Seq Library and NextSeq 75_sequencing) and whole transcriptome (Illumina TruSeq Stranded mRNA HT Library followed by NextSeq 75_sequencing) profiling. The two groups were matched according to age, racial background, and menopausal status. The cohort included 131 pre-menopausal and 29 post-menopausal women (mean age 38.6, SD 12.1). Frozen breast tissue cores with either abundant or moderate epithelial cellularity were selected. For the gene expression analysis, the reads were mapped to the human genome reference, GRCh38.p12 using the alignment software STAR ver. 2.5.2b and the read counts per gene were obtained using featureCounts ver. 1.6.3. For the evaluation of the epigenetically regulated genes, we focused our investigation on two types of functionally relevant epigenetic panels: the first panel includes 666 cancer-related markers (or pan-cancer) described by Shegafinia et al 2018, and the second panel includes 16 epigenetic markers identified in normal breast tissues-derived cells as connected with breast epithelial cell differentiation*. Results: In a preliminary analysis, we searched for transcriptomic differences in the breast tissues of high-risk women (N=43) as compared with that of matched average-risk subjects (N=48).The differential expression analysis identified 126 differentially expressed genes (DEGs, FDR2), including 117 upregulated and 9 downregulated in the high-risk group. Interestingly, pathway analysis (Ingenuity Pathway Analysis, IPA v06_01) showed that most of these genes are involved with cancer. The sub-category “tumorigenesis of tissues” as defined by IPA included 71 genes. Through TCGA database analysis, using the UALCAN portal, we searched among our candidate genes for those targets that showed both overexpression and hypomethylation in breast tumors as compared with the normal breast, or vice versa, downregulation and hypermethylation in breast cancer. This pipeline allowed us to identify 17 genes potentially epigenetically regulated and involved in breast cancer susceptibility and the early phases of cancer development. Among those, two genes, FAM83A and PLA2G3, belong to a family of genes previously described as epigenetically dysregulated in multiple cancers (TCGA dataset, and Shegafinia et al 2018) and transform breast epithelial cell in vitro (FAM83A), thus reaffirming robustness of our approach in identifying genes associated with breast cancer risk Conclusion: The discovery of epigenetically regulated genes associated with breast cancer risk will open the doors to a deeper understanding of the process of cancer initiation and progression. The present study identified 17 gene alterations, two of which are epigenetically regulated in cancer. Our findings highlight an opportunity to address molecular alterations potentially linked with breast cancer susceptibility and risk, using a unique resource of normal breast samples. *Unpublished data by Dr. H. Nakshatri Citation Format: Natascia Marino, Rana German, Harikrishna Nakshatri, Ashley Vode, Jun Liu, Jie Huang, Douglas B Rush, Sha Cao, Anna Maria V Storniolo. Molecular landscape of the breasts of women at high risk for breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-08-16.

Background: In several epidemiologic studies, the oral anti-diabetic medication metformin is associated with lower cancer incidence and diminished tumor progression. The molecular mechanism of metformin anti-cancer activity remains unclear, particularly in the preventive setting. This study investigated the effect of metformin on inflammatory and proliferative pathways in cancer-free breast tissue of overweight and obese women at high risk for breast cancer, in an effort to better understand how we might prevent obesity- related carcinogenesis in the breast. Methods: The study cohort included 24 obese or overweight (BMI ≥ 25), postmenopausal women at increased risk for breast cancer based on family history, risk models indicating lifetime risk ≥ 20%, atypical hyperplasia, or history of DCIS or breast cancer treated over 5 years ago. Subjects were randomized to metformin (850 mg BID) or placebo for 12 months. Breast tissue core biopsy and blood collection were performed at baseline and after 12 months. Reverse Phase Protein Array (MD Anderson RPPA Core Facility) was used to determine the proteomic profiling of the breast tissue biopsies (5 replicates for each subject) at both time points. 297 unique antibodies were analyzed by Array-Pro Analyzer 6.3 then by SuperCurve_1.5.0 via SuperCurveGUI_2.1.1. All relative protein level data points were normalized for protein loading and transformed to linear values. Serum biomarkers including IGF-I, IGF-II, Leptin, IL6, IL10, adiponectin, leptin, and IGFBP3 were measured using commercial enzyme-linked immunosorbent assays (ELISA). Statistical analysis was performed using R platform. Wilcox and T tests are used to check the difference for each protein between the two time points in each group. P values below 0.05 were deemed significant. Results: Of 24 women randomized, 18 completed the study, including 9 treated with metformin and 9 treated with placebo. Participants receiving metformin showed no significant changes in the measured serum biomarkers between pre- and post-treatment, or in comparison with the placebo group. Proteomic analysis revealed a change in both protein level as well as protein phosphorylation status in the metformin-treated group. Metformin treatment induced downregulation of regulators of cell proliferation and survival (Ets1, P=0.02; Abl1, P=0.04; Smad3, P=0.01; 14-3-3β, P=0.02; TG2, P=0.03, Beclin1, P=0.04; pT1135-RICTOR, P=0.03) in breast tissue. Several of the downregulated targets belong to two major pathways: mTOR and TGFβ. Increase in proteins involved in metabolic and AMPK pathways (PDHK1, P=0.007; pS79-ACC, P=0.02; Mnk1, P=0.02) was detected in the metformin-treated group post- treatment; however, no change was detected in the placebo-treated group. Conclusion: Our preliminary data suggest that metformin treatment has a direct effect on breast tissue of overweight or obese, postmenopausal woman at increased risk for breast cancer by reducing pro-survival pathways, such as mTOR and TGFβ, and activating metabolic pathways, such as AMPK. Further investigation of the specific targets activated or inhibited in the cancer-free breast tissue by metformin is being performed to broaden our understanding of the protective mechanism of this antidiabetic agent. Citation Format: Tarah Ballinger, Natascia Marino, Rana German, Jason True, Xiaoyu Lu, Sha Cao, Anna Maria Storniolo, Lida Mina. Prospective, placebo-controlled, randomized study of metformin for breast cancer prevention in overweight/ obese women [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-13-03.

