Rat Mammary Extracellular Matrix Composition and Response to Ibuprofen Treatment During Postpartum Involution by Differential GeLC–MS/MS Analysis

Breast cancer patients diagnosed within five years following
pregnancy
have increased metastasis and decreased survival. A hallmark of postpartum
biology that may contribute to this poor prognosis is mammary gland
involution, involving massive epithelial cell death and dramatic stromal
remodeling. Previous studies show pro-tumorigenic properties of extracellular
matrix (ECM) isolated from rodent mammary glands undergoing postpartum
involution. More recent work demonstrates systemic ibuprofen treatment
during involution decreases its tumor-promotional nature. Utilizing
a proteomics approach, we identified relative differences in the composition
of mammary ECM isolated from nulliparous rats and those undergoing
postpartum involution, with and without ibuprofen treatment. GeLC–MS/MS
experiments resulted in 20327 peptide identifications that mapped
to 884 proteins with a <0.02% false discovery rate. Label-free
quantification yielded several ECM differences between nulliparous
and involuting glands related to collagen-fiber organization, cell
motility and attachment, and cytokine regulation. Increases in known
pro-tumorigenic ECM proteins osteopontin, tenascin-C, and laminin-α1
and pro-inflammatory proteins STAT3 and CD68 further identify candidate
mediators of breast cancer progression specific to the involution
window. With postpartum ibuprofen treatment, decreