Abstract Background. In observational studies, regular use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced breast cancer risk and disease recurrence with evidence that the benefit is greater in overweight/obese women. This parallels findings that obesity is associated with an increase in proinflammatory processes with possible effects on the breast stroma and extracellular matrix. Methods. To assess if body mass index (BMI kg/m2) alters breast tissue response to NSAIDs, we conducted exploratory analyses of change in breast tissue collagen-associated peptides following 6 months of treatment with the NSAID sulindac (150 mg bid) by BMI category: normal (18.5-24.9 n=6), overweight (25-29.9 n=14) and obese (≥30 n=11). Samples for this study were non-cancer core needle breast biopsies from postmenopausal women with a history of hormone receptor positive breast cancer enrolled to study sulindac effect on breast tissue biomarkers. At completion, 36 of 50 patients underwent baseline biopsy and 31 had sufficient tissue for paired analyses. Tissue collagen-associated peptides were studied using whole slide tissue imaging mass spectrometry proteomics. Individual peptide signals were normalized to total ion current and mean peak intensity per area across the entire biological specimen were used to generate a score per patient. Change in stroma peptides was evaluated by BMI status using the Wilcoxon matched pairs signed rank test. Unsupervised hierarchical clustering and heatmap visualization were used to assess differential expression of the peaks. Results. Approximately, 550 peptide peaks were found by targeted collagen tissue imaging proteomics. Striking differences in response to sulindac were observed by BMI. In overweight patients, 6 peptides related to COL1A1, COL6A1, COL6A3 and VIM on database matching differed significantly before and after sulindac treatment. Three were also altered in obese women. Two of the peptides overlapped 10 peptide changes identified in unstratified analyses reported in a separate submitted abstract on the main effect of sulindac on tissue collagen. Interestingly, these peptides were not altered in patients with normal BMI. In addition, on study 15 patients experienced a decrease in BMI and 12 experienced an increase. Two collagen peptides showed inverse relationships dependent on change in BMI status during the study. Conclusion. Six-month treatment with the non-selective NSAID sulindac was associated with changes in collagen-associated peptide differently by BMI status and by weight change. Our findings are most consistent with changes in post-translational hydroxylated proline modifications of collagen variation in the triple helical region. Ongoing work may provide insights on inflammation/adiposity-associated inflammation and effects on breast tissue collagen. Citation Format: Denys Rujchanarong, Peggi M. Angel, Alison Stopeck, Christina Preece, Pavani Chalasani, Patricia A. Thompson. Evidence that body mass index modifies breast tissue collagen peptide response pattern to treatment with the non-steroidal anti-inflammatory drug sulindac [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 P1-10-05.
Abstract Higher breast cancer mortality rates continue to disproportionally affect black women (BW) compared to white women (WW). This disparity is largely due to differences in tumor aggressiveness that can be related to distinct ancestry-associated breast tumor microenvironments (TMEs). Yet, characterization of the normal microenvironment (NME) in breast tissue and how they associate with breast cancer risk factors remains unknown. N-glycans, a glucose metabolism-linked post-translational modification, has not been characterized in normal breast tissue. We hypothesized that normal female breast tissue with distinct Breast Imaging and Reporting Data Systems (BI-RADS) categories have unique microenvironments based on N-glycan signatures that varies with genetic ancestries. Profiles of N-glycans were characterized in normal breast tissue from BW (n = 20) and WW (n = 20) at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). A total of 176 N-glycans (32 core-fucosylated and 144 noncore-fucosylated) were identified in the NME. We found that certain core-fucosylated, outer-arm fucosylated and high-mannose N-glycan structures had specific intensity patterns and histological distributions in the breast NME dependent on BI-RADS densities and ancestry. Normal breast tissue from BW, and not WW, with heterogeneously dense breast densities followed high-mannose patterns as seen in invasive ductal and lobular carcinomas. Lastly, lifestyles factors (e.g. age, menopausal status, Gail score, BMI, BI-RADS) differentially associated with fucosylated and high-mannose N-glycans based on ancestry. This study aims to decipher the molecular signatures in the breast NME from distinct ancestries towards improving the overall disparities in breast cancer burden.
A primary difference between black women (BW) and white women (WW) diagnosed with breast cancer is aggressiveness of the tumor. Black women have higher mortalities with similar incidence of breast cancer compared to other race/ethnicities, and they are diagnosed at a younger age with more advanced tumors with double the rate of lethal, triple negative breast cancers. One hypothesis is that chronic social and economic stressors result in ancestry-dependent molecular responses that create a tumor permissive tissue microenvironment in normal breast tissue. Altered regulation of N-glycosylation of proteins, a glucose metabolism-linked post-translational modification attached to an asparagine (N) residue, has been associated with two strong independent risk factors for breast cancer: increased breast density and body mass index (BMI). Interestingly, high body mass index (BMI) levels have been reported to associate with increases of cancer-associated N-glycan signatures. In this study, we used matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) to investigate molecular pattern changes of N-glycosylation in ancestry defined normal breast tissue from BW and WW with significant 5-year risk of breast cancer by Gail score. N-glycosylation was tested against social stressors including marital status, single, education, economic status (income), personal reproductive history, the risk factors BMI and age. Normal breast tissue microarrays from the Susan G. Komen tissue bank (BW=43; WW= 43) were used to evaluate glycosylation against socioeconomic stress and risk factors. One specific N-glycan (2158 m/z) appeared dependent on ancestry with high sensitivity and specificity (AUC 0.77, Brown/Wilson p-value<0.0001). Application of a linear regression model with ancestry as group variable and socioeconomic covariates as predictors identified a specific N-glycan signature associated with different socioeconomic stresses. For WW, household income was strongly associated to certain N-glycans, while for BW, marital status (married and single) was strongly associated with the same N-glycan signature. Current work focuses on understanding if combined N-glycan biosignatures can further help understand normal breast tissue at risk. This study lays the foundation for understanding the complexities linking socioeconomic stresses and molecular factors to their role in ancestry dependent breast cancer risk.
Abstract Breast cancer risk increases significantly in women who maintain higher radiologically dense breast tissue as they age. However, the mechanisms that underlie this association remain poorly understood, especially in postmenopausal women. A predominant contributor to dense breasts and differences between women is the relative amount of fibrillar collagens. Experimental models suggest that changes in collagen properties such as fiber alignment and stiffness promote local stromal fibrosis, which we hypothesize may contribute to the relationship between breast density, aging, and tumorigenesis. Normal breast biopsy samples from the contralateral, healthy breast of 32 postmenopausal breast cancer patients participating in a clinical trial for drug effect on breast density were examined and scored for areas of fibrosis and analyzed using second harmonic generation microscopy (SHG) and mass spectrometry proteomics. Our aim was to identify areas of fibrosis and relate the severity of focal fibrosis to the physical properties and peptide composition of collagen fibers. Using GraphPad Prism, we conducted one-way ANOVA and Kruskal-Wallis tests and post-hoc multiple comparison tests. H&E-stained breast tissue samples were scored according to percent fibrosis. The minimum and maximum percent fibrosis were 5% and 100%, respectively, with a median of 60% (95% CI: 97.06%). Out of 31 people, 16 had a higher fibrosis score than the median. Consistent with pathologic scoring of the presence and extent of fibrosis, fiber counts were significantly lower in tissue with lower fibrosis scores than samples with more highly scored fibrosis (p-value <0.001). Distance to the nearest fiber was greater in samples with < 20% fibrosis, whereas greater alignment to the nearest fiber was observed in higher fibrosis percentages. At the molecular level, 22 peptides demonstrated a strong correlation with percent fibrosis (spearman r ≥ 60), of which 13 peptides were statistically significant (p-value <0.0001). The putatively identified peptides belong to type I, III, IV and VI collagens. Fiber length and straightness, but not fiber width, were higher in tissues with greater percent fibrosis. Significant correlations were identified between collagen physical features and peptides: two distinct peptides correlated with collagen fiber length and straightness while 20 peptides correlated with fiber width. These putative peptides belong to types I, III, IV and V collagens with ~70% of the peptides containing at least one hydroxyproline modification. This study is the first to explore the link between focal fibrosis and collagen-associated peptides in healthy breast tissue of high-risk postmenopausal women. These findings provide novel insights on stromal collagens associated with fibrotic changes in normal at-risk breast tissue that may act in tissue susceptibility to tumor formation. Citation Format: Denys Rujchanarong, Alison T. Stopeck, Christina Preece, Ren Sun, Pavani Chalasani, Sean Brown, Ji D. Bai, Heather Jensen Smith, Sungyong You, Peggi M. Angel, Patricia Thompson. Correlation between collagen peptides and physical features of fibrosis in breast tissue from women at increased risk of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 782.