Obesity is a risk factor for postmenopausal estrogen receptor alpha (ERα)-positive (ER+) breast cancer. Molecular mechanisms underlying factors from plasma that contribute to this risk and how these mechanisms affect ERα signaling have yet to be elucidated. To identify such mechanisms, we performed whole metabolite and protein profiling in plasma samples from women at high risk for breast cancer, which led us to focus on factors that were differentially present in plasma of obese versus nonobese postmenopausal women. These studies, combined with in vitro assays, identified free fatty acids (FFA) as circulating plasma factors that correlated with increased proliferation and aggressiveness in ER+ breast cancer cells. FFAs activated both the ERα and mTOR pathways and rewired metabolism in breast cancer cells. Pathway preferential estrogen-1 (PaPE-1), which targets ERα and mTOR signaling, was able to block changes induced by FFA and was more effective in the presence of FFA. Collectively, these data suggest a role for obesity-associated gene and metabolic rewiring in providing new targetable vulnerabilities for ER+ breast cancer in postmenopausal women. Furthermore, they provide a basis for preclinical and clinical trials where the impact of agents that target ERα and mTOR signaling cross-talk would be tested to prevent ER+ breast cancers in obese postmenopausal women.Significance:These findings show that obesity-associated changes in certain blood metabolites rewire metabolic programs in cancer cells, influence mammary epithelial cell tumorigenicity and aggressiveness, and increase breast cancer risk.

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca(2+) oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca(2+)-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Histologically normal tissue adjacent to the tumor can provide insight of the microenvironmental alterations surrounding the cancerous lesion and affecting the progression of the disease. However, little is known about the molecular changes governing cancer initiation in cancer-free breast tissue. Here, we employed laser microdissection and whole-transcriptome profiling of the breast epithelium prior to and post tumor diagnosis to identify the earliest alterations in breast carcinogenesis. Furthermore, a comprehensive analysis of the three tissue compartments (microdissected epithelium, stroma, and adipose tissue) was performed on the breast donated by either healthy subjects or women prior to the clinical manifestation of cancer (labeled “susceptible normal tissue”). Although both susceptible and healthy breast tissues appeared histologically normal, the susceptible breast epithelium displayed a significant upregulation of genes involved in fatty acid uptake/transport (CD36 and AQP7), lipolysis (LIPE), and lipid peroxidation (AKR1C1). Upregulation of lipid metabolism- and fatty acid transport-related genes was observed also in the microdissected susceptible stromal and adipose tissue compartments, respectively, when compared with the matched healthy controls. Moreover, inter-compartmental co-expression analysis showed increased epithelium-adipose tissue crosstalk in the susceptible breasts as compared with healthy controls. Interestingly, reductions in natural killer (NK)-related gene signature and CD45+/CD20+ cell staining were also observed in the stromal compartment of susceptible breasts. Our study yields new insights into the cancer initiation process in the breast. The data suggest that in the early phase of cancer development, metabolic activation of the breast, together with increased epithelium-adipose tissue crosstalk may create a favorable environment for final cell transformation, proliferation, and survival.

A mother's ability to produce a nutritionally-complete neonatal food source has provided a powerful evolutionary advantage to mammals. Milk production by mammary epithelial cells is adaptive, its release is exquisitely-timed and its own glandular stagnation with the permanent cessation of suckling triggers the cell death and tissue remodeling that enables female mammals to nurse successive progeny. Both chemical and mechanical signals play a role in this process. Despite this duality of input, however, much remains unknown about the nature and function of mechanical forces in this organ. Here, we characterize the force landscape in the functionally-mature gland and the capacity of luminal and basal cells to experience and exert force. We explore molecular instruments for force-sensing, in particular channel-mediated mechanotransduction, revealing increased expression of Piezo1 in mammary tissue in lactation and confirming functional expression in luminal cells. We also reveal, however, that lactation and involution proceed normally in mice with luminal-specific Piezo1 deletion. These findings support a multifaceted system of chemical and mechanical sensing in the mammary gland, and a protective redundancy that ensures continued lactational competence and offspring survival.

