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Supplementary Table S2 from Interactions with Fibroblasts Are Distinct in Basal-Like and Luminal Breast Cancers

Supplementary Figure S1 from Interactions with Fibroblasts Are Distinct in Basal-Like and Luminal Breast Cancers

Supplementary Table 1. Mutations in LSR from Human Cancer Samples. Supplementary Table 2. Potential post-translational modification sites of LSR transcript variants.

Supplementary Figure 1 Legend.

Legends for supplementary figures and tables.

AVI file - 3MB, Movie of THP-1 cells engulfing latex microparticles after coculturing with SUM149 basal-like breast cancer cells

AVI file - 3MB, Movie of THP-1 cells engulfing latex microparticles after coculturing with MCF-7 luminal breast cancer cells.

Supplementary Table 1

Supplemental Table 1 shows univariate odds ratios, Supplemental Table 2 shows adjusted odds ratios stratified by race, and SupplementalTable 3 compares associations using mode of detection derived using a 1 year vs. 2 year interval.

Supplementary Table 2B

Supplementary Figure 2

Flowchart of eligibility criteria.

PDF file - 103K, List of genes defined in the literature as significant in monocyte-to-macrophage differentiation and polarization.

Mouse Mapping Statistics, n=3/group.

PDF file - 128K, Control system-generated M1 and M2 macrophage significantly expressed genes involved in macrophage differentiation and polarization defined by 4x44k microarray analysis.

Lipolysis-stimulated lipoprotein receptor (LSR) has been found in the plasma membrane and is believed to function in lipoprotein endocytosis and tight junctions. Given the impact of cellular metabolism and junction signaling pathways on tumor phenotypes and patient outcome, it is important to understand how LSR cellular localization mediates its functions. We conducted localization studies, evaluated DNA binding, and examined the effects of nuclear LSR in cells, xenografts, and clinical specimens. We found LSR within the membrane, cytoplasm, and the nucleus of breast cancer cells representing multiple intrinsic subtypes. Chromatin immunoprecipitation (ChIP) showed direct binding of LSR to DNA, and sequence analysis identified putative functional motifs and post-translational modifications of the LSR protein. While neither overexpression of transcript variants, nor pharmacologic manipulation of post-translational modification significantly altered localization, inhibition of nuclear export enhanced nuclear localization, suggesting a mechanism for nuclear retention. Coimmunoprecipitation and proximal ligation assays indicated LSR–pericentrin interactions, presenting potential mechanisms for nuclear-localized LSR. The clinical significance of LSR was evaluated using data from over 1,100 primary breast tumors, which showed high LSR levels in basal-like tumors and tumors from African-Americans. In tumors histosections, nuclear localization was significantly associated with poor outcomes. Finally, in vivo xenograft studies revealed that basal-like breast cancer cells that overexpress LSR exhibited both membrane and nuclear localization, and developed tumors with 100% penetrance, while control cells lacking LSR developed no tumors. These results show that nuclear LSR alters gene expression and may promote aggressive cancer phenotypes.Implications: LSR functions in the promotion of aggressive breast cancer phenotypes and poor patient outcome via differential subcellular localization to alter cell signaling, bioenergetics, and gene expression. Mol Cancer Res; 15(2); 165–78. ©2016 AACR.

Delayed terminal duct lobular unit (TDLU) involution is associated with elevated mammographic breast density (MD). Both are independent breast cancer risk factors among women with benign breast disease (BBD). Prior digital analyses of normal breast tissues revealed that epithelial nuclear density (END) and TDLU involution are inversely correlated. Accordingly, we examined associations of END, TDLU involution, and MD in BBD clinical biopsies. This study included digitized images of 262 representative image-guided hematoxylin and eosin–stained biopsies from 224 women diagnosed with BBD, enrolled within the cross-sectional BREAST-Stamp project that were visually assessed for TDLU involution (TDLU count/100 mm2, median TDLU span and median acini count per TDLU). A digital algorithm estimated nuclei count per unit epithelial area, or END. Single X-ray absorptiometry of prebiopsy ipsilateral craniocaudal digital mammograms measured global and localized MD surrounding the biopsy region. Adjusted ordinal logistic regression models assessed relationships between tertiles of TDLU and END measures. Analysis of covariance examined mean differences in MD across END tertiles. TDLU measures were positively associated with increasing END tertiles [TDLU count/100 mm2, ORT3vsT1: 3.42, 95% confidence interval (CI), 1.87–6.28; acini count/TDLUT3vsT1, OR: 2.40, 95% CI, 1.39–4.15]. END was significantly associated with localized, but not, global MD. Relationships were most apparent among patients with nonproliferative BBD. These findings suggest that quantitative END reflects different but complementary information to the histologic information captured by visual TDLU and radiologic MD measures and merits continued evaluation in assessing cellularity of breast parenchyma to understand the etiology of BBD.

Human Energy Responsive Methylation Changes in Normal Breast Tissue.

IPA Biofunction Analysis. Significant disease or biofunction annotation implicated by IPA software.

Regulation of pro-growth and inflammatory gene expression.

Using a murine model of basal-like breast cancer, we tested the hypothesis that chronic obesity, an established breast cancer risk and progression factor in women, induces mammary gland epigenetic reprogramming and increases mammary tumor growth. Moreover, we assessed whether the obesity-induced epigenetic and protumor effects are reversed by weight normalization. Ovariectomized female C57BL/6 mice were fed a control diet or diet-induced obesity (DIO) regimen for 17 weeks, resulting in a normal weight or obese phenotype, respectively. Mice on the DIO regimen were then randomized to continue the DIO diet or were switched to the control diet, resulting in formerly obese (FOb) mice with weights comparable with control mice. At week 24, all mice were orthotopically injected with MMTV-Wnt-1 mouse mammary tumor cells. Mean tumor volume, serum IL6 levels, expression of proinflammatory genes in the mammary fat pad, and mammary DNA methylation profiles were similar in DIO and FOb mice and higher than in controls. Many of the genes found to have obesity-associated hypermethylation in mice were also found to be hypermethylated in the normal breast tissue of obese versus nonobese human subjects, and nearly all of these concordant genes remained hypermethylated after significant weight loss in the FOb mice. Our findings suggest that weight normalization may not be sufficient to reverse the effects of chronic obesity on epigenetic reprogramming and inflammatory signals in the microenvironment that are associated with breast cancer progression. Cancer Prev Res; 9(5); 339–48. ©2016 AACR.

PDF file - 64K, Gene expression heatmap data of THP-1 macrophages differentiated by classical stimuli on Agilent 4x44k microarrays.

Supplementary Table 3

S1. QPCR analysis of luminal progenitor and cancer stem cell genes. Primers were designed against the indicated genes. S2. Representative Z-stack projections of MCF7 breast cancer and MCF10AI breast epithelial cells generated from deconvolved slices using the maximum intensity criteria. S3. Ingenuity Pathway Analysis identification of the top biological functions and disorders associated with LSR target genes. S4. Top network generated via Path Designer of Ingenuity Pathway Analysis with LSR target genes. S5. ChIP-QPCR analysis of LSR target genes. S6. LSR expression in breast tumors stratified by menopause status. S7. Analysis of LSR expression in breast tumors. S8. Integrative Genomics Viewer snapshot image of RNA-seq data from MCF7, SUM149, SUM!59 and MDA-MB231 breast cancer cell lines. S9. Evolutionary conservation of LSR. S10. Predicted phosphorylation sites of full-length LSR. S11. Subcellular localization of LSR protein upon treatment with posttranslation modification pharmacologicals. S12. Localization of LSR binding sites on chromosomes.

PDF file - 62K, Ingenuity Pathway Analysis on M1- and M2-specific gene clusters from THP-1expression responses to classical stimuli.

Supplementary Figure 1

Supplementary Information including supplementary figure legends and supplementary table titles

Figure S1. A. PBRM1 and SETD2 expression between CC1 and CC2; B. PBRM1 and SETD2 expression between NCC1 and NCC2.

Figure S2. A. Contributions of small indel (ID) signatures (A and E) between CC1 and CC2; B. Number of mutations in small indel (ID) signatures (A and E) between CC1 and CC2.

Figure S5. SNAI1, SNAI3, and TWIST expression in CC1 and CC2 tumors.

Supplementary Legend

Figure S3. Gene expression profiling based on the NanoString panel in 23 skull-base (top) and 25 non-skull-base (bottom) chordoma patients from North America.

Table S1. Genes included in the targeted DNA panel sequencing. Table S2. Differences in clinical characteristics by molecular subtype. Table S3. Distributions of mutation status in PBRM1 and SETD2 by molecular subtype in RNASeq and Nanostring datasets. Table S4. Differentially expressed genes among molecular subtypes. Table S5. GSEA results for up- and down-regulated gene sets comparing CC2 to CC1.