Breast stroma plays a significant role in breast cancer risk and progression yet remains poorly understood. In breast stroma, collagen is the most abundantly expressed protein and its increased deposition and alignment contributes to progression and poor prognosis. Collagen post-translation modifications such as hydroxylated-proline (HYP) control deposition and stromal organization. The clinical relevance of collagen HYP site modifications in cancer processes remains undefined due to technical issues accessing collagen from formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a targeted approach for investigating collagen and other extracellular matrix proteins from FFPE tissue. Here, we hypothesized that immunohistochemistry staining for fibroblastic markers would not interfere with targeted detection of collagen stroma peptides and could reveal peptide regulation influenced by specific cell types. Our initial work demonstrated that stromal peptide peak intensities when using MALD-IMS following IHC staining (αSMA, FAP, P4HA3 and PTEN) were comparable to serial sections of nonstained tissue. Analysis of histology-directed IMS using PTEN on breast tissues and TMAs revealed heterogeneous PTEN staining patterns and suggestive roles in stromal protein regulation. This study sets the foundation for investigations of target cell types and their unique contribution to collagen regulation within extracellular matrix niches.
Obesity is a major health problem and has been linked to a variety of factors including prenatal and juvenile nutritional environment. Nutritional environmental effects have been shown to have epigenetic effects in humans, mice, and Drosophila melanogaster . However, the degree to which a high‐sucrose diet influences obesity and has an epigenetic impact on offspring has not been as thoroughly investigated in males as in females. We therefore examined the effects of control, low‐sucrose, and high‐sucrose diets on both maternal and paternal obesity and epigenetic effects in the male and female members of the F1 and F2 generations. This study demonstrates that variation in sucrose induced changes in parental and offspring fecundity and size. Support or Funding Information This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers; 8UL1GM118979‐02; 8TL4GM118980‐02; 8RL5GM118978‐02. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Health. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Fibroblasts maintain tissue and organ homeostasis through output of extracellular matrix that affects nearby cell signaling within the stroma. Altered fibroblast signaling contributes to many disease states and extracellular matrix secreted by fibroblasts has been used to stratify patient by outcome, recurrence, and therapeutic resistance. Recent advances in imaging mass spectrometry allow access to single cell fibroblasts and their ECM niche within clinically relevant tissue samples.We review biological and technical challenges as well as new solutions to proteomic access of fibroblast expression within the complex tissue microenvironment. Review topics cover conventional proteomic methods for single fibroblast analysis and current approaches to accessing single fibroblast proteomes by imaging mass spectrometry approaches. Strategies to target and evaluate the single cell stroma proteome on the basis of cell signaling are presented.The promise of defining proteomic signatures from fibroblasts and their extracellular matrix niches is the discovery of new disease markers and the ability to refine therapeutic treatments. Several imaging mass spectrometry approaches exist to define the fibroblast in the setting of pathological changes from clinically acquired samples. Continued technology advances are needed to access and understand the stromal proteome and apply testing to the clinic.