// Shatovisha Dey 1 , Natascia Marino 2, 3 , Kanokwan Bishop 1 , Paige N. Dahlgren 1 , Aditi Shendre 4 , Anna Maria Storniolo 2, 3 , Chunyan He 5 and Hiromi Tanaka 1 1 Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA 2 Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA 3 Susan G. Komen Tissue Bank at IU Simon Cancer Center, Indianapolis, IN, USA 4 Department of Epidemiology, Richard M. Fairbanks School of Public Health, Indiana University, Indianapolis, IN, USA 5 Department of Internal Medicine, College of Medicine, Markey Cancer Center, University of Kentucky, Lexington, KY, USA Correspondence to: Hiromi Tanaka, email: hirtanak@iu.edu Keywords: BRCA1; BRCA2; telomere; circulating cell-free DNA; qPCR Received: November 03, 2017 Accepted: December 21, 2017 Published: December 29, 2017 ABSTRACT Plasma cell-free DNA (cfDNA) is a small DNA fragment circulating in the bloodstream originating from both non-tumor- and tumor-derived cells. A previous study showed that a plasma telomeric cfDNA level decreases in sporadic breast cancer patients compared to controls. Tumor suppressor gene products including BRCA1 and BRCA2 (BRCA1&2) play an important role in telomere maintenance. In this study, we hypothesized that the plasma telomeric cfDNA level is associated with the mutation status of BRCA1&2 genes. To test this hypothesis, we performed plasma telomeric cfDNA quantitative PCR (qPCR)-based assays to compare 28 women carriers of the BRCA1&2 mutation with age-matched controls of 28 healthy women. The results showed that the plasma telomeric cfDNA level was lower in unaffected BRCA1&2 mutation carriers than in age-matched controls from non-obese women (BMI 30). Moreover, the plasma telomeric cfDNA level applied aptly to the Tyrer-Cuzick model in non-obese women. These findings suggest that circulating cfDNA may detect dysfunctional telomeres derived from cells with BRCA1&2 mutations and, therefore, its level is associated with breast cancer susceptibility. This pilot study warrants further investigation to elucidate the implication of plasma telomeric cfDNA levels in relation to cancer and obesity.

Breast cancer is the second leading cause of cancer mortality among women. Mammography and tumor biopsy followed by histopathological analysis are the current methods to diagnose breast cancer. Mammography does not detect all breast tumor subtypes, especially those that arise in younger women or women with dense breast tissue, and are more aggressive. There is an urgent need to find circulating prognostic molecules and liquid biopsy methods for breast cancer diagnosis and reducing the mortality rate. In this study, we systematically evaluated metabolites and proteins in blood to develop a pipeline to identify potential circulating biomarkers for breast cancer risk. Our aim is to identify a group of molecules to be used in the design of portable and low-cost biomarker detection devices. We obtained plasma samples from women who are cancer free (healthy) and women who were cancer free at the time of blood collection but developed breast cancer later (susceptible). We extracted potential prognostic biomarkers for breast cancer risk from plasma metabolomics and proteomics data using statistical and discriminative power analyses. We pre-processed the data to ensure the quality of subsequent analyses, and used two main feature selection methods to determine the importance of each molecule. After further feature elimination based on pairwise dependencies, we measured the performance of logistic regression classifier on the remaining molecules and compared their biological relevance. We identified six signatures that predicted breast cancer risk with different specificity and selectivity. The best performing signature had 13 factors. We validated the difference in level of one of the biomarkers, SCF/KITLG, in plasma from healthy and susceptible individuals. These biomarkers will be used to develop low-cost liquid biopsy methods toward early identification of breast cancer risk and hence decreased mortality. Our findings provide the knowledge basis needed to proceed in this direction.

The mammary epithelium is indispensable for the continued survival of more than 5000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate milk ejection. Using quantitative, multidimensional imaging of mammary cell ensembles, we reveal how stimulus evoked Ca2+oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive displacement pumps, each linked to a contractile ductal network, milk is delivered into the mouth of the dependent neonate, seconds after the command.Significance StatementThe mammary gland is functional for only a brief period of a female’s lifetime—if at all. During this time, it operates not for the survival of the individual, but for the survival of her species. Here, we visualize the nature of alveolar contractions in the functionally-mature mammary gland, revealing how specialized epithelial cells, which possess the ability to behave like smooth muscle cells, undergo Ca2+-dependent contractions. We demonstrate that individual oscillators can be electrically coupled to achieve global synchrony, a phenomenon that has not yet been observed in the mammary gland. By imaging activity across scales, we provide a window into the organization, dynamics and role of epithelial Ca2+oscillations in the organ principally responsible for sustaining neonatal life in mammals.