Purpose:Chordoma is a rare bone tumor with a high recurrence rate and limited treatment options. The aim of this study was to identify molecular subtypes of chordoma that may improve clinical management.Experimental Design:We conducted RNA sequencing in 48 tumors from patients with Chinese skull-base chordoma and identified two major molecular subtypes. We then replicated the classification using a NanoString panel in 48 patients with chordoma from North America.Results:Tumors in one subtype were more likely to have somatic mutations and reduced expression in chromatin remodeling genes, such as PBRM1 and SETD2, whereas the other subtype was characterized by the upregulation of genes in epithelial–mesenchymal transition and Sonic Hedgehog pathways. IHC staining of top differentially expressed genes between the two subtypes in 312 patients with Chinese chordoma with long-term follow-up data showed that the expression of some markers such as PTCH1 was significantly associated with survival outcomes.Conclusions:Our findings may improve the understanding of subtype-specific tumorigenesis of chordoma and inform clinical prognostication and targeted options.

Figure S4. Expression of selected markers in relation to overall survival (left) and recurrence-free survival (right) in Chinese chordoma patients with long-term follow-up data. A. CLDN11; B. GLI1; C. EPHA3; D. LEF1.

Supplemental Figure 2. The association between smoking and ESR1-defined breast cancer subtypes.

Supplementary Table 5 - PDF file 46K, Clinicopathologic characteristics by the Active/Inactive extratumoral microenvironment subtype

PDF file, 59K, The complete list of genes significantly associated with age are given along with the average fold change comparing the median centered data for the youngest patients (49, n=6).

PDF file - 625K, Raw metabolomics data for all samples.

Supplementary Table 3 - PDF file 23K, Overlaps in genes among biologically relevant signatures

HRs of all-cause mortality associated with parity and birth recency, in the CBCS Phases I and II

Supplemental Figure 3. The association between smoking and LS-defined breast cancer subtypes.

Agreement between manual and automated methods for quantification of biomarker expression data in tissue microarrays.

Agreement between IHC-based and RNA-based intrinsic subtyping

Background: Age is the strongest breast cancer risk factor, with overall breast cancer risk increasing steadily beginning at approximately 30 years of age. However, while breast cancer risk is lower among younger women, young women's breast cancer may be more aggressive. Although, several genomic and epidemiologic studies have shown higher prevalence of aggressive, estrogen-receptor negative breast cancer in younger women, the age-related gene expression that predisposes to these tumors is poorly understood. Characterizing age-related patterns of gene expression in normal breast tissues may provide insights on etiology of distinct breast cancer subtypes that arise from these tissues.Methods: To identify age-related changes in normal breast tissue, 96 tissue specimens from patients with reduction mammoplasty, ages 14 to 70 years, were assayed by gene expression microarray.Results: Significant associations between gene expression levels and age were identified for 802 probes (481 increased, 321 decreased with increasing age). Enriched functions included “aging of cells,” “shape change,” and “chemotaxis,” and enriched pathways included Wnt/beta-catenin signaling, Ephrin receptor signaling, and JAK/Stat signaling. Applying the age-associated genes to publicly available tumor datasets, the age-associated pathways defined two groups of tumors with distinct survival.Conclusion: The hazard rates of young-like tumors mirrored that of high-grade tumors in the Surveillance, Epidemiology, and End Results Program, providing a biologic link between normal aging and age-related tumor aggressiveness.Impact: These data show that studies of normal tissue gene expression can yield important insights about the pathways and biologic pressures that are relevant during tumor etiology and progression. Cancer Epidemiol Biomarkers Prev; 21(10); 1735–44. ©2012 AACR.

Supplementary Figure 1 - Principle component analysis showed considerable homogeneity of global gene expression in the test and training data sets.

Background: Parity and time since last birth influence breast cancer risk and vary by intrinsic tumor subtype, but the independent effects of these factors on prognosis have received limited attention.Methods: Study participants were 1,140 invasive breast cancer patients from phases I and II of the population-based Carolina Breast Cancer Study, with tissue blocks available for subtyping using immunohistochemical markers. Breast cancer risk factors, including pregnancy history, were collected via in-person interviews administered shortly after diagnosis. Vital status was determined using the National Death Index. The association of parity and birth recency with breast cancer–specific and overall survival was assessed using Cox proportional hazards models.Results: During follow-up (median = 13.5 years), 450 patients died, 61% due to breast cancer (n = 276). High parity (3+ births) and recent birth (Conclusions: Parity and recent birth are associated with worse survival among breast cancer patients, particularly among luminal breast cancers and long-term survivors.Impact: The biologic effects of parity and birth recency may extend from etiology to tumor promotion and progression. Cancer Epidemiol Biomarkers Prev; 25(1); 60–67. ©2015 AACR.

