Abstract P1-09-06: Gene-set enrichment analysis (GSEA) of breast tissue from healthy women with less than six months history of breastfeeding shows enrichment in Hedgehog signaling, notch signaling and luminal progenitor gene signatures
Authors: Basree MM , Shinde N , Palettas M , Weng D , Stover DG , Sizemore GM , Shields P , Majumder S , Ramaswamy B 
Published: May 2019

Introduction: Multiple epidemiological studies have shown that prolonged breastfeeding is associated with a reduced risk of developing triple negative/basal-like breast cancer (TN/BLBC). We have modeled abrupt involution (AI) due to lack of breastfeeding and gradual involution (GI) of the mammary gland that occurs over time upon prolonged breastfeeding in wild-type FVB/N mice and discovered prominent histological and molecular changes in the AI glands over time. Our studies revealed for the first time a clear and persistent expansion of mammary luminal progenitor (LP) epithelial cells in AI glands (AACR abstract#2242, 2018). Here, we corroborate animal studies using normal human breast tissue obtained from a reduction mammoplasty tissue collection study (OSU-2011C0094).

Methods: Breast tissue obtained from parous premenopausal women with no history of breast cancer who breastfed for ≥6 months (GI, n=16) versus those who breastfed for &lt;6 months (AI, n=16) (OSU-2011C0094) was used for gene expression analysis. RNA isolated from these normal mammary tissues was analyzed using Affymatrix Gene ChIP Human Transcriptome array 2.0; Gene Set Enrichment Analysis (GSEA) was used to analyze the microarray data. Molecular Signatures Database was used in GSEA querying C2 curated gene sets, Hallmark gene sets, and Lim-Mammary-Luminal-Progenitor gene sets. H&E sections of the breast tissue were used to assess lobular type by counting number of ductules per terminal ductal lobular unit (TDLU). False discovery rate (FDR) q-values and p-values were used for multiple comparison adjustment.

Results: GSEA revealed that breast tissue obtained from women in the AI cohort exhibited strong positive enrichment for Notch and Hedgehog Signaling (Hhg) pathways (FDR q-value= 0.20 and 0.12, respectively). In GI women, GSEA showed an overall trend towards enrichment in metabolic pathways and immune system functions. Moreover, there was non-significant trend towards positive enrichment of mouse LP gene signature in AI women only (FDR q-value= 0.30). Age and BMI were not statistically different between AI and GI cohorts. Analysis of TDLU, the primary anatomical source of most breast cancers, revealed that breast tissue from AI women had proportionally higher lobular type 1 only epithelium than GI women who exhibited more differentiated lobular epithelium (p-value= 0.049).

Conclusion: We report here for the first time that mammary glands from women who breastfed &lt;6 months were enriched for stem-cell signaling pathways and LP gene signature. This reflects some similarity to BRCA1 mutation carriers, who demonstrate expanded luminal progenitor population. In addition, higher Type 1 TDLU's are seen in breast tissue from parous women who breastfed &lt;6 months. Together, these data demonstrate features for TN/BLBC precursors enriched in patients who breastfed for &lt;6 months. Understanding this mechanistic link will help in developing prevention strategies, particularly for African-American women who have lower prevalence of breastfeeding and higher incidence of TN/BLBC.

Citation Format: Basree MM, Shinde N, Palettas M, Weng D, Stover DG, Sizemore GM, Shields P, Majumder S, Ramaswamy B. Gene-set enrichment analysis (GSEA) of breast tissue from healthy women with less than six months history of breastfeeding shows enrichment in Hedgehog signaling, notch signaling and luminal progenitor gene signatures [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-09-06.