A mother’s ability to produce a nutritionally-complete neonatal food source has provided a powerful evolutionary advantage to mammals. Milk production by secretory mammary epithelial cells is adaptive, its release is exquisitely timed and its own glandular stagnation with the permanent cessation of suckling triggers the programmed cell death and tissue remodeling that enables female mammals to nurse successive progeny. Both chemical and mechanical signals control epithelial expansion, function and remodeling. Despite this duality of input, however, the nature and function of mechanical forces in the mammary gland remain unknown. Here, we characterize the mammary force landscape and the capacity of luminal and basal epithelial cells to experience and exert force. We explore the molecular instruments for force-sensing in the mammary gland and the physiological requirement for PIEZO1 in lactation and involution. Our study supports the existence of a multifaceted system of chemical and mechanical sensing in the mammary gland, and a protective redundancy that ensures continued lactational competence and offspring survival.

1560 Background: Epigenetic mechanisms such as DNA methylation are important regulators of gene expression and are frequently dysregulated early in breast carcinogenesis. The relationship between DNA methylation aberrations in normal breast tissue and breast cancer risk remains unclear. Methods: Disease-free breast tissue cores donated by 71 high-risk (Tyrer-Cuzick lifetime risk ≥20%) and 79 average-risk women were obtained from the Komen Tissue Bank and processed for whole methylome (Diagenode's MethylCap Library and single-end 75-bp sequencing on Illumina Nextseq) and whole transcriptome (Illumina Nextseq) profiling. Reads from RNA-seq data were aligned to the human genome reference, GRCh38.p12 using STAR v.2.5.2b and tested for differential gene expression using DESeq2 ver. 1.24.0. For DNA methylation data, difference of variation in deduplicated read coverage among 250-bp fixed sized bins spanning CpG islands between high- and average-risk libraries was computed as z-ratios to identify differentially methylated regions. Pathway analysis was performed using IPA v06_01. Results: We identified 1355 CpGs that were differentially methylated between high- and average-risk breast tissues (ΔZ > 0.5, FDR < 0.05). Hypomethylated CpGs were overrepresented in high-risk tissue and were found predominantly (68%) in non-coding regions. Hypermethylated CpG sites were found equally in the gene body and non-coding regions. Transcriptomic analysis identified 112 differentially expressed genes (fold change≥2, FDR < 0.05), involved in chemokines signaling, metabolism and estrogen biosynthesis. Among those, FAM83A (logfc = 2.3, FDR = 0.004) was previously described as epigenetically dysregulated in multiple cancers and transforms breast epithelial cell in vitro. Methylation-expression correlations revealed 11 epigenetically regulated genes including cellular transformation-associated BMPR1B. Two hypomethylated/upregulated long non-coding RNAs were also identified in high-risk breasts. Conclusions: This is the first gene expression/DNA methylation analysis of normal breasts from women at either high or average risk of breast cancer. Our discovery of epigenetically regulated genes associated with breast cancer risk provides an opportunity to mechanistically dissect breast cancer susceptibility and risk-associated molecular alterations. Unlike the current focus of identifying germline mutations or single nucleotide polymorphisms responsible for higher risk, our studies reveal an epigenetic mechanism, which is not discernable through simple genomic sequencing.

Introduction: Breast cancer is the most prevalent type of cancer among women (other than some nonmelanoma skin cancers), with approximately one in eight women diagnosed with breast cancer during their lifetime. The mammary tissue microbiome has recently been characterized, and shifts in the abundance of mammary tissue bacterial taxa have been associated with benign and malignant breast tumors. This suggests particular bacterial taxa as oncogenic and key in driving breast cancer development. However, these studies utilized mammary tissue obtained from breast reduction or enhancement surgery as healthy control tissue, which has significant histologic abnormalities compared to tissue from normal healthy donors. The Susan G. Komen Tissue Bank (KTB) at the Indiana University Simon Cancer Center (IUSCC) is a unique and precious repository of normal breast tissue, ideal for compositional and functional analyses of the mammary tissue microbiome. We have also identified a subset of women who donated healthy tissue to the KTB and later developed breast cancer. This provides us with a rare opportunity to study shifts in the mammary microbiome composition that may preclude breast cancer development. Our research objective is to compare the composition of the mammary microbiome in healthy and prediagnostic tissue to that of cancerous tissue. Methods: Our cohort comprises four tissue subsets: healthy (n = 50), prediagnostic (n = 15), adjacent normal (n = 50), and tumor (n = 50) selected from the KTB and IUSCC Tissue Banks. DNA was isolated from all 165 tissue samples. DNA from three of these samples, two positive controls, and two negative controls was submitted for Illumina Miseq paired-end sequencing of the V4 region of the 16S gene at the University of California, Davis Host Microbe Systems Core Lab. Results/Conclusions: The breast tissue samples display microbiome compositions dominated by three phyla: Proteobacteria, Firmicutes, and Bacteroidetes. Negative controls displayed fewer than 10 reads and positive controls yielded accurate percentages of several difficult-to-lyse bacteria. In January 2020, all 165 DNA samples will be submitted for 16S rRNA metagenomic sequencing for analysis of the microbiome composition among the four tissue subsets. We will use multivariate linear modeling to adjust for clinical factors such as age, BMI, and menopausal status and to determine if microbiome composition is modified by cancer status. This work will be fundamental in characterizing the composition of the healthy mammary tissue microbiome and identifying microbial determinants of breast cancer. Citation Format: Jaelyn Gabel, Courtney Hoskinson, Annie Kump, Karin B. Michels, Natascia Marino, Leah T. Stiemsma. The mammary tissue microbiome in breast cancer development [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B23.