PDF file - 102K, Glucose metabolism is elevated in basal-like breast cancer with lactate production increasing with stroma presence. Glucose uptake, oxidation, and glycogen synthesis in increased in breast cancer cells (MCF7 and SUM149) when compared to CAFs (LCAF and BCAF) as shown in A. However, SUM149, basal-like breast cancer cells, exhibit higher levels of glucose metabolism than MCF7, luminal cells (A). Lactate production is increased in all coculture conditions regardless of luminal or basal-like epithelial or fibroblast cells (B).

Introduction: Overall survival of early-stage breast cancer patients is similar for those who undergo breast-conserving therapy (BCT) and mastectomy; however, 10% to 15% of women undergoing BCT suffer ipsilateral breast tumor recurrence. The risk of recurrence may vary with breast cancer subtype. Understanding the gene expression of the cancer-adjacent tissue and the stromal response to specific tumor subtypes is important for developing clinical strategies to reduce recurrence risk.Methods: We utilized two independent datasets to study gene expression data in cancer-adjacent tissue from invasive breast cancer patients. Complementary in vitro cocultures were used to study cell–cell communication between fibroblasts and specific breast cancer subtypes.Results: Our results suggest that intrinsic tumor subtypes are reflected in histologically normal cancer-adjacent tissue. Gene expression of cancer-adjacent tissues shows that triple-negative (Claudin-low or basal-like) tumors exhibit increased expression of genes involved in inflammation and immune response. Although such changes could reflect distinct immune populations present in the microenvironment, altered immune response gene expression was also observed in cocultures in the absence of immune cell infiltrates, emphasizing that these inflammatory mediators are secreted by breast-specific cells. In addition, although triple-negative breast cancers are associated with upregulated immune response genes, luminal breast cancers are more commonly associated with estrogen-response pathways in adjacent tissues.Conclusions: Specific characteristics of breast cancers are reflected in the surrounding histologically normal tissue. This commonality between tumor and cancer-adjacent tissue may underlie second primaries and local recurrences.Impact: Biomarkers derived from cancer-adjacent tissue may be helpful in defining personalized surgical strategies or in predicting recurrence risk. Cancer Epidemiol Biomarkers Prev; 24(2); 406–14. ©2014 AACR.

Purpose: Cancer cells have altered metabolism, with increased glucose uptake, glycolysis, and biomass production. This study conducted genomic and metabolomic analyses to elucidate how tumor and stromal genomic characteristics influence tumor metabolism.Experimental Design: Thirty-three breast tumors and six normal breast tissues were analyzed by gene expression microarray and by mass spectrometry for metabolites. Gene expression data and clinical characteristics were evaluated in association with metabolic phenotype. To evaluate the role of stromal interactions in altered metabolism, cocultures were conducted using breast cancer cells and primary cancer-associated fibroblasts (CAF).Results: Across all metabolites, unsupervised clustering resulted in two main sample clusters. Normal breast tissue and a subset of tumors with less aggressive clinical characteristics had lower levels of nucleic and amino acids and glycolysis byproducts, whereas more aggressive tumors had higher levels of these Warburg-associated metabolites. While tumor-intrinsic subtype did not predict metabolic phenotype, metabolic cluster was significantly associated with expression of a wound response signature. In cocultures, CAFs from basal-like breast cancers increased glucose uptake and basal-like epithelial cells increased glucose oxidation and glycogen synthesis, suggesting interplay of stromal and epithelial phenotypes on metabolism. Cytokine arrays identified hepatocyte growth factor (HGF) as a potential mediator of stromal–epithelial interaction and antibody neutralization of HGF resulted in reduced expression of glucose transporter 1 (GLUT1) and decreased glucose uptake by epithelium.Conclusions: Both tumor/epithelial and stromal characteristics play important roles in metabolism. Warburg-like metabolism is influenced by changes in stromal–epithelial interactions, including altered expression of HGF/Met pathway and GLUT1 expression. Clin Cancer Res; 19(3); 571–85. ©2012 AACR.