Abstract Despite having similar incidence of breast cancer, black women (BW) are typically diagnosed with more aggressive subtypes of breast cancer compared to white women (WW). One mediator contributing to the disparity in breast cancer mortality rates is the distinct tumor microenvironments (TME) associated with BW and WW. Yet, characterization of the normal microenvironment (NME) in breast tissue and how they may associate with breast cancer risk factors remains unknown. Contemporary literature reports that TMEs from BW and WW with breast cancer are characterized with higher densities of pro-tumorigenic macrophages (M2) and cancer-associated adipocytes have been observed in BW compared to WW, and typically associated with worse breast cancer prognosis. While the TME has been shown to be critical for breast cancer progression, the lack of characterization of the NME is a limiting factor towards understanding the molecular differences between a normal and cancerous breast microenvironment. We hypothesize that characterizing the tissue compositions in the breast NME could elucidate unique molecular signatures that may be contributing to distinct alterations that are driving the disparity in breast cancer aggressiveness in BW. The cohorts consisted of normal breast tissue from BW (n=20) and WW (n=20) with breast cancer risk determined by Gail score. The Gail model is a well-established tool used to assess the 5-year risk of developing invasive breast cancer based on demographic and clinical data. All tissues were obtained from the Susan G. Komen tissue bank and used with institutional permission. Profiles of N-glycans, a glucose metabolism-linked post-translational modification, were characterized in normal breast tissue from BW and WW at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Tissue from both cohorts were characterized based on their tissue compositions including areas rich in stroma, adipocytes, and crown like structures (CLSs), a pro-inflammatory process consisting of dying adipocytes surrounded by stroma and macrophages. Nearly 70 N-glycans were identified in the NME from both BW and WW. When comparing N-glycan peaks between premenopausal and postmenopausal women, one N-glycan peak (m/z 1905.633) was significantly higher in the premenopausal group (p<0.0001) regardless of genetic ancestry. However, when comparing peak m/z 1905.633 based on genetic ancestry, premenopausal and postmenopausal BW had significantly higher peak intensities compared to premenopausal and postmenopausal WW (premenopausal BW vs WW, p = 0.024; postmenopausal BW vs WW, p = 0.028). Overall, the data suggests that there may be distinct N-glycan structures associated with premenopausal and postmenopausal women based on genetic ancestry. Characterizing the NME from BW and WW at risk for breast cancer may help towards understanding potential breast stroma priming molecular signatures contributing to differences in breast cancer aggressiveness observed in BW compared to WW. Citation Format: Denys Rujchanarong, Anand Mehta, Richard R. Drake, Marvella E. Ford, Peggi M. Angel. Deciphering the normal breast microenvironment in Black women and White women [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A071.
Abstract A primary difference between black women (BW) and white women (WW) diagnosed with breast cancer is not incidence, but aggressiveness of the tumor. Black women have higher mortalities with similar incidence of breast cancer compared to other race/ethnicities, and are diagnosed at a younger age with more advanced tumors with double the rate of lethal, triple negative breast cancers. There is ongoing debate regarding whether the underlying cause of higher mortality is related to healthcare inequalities or due to ancestry dependent molecular features found in normal breast tissue that facilitate the aggressive phenotype found in black women. One hypothesis is that chronic social and economic stressors result in molecular responses that create a tumor permissive tissue microenvironment in normal breast tissue, and these chronic stresses differ by race/ethnicity. In this study, we investigate molecular pattern changes in tissue N-glycosylation in a cohort of ancestry defined normal breast tissue from BW and WW with significant 5-year risk of breast cancer by Gail score. N-glycosylation, a glucose metabolism-linked post-translational modification attached to an asparagine (N) residue, was tested against social stressors. Social stresses included marital status, single, education, economic status (income), personal reproductive history, the risk factors BMI and age. Normal