In addition to the accumulation of pro-oncogenic mutations in the epithelial cells, the tumorigenic process involves the dysregulation of the interactions between the epithelial cells and their microenvironment, as well as alterations within the microenvironment itself. The latter is composed of endothelial cells, immune cells, fibroblasts, unaffected epithelium, and adipocytes. Increasing evidences support that cell transformation progresses in a potentially cancer-promoting microenvironment context, also known as field cancerization model. Therefore, it is critical to investigate changes occurring in both the epithelial compartment and the surrounding microenvironment to understand how breast cancer initiates. In our preliminary study, we analyzed the transcriptome profile of microdissected breast tissue compartments (epithelium, stroma and adipose tissue) from tissue biopsies obtained from women who donated their histologically normal tissue two-to-five years before breast cancer diagnosis (here labeled pre-cancer), and matched healthy control donors. In the pre-cancer breast epithelium we detected a significant increase in lipid metabolism-related genes including lipases (HSL, LPL) and perilipins (PLIN1, PLIN4), which mediate the release of fatty acids from triacylglycerol storage, and ELOVL5 and ELOVL7, which generate oleic acid. Moreover, Acyl-CoA Synthetase Medium Chain Family Member 1, ACSM1, involved in the activation of lipoic acid, an essential cofactor for mitochondrial metabolism, is also upregulated in pre-cancer breast tissue. Upregulation of genes involved in metabolic activation is observed also in the stroma and adipose tissue compartments. Interestingly, the transcriptome profiling of the pre-cancer breast stroma shows the downregulation of genes coding for several immune cell markers as compared with the healthy controls. Immunohistochemical staining of CD45 confirms a significant reduction in immune cells in the pre-cancer versus matched healthy control breasts. This finding suggests the prevalence of an immune-suppressive environment in the breast long before the clinical diagnosis of breast cancer. Understanding the changes occurring in the cancerized field is critical for elucidating 1) the involvement of microenvironment in cancer initiation 2) the role of histologically normal yet genetically altered surgical margins in risk of disease recurrence upon lumpectomy for early breast cancer diagnosis, and 3) the mechanisms of field cancerization in the contralateral breast. Citation Format: Natascia Marino, Rana German, Xi Rao, George Sandusky, Max Jacobsen, Sha Cao, Anna Maria Storniolo. Metabolic reprogramming of the breast contributes to a cancer promoting milieu [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 4641.

Despite important progress in adjuvant and neoadjuvant therapies, metastatic disease often develops in breast cancer patients and remains the leading cause of their deaths. For patients with established metastatic disease, therapy is palliative, with few breaks and with mounting adverse effects. Many have hypothesized that a personalized or precision approach (the terms are used interchangeably) to cancer therapy, in which treatment is based on the individual characteristics of each patient, will provide better outcomes. Here, we discuss the molecular basis of breast cancer metastasis and the challenges in personalization of treatment. The instability of metastatic tumors remains a leading obstacle to personalization, because information from a patient's primary tumor may not accurately reflect the metastasis, and one metastasis may vary from another. Furthermore, the variable presence of tumor subpopulations, such as stem cells and dormant cells, may increase the complexity of the targeted treatments needed. Although molecular signatures and circulating biomarkers have been identified in breast cancer, there is lack of validated predictive molecular markers to optimize treatment choices for either prevention or treatment of metastatic disease. Finally, to maximize the information that can be obtained, increased attention to clinical trial design in the metastasis preventive setting is needed.

Nm23-H1 has been identified as a metastasis suppressor gene, but its protein interactions have yet to be understood with any mechanistic clarity. In this study, we evaluated the proteomic spectrum of interactions made by Nm23-H1 in 4T1 murine breast cancer cells derived from tissue culture, primary mammary tumors, and pulmonary metastases. By this approach, we identified the actin-severing protein Gelsolin as binding partner for Nm23-H1, verifying their interaction by coimmunoprecipitation in 4T1 cells as well as in human MCF7, MDA-MB-231T, and MDA-MB-435 breast cancer cells. In Gelsolin-transfected cells, coexpression of Nm23-H1 abrogated the actin-severing activity of Gelsolin. Conversely, actin severing by Gelsolin was abrogated by RNA interference–mediated silencing of endogenous Nm23-H1. Tumor cell motility was negatively affected in parallel with Gelsolin activity, suggesting that Nm23-H1 binding inactivated the actin-depolymerizing function of Gelsolin to inhibit cell motility. Using indirect immunoflourescence to monitor complexes formed by Gelsolin and Nm23-H1 in living cells, we observed their colocalization in a perinuclear cytoplasmic compartment that was associated with the presence of disrupted actin stress fibers. In vivo analyses revealed that Gelsolin overexpression increased the metastasis of orthotopically implanted 4T1 or tail vein–injected MDA-MB-231T cells (P = 0.001 and 0.04, respectively), along with the proportion of mice with diffuse liver metastases, an effect ablated by coexpression of Nm23-H1. We observed no variation in proliferation among lung metastases. Our findings suggest a new actin-based mechanism that can suppress tumor metastasis. Cancer Res; 73(19); 5949–62. ©2013 AACR.

Nm23-H1, also known as NDPK-A, was the first of a class of metastasis suppressor genes to be identified. Overexpression of Nm23-H1 in metastatic cell lines (melanoma, breast carcinoma, prostate, colon, hepatocellular, and oral squamous cell carcinoma) reduced cell motility in in vitro assays and metastatic potential in xenograft models, without a significant effect on primary tumor size. The mechanism of Nm23-H1 suppression of metastasis, however, is incompletely understood. Nm23-H1 has been reported to bind proteins, including those in small G-protein complexes, transcriptional complexes, the Map kinase, the TGF-β signaling pathways and the cytoskeleton. Evidence supporting these associations is presented together with evidence of resultant biochemical and phenotypic consequences of association. Cumulatively, the data suggest that part of the anti-metastatic function of Nm23-H1 lies in pathways that it interrupts via binding and inactivation of proteins.

Breast cancers are classified into five intrinsic subtypes based on gene expression profile. It is suggested that these intrinsic subtypes originate from specific developmental stage of breast epithelial cell hierarchy, stem-progenitor-mature cell. However, normal breast epithelial cell lines representing these intrinsic subtypes are yet to be created. Using normal breast tissues of ancestry-mapped Caucasian, African American, and Hispanic women and a primary cell culturing system that allows growth of normal epithelial cells of different developmental stages including estrogen receptor-positive mature luminal cells, we created 15 human telomerase-immortalized breast epithelial cell lines. These cells formed acini on a matrigel and ductal structures on 3-dimensional collagen or hydrogel, indicating that these cell lines have retained characteristics of normal breast epithelial cells. RNA sequencing and PAM50 intrinsic subtype clustering algorithms were used to identify the intrinsic subtypes of the immortalized cell lines together with two well characterized “normal” breast epithelial cell lines MCF10A and HMEC as well as luminal breast cancer cell line MCF-7. Unlike MCF10A and HMEC, which are enriched for basal-like gene expression pattern, our cell lines classified into luminal A, basal, and normal-like subtypes. This was also reflected in the immunofluorescence staining with basal marker KRT14 and luminal marker KRT19. Few of these cell lines were dual positive for KRT14 and KRT19, but in varying proportions. Cell lines representing claudin-low subtypes were also created, which are phenotypically (CD201+/EpCAM-) different from the above cell lines. Cell lines showed inter-individual differences in stemness/differentiation capabilities and variable basal activity of signaling molecules such as NF-kB, AP-1 and pERK, which is consistent with, possibly reflecting, recent discoveries of genetic variations in gene regulatory regions among general population that contribute to widespread differences in gene expression/signaling under “normal” state. As majority of breast cancers are believed to originate from luminal progenitor cells, which are well represented our cell lines, these cell lines are ideal to delineate the impact of inter-individual and ethnic differences in normal breast biology on breast cancer initiation and progression as well as to determine whether cell-type-origin instead of genomic aberration drives intrinsic subtype-enriched gene expression patterns in breast tumors. Citation Format: Brijesh Kumar, Mayuri Prasad, Manjushree Anjanappa, Poornima Nakshatri, Natascia Marino, Anna Maria Storniolo, Xi Rao, Sheng Liu, Jun Wan, Yunlong Liu, Harikrishna Nakshatri. Breast epithelial cell lines from normal breast with luminal and intrinsic subtypes -enriched gene expression document inter-individual differences in differentiation cascade [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 4993.