breast tissue microarrays from the Susan G. Komen tissue bank (WB=43; WW= 43) were used to evaluate glycosylation against socioeconomic stress and risk factors. There was no significant difference in age (Median age BW 42.5, 95% CI [40.0, 45.0]; WW 42.0, 95% CI [39.7, 44.3]). A subgroup of women had similar BMI (BW, n=24 Median BMI 29.5, 95% CI [27.6, 32.3]; WW, n=24 28.3, 95% CI [23.5, 31.6]. BW women had an overall lower median risk by 5-year Gail score, which, (BW 9.4, 95% CI [8.9, 9.9]; WW 10.4, 95% CI [8.1, 12.6]). Area under the receiver operating curve (AUC) ≥0.70, Brown/Wilson p-value <0.001 was used to assess for individual glycosylation differences in normal breast tissue at risk for breast cancer. Out of 55 N-glycans profiled in normal breast by glycomics mass spectrometry, one N-glycan appeared dependent on ancestry with high sensitivity and specificity (AUC 0.788, Brown/Wilson p-value<0.0001). For women of the same BMI, a total of 12 N-glycans could report potential ancestry-dependent differences. Interestingly, when we fit a linear regression model with ancestry as group variable and socioeconomic covariates as predictors, different N-glycans associated with different socioeconomic stresses. Five N-glycans in particular linked to different socioeconomic stresses for both BW and WW. For white women, household income was strongly associated to certain N-glycans, while for black women, marriage status (married and single) was strongly associated with the same N-glycan signature. Current work focuses on understanding if combined N-glycan biosignatures can further help understand normal breast tissue at risk. The data suggests that metabolic patterns linked to socioeconomic stresses may contribute to breast cancer risk dependent on ancestry. This study lays the foundation for understanding the complexities linking socioeconomic stresses and molecular factors to their role in ancestry dependent breast cancer risk and aggressiveness in black women. Citation Format: Peggi M Angel, Yeonhee Park, Danielle A Scott, Denys Rujchanarong, Sean Brown, Richard R Drake, George E Sandusky, Harikrishna Nakshatri. Metabolic links to socioeconomic stresses uniquely affecting race in normal breast tissue at risk for breast cancer [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 P3-14-13.
Collagen stroma interactions within the extracellular microenvironment of breast tissue play a significant role in breast cancer, including risk, progression, and outcomes. Hydroxylation of proline (HYP) is a common post-translational modification directly linked to breast cancer survival and progression. Changes in HYP status lead to alterations in epithelial cell signaling, extracellular matrix remodeling, and immune cell recruitment. In the present study, we test the hypothesis that the breast cancer microenvironment presents unique PTMs of collagen, which form bioactive domains at these sites that are associated with spatial histopathological characteristics and influence breast epithelial cell signaling. Mass spectrometry imaging proteomics targeting collagens were paired with comprehensive proteomic methods to identify novel breast cancer-related collagen domains based on spatial localization and regulation in 260 breast tissue samples. As ancestry plays a significant role in breast cancer outcomes, these methods were performed on ancestry diverse breast cancer tissues. Lumpectomies from the Cancer Genome Atlas (TCGA; n=10) reported increased levels of prolyl 4-hydroxylase subunit alpha-3 (P4HA3) accompanied by spatial regulation of fibrillar collagen protein sequences. A concise set of triple negative breast cancer lumpectomies (n=10) showed spatial regulation of specific domain sites from collagen alpha-1(I) chain. Tissue microarrays identified proteomic alterations around post-translationally modified collagen sites in healthy breast (n=81) and patient matched normal adjacent (NAT; n=76) and invasive ductal carcinoma (n=83). A collagen alpha-1(I) chain domain encompassing amino acids 506-514 with site-specific proline hydroxylation reported significant alteration between patient matched normal adjacent tissue and invasive breast cancer. Functional testing of domain 506-514 on breast cancer epithelial cells showed proliferation, chemotaxis and cell signaling response dependent on site localization of proline hydroxylation within domain 506-514 variants. These findings support site localized collagen HYP forms novel bioactive domains that are spatially distributed within the breast cancer microenvironment and may play a role in ancestral traits of breast cancer.