Background: Identifying the earliest stages of breast cancer carcinogenesis, present well before any clinical signs of disease, is the necessary underpinning of an effective breast cancer prevention strategy. We propose to elucidate transcriptomic changes occurring in the breast tissue during cancer initiation. by analyzing biospecimens donated by women before any clinical sign of sporadic breast tumor (here labeled “susceptible”). In the present work, we report on the transcriptome differences in the microdissected breast compartments (epithelium, stroma and fat) of susceptible versus healthy premenopausal women. Methods: The specimens were obtained from the Susan G. Komen Tissue Bank at IU Simon Cancer Center. We compared the transcriptome profiles of breast tissues from 7 susceptible and 17 healthy premenopausal women between the age of 34 and 52 years, who were free of breast pathology at the time of donation. Donors in the two experimental groups were matched according to age, racial background and menstrual phase. Differential expression analysis was performed using EdegR. False discovery rate (FDR) was computed from p-values using the Benjamini-Hochberg procedure. Ingenuity Pathway Analysis was used to identify relevant signaling pathways. Because circulating hormones variations during menstrual cycle affect the breast epithelium gene expression, we also examined transcriptome differences independently from the menstrual phase. Results/Discussion: We found 536 transcripts differentially expressed between the two groups (p 2 and FDR Conclusion: This study shows that earliest alterations in breast cancer initiation affect metabolic pathway as well as transcriptional regulation. Interestingly, cell adhesion signaling may also be dysregulated at this early stage of cancer development. The findings will contribute to our understanding of the mechanisms of cancer initiation, as well as the identification of new therapeutic targets and thus, improvement of preventive interventions. Citation Format: Natascia Marino, Rana German, Mariah L. Johnson, Xi Rao, Xiaoling Xuei, Jun Wan, Anna Maria V. Storniolo. Transcriptional changes in breast cancer initiation [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 5054.

e13548Background: MicroRNAs (miRs) and their distribution in circulation have been implicated in physiological and pathological processes. Recently, circulating miRs have emerged as potential bioma...

Nm23 was the first of what has become a field of over 20 known metastasis suppressor genes. Since the discovery of Nm23 in 1988, a variety of mechanisms have been attributed to its activity, including a histidine kinase activity, binding of other proteins to regulate metastatic formation, and altered gene expression downstream of Nm23. Here, we will review current efforts to translate the previous work done on this metastasis suppressor gene into the clinic, including high-dose medroxyprogesterone acetate (MPA), which has been shown to upregulate Nm23 expression. In addition, we will detail a new potential target downstream of Nm23. LPA1 is one of a group of known cell surface receptors for lysophosphatidic acid (LPA), which has been shown to be inversely correlated with Nm23 expression. A specific LPA1 antagonist could conceivably mimic the effects of Nm23 by downregulating the activity of the LPA1 pathway, which would be of considerable interest for potential clinical use.

The h-prune protein is a member of the DHH protein superfamily, and its overexpression in breast, colorectal and gastric cancers correlates with depth of invasion and degree of lymph-node metastasis. Taken together with the observation that h-prune is highly expressed in metastatic breast cancer, this suggests that h-prune can be used as a marker for the identification of subsets of cancer patients with highly aggressive tumours. H-prune possesses a phosphodiesterase (cAMP-PDE) activity, and inhibition of PDE activity with dipyridamole suppresses cell motility. H-prune interacts with nm23-H1, GSK-3beta and gelsolin. Although a correlation between an h-prune PDE activity and cellular motility has been shown, GSK-3beta does not affect the PDE activity of h-prune. Inhibition of the interactions between h-prune and GSK-3beta and nm23-H1 additively suppresses the migration of colon cancer and breast cancer cells, thus suggesting that h-prune regulates cell motility by two different means of action: through its PDE activity and in its interactions with protein partners. Therefore, the identification of highly specific inhibitors of h-prune should be useful in the development of drugs to treat cancer metastasis.

A genetic interaction between PRUNE and NM23/NDPK has been postulated in Drosophila melanogaster. Many have focused on Drosophila for the genetic combination between PRUNE "knock down" and AWD/NM23 fly mutants bearing the P97S mutation (K-pn, Killer of PRUNE mutation). We postulated a role for PRUNE-NM23 interactions in vertebrate development, demonstrating a physical interaction between the human PRUNE and NM23-H1 proteins, and partially characterizing their functional significance in cancer progression. Here, we present an initial analysis towards the functional characterization of the PRUNE-NM23 interaction during mammalian embryogenesis. Our working hypothesis is that PRUNE, NM23-H1 and their protein-protein interaction partners have important roles in mammalian brain development and adult brain function. Detailed expression analyses from early mouse brain development to adulthood show significant co-expression of these two genes during embryonic stages of brain development, especially focusing on the cortex, hippocampus, midbrain and cerebellum. We hypothesize that their abnormal expression results in an altered pathway of activation, influencing protein complex formation and its protein partner interactions in early embryogenesis. In the adult brain, their function appears concentrated towards their enzyme activities, wherein biochemical variations can result in brain dysfunction.

The human (h)-prune protein is a member of the DHH protein superfamily and it has a cAMP phosphodiesterase activity. Its overexpression in breast, colorectal and gastric cancers correlates with depth of invasion and a high degree of lymph-node metastasis. One mechanism by which h-prune stimulates cell motility and metastasis processes is through its phosphodiesterase activity, which can be suppressed by dipyridamole, a pyrimido[5,4-d]pyrimidine analogue. To obtain new and more potent agents that have high specificity towards inhibition of this h-prune activity, we followed structure activity-relationship methodologies starting from dipyridamole and synthesised eight new pyrimido pyrimidine derivatives. We analysed these newly generated compounds for specificity towards h-prune activities in vitro in cellular models using scintillation proximity assay for cAMP-PDE activity, cell index in cell proliferation assays and transwell methodology for two-dimensional cell migration in a top-down strategy of selection. Our findings show that two pyrimido[5,4-d]pyrimidine compounds are more effective than dipyridamole in two highly metastatic cellular models of breast cancer in vitro. Future studies will assess their therapeutic effectiveness against breast and other cancers where there is over-expression of h-prune, and in ad-hoc, proof of concept, animal models. (C) 2012 Elsevier Masson SAS. All rights reserved.

Background: Several evidences indicate that different racial and ethnic backgrounds affect the incidence and severity of diseases such as breast cancer and diabetes, and the response to therapy. For example White and African American women are more likely to develop breast cancer than Hispanic and Asian women. Moreover, African American women are more likely to develop more aggressive (Triple Negative Breast Cancer), more advanced-stage breast cancer at a young age. Therefore, given the health disparity and with the advance of personalized medicine, it is becoming critical to address population stratification. The Komen Tissue Bank at IU Simon Cancer Center (KTB), the only biobank of normal breast tissue from healthy women, is employing a more accurate approach to detect population stratification through the use of Ancestry Informative Markers (AIMs). Methodology: A total of 2,973 DNA samples were obtained from the KTB. Genotyping was performed using the KASP technology (LGC Genomics) and a 41-SNP panel (labeled 41-AIM panel) selected from Nievergelt et al, 2013. Genotype analysis using the 41-AIM panel along with a Bayesian clustering method (STRUCTURE) was able to discern continental origins including European/ Middle East (Caucasian), East Asia, Central/South Asia, Africa, Americas, and Oceania. A reference set was obtained from the HeatMap. The results were compared with the self-reported data. Results/Conclusion: Our genetic ancestry analysis indicates that out of 2,973 KTB tissue donors, 2,132 are primarily Caucasian, 532 African, 234 of Asian, 72 Latino, 3 Oceanic origins. High mixture of racial markers was observed in several samples. Moreover, when we compare these findings with the self-reported data we find a 10.9% of discrepancies indicating the imprecision of the self-reported information. The implementation of the genetic ancestry data into the KTB creates the potential for a more accurate study of health disparities. Citation Format: Julia R. McCarty, Guanlong Jiang, Mariah L. Johnson, Teresa Mahin, Nicholas Scherer, Jonathon Dunn, Anna Maria Storniolo, Natascia Marino. Komen Tissue Bank donors: Genetically determined ethnicity and race [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 5281. doi:10.1158/1538-7445.AM2017-5281

Background: Menarche, the onset of the female menstruation, is a marker of pubertal timing. Age at menarche varies widely between girls and is highly dependent on nutritional status and body fat accumulation. The occurrence of menarche at an early age is linked to an increased risk of several adverse health conditions later in life, such as obesity, type-2 diabetes, breast and endometrial cancer, and cardiovascular disease. Indeed, for every one year decrease in age at menarche (from an average age at menarche of 12.5 years), breast cancer risk is increased by 5%. Several genome wide association studies (GWAS) have identified genetic variants (i.e. in the LIN28B gene) that are associated with early age at menarche, however little is known about the changes occurring in the breast tissue of women with early menarche. We hypothesize that early age at menarche results in permanent molecular alterations in the breast tissue and that those abnormalities may contribute to the tissue’s susceptibility to carcinogens and breast cancer development. Methods: To test our hypothesis we used the resources available at the Susan G. Komen Tissue Bank at the Indiana University Simon Cancer Center (KTB). We selected histologically normal breast tissue from healthy, young women with either early (age ≤ 10 years) or late menarche (age ≥ 15 years), and matched for age, race, BMI, and menstrual phase. Breast tissue biopsies from these women were microdissected to isolate the breast epithelium and next generation RNA-sequencing was used to generate a transcriptome profile for each sample. Differential expression was performed using DESeq2 in R. The tissue samples were also evaluated using immunostaining. Results/Conclusions: Preliminary data show significant differences when comparing the transcriptome profiles of the microdissected breast epithelium from the early and late menarche sample cohorts. The tissue from women with early menarche had upregulation of genes associated with defense against oxidative stress and/or infectious bacteria (lactotransferrin [LTF], ceruloplasmin [CP]), cell adhesion, (ITGα11, ITGαX, ITGαM, ITGαL, ITGβ2), immune response (CARD9, LAIR1), and had downregulation of ubiquitination pathways (USP40, AMFR) and lipoprotein metabolism (OSBPL1A, LIPH, PIGN). Immunohistochemical evaluation of markers of oxidative stress (LTF, CP), cell proliferation (Ki67), and immune infiltrates (CD45, CD20, CD8, CD68) is underway. Together, this information will give us the opportunity to better understand early age at menarche as a breast cancer risk factor and advance research for women’s health. Citation Format: Mariah L. Johnson, Natascia Marino, Anna Maria V. Storniolo, Bradley A. Hancock, Milan Radovich, George E. Sandusky. Molecular alterations in the breast associated with early menarche [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 4250. doi:10.1158/1538-7445.AM2017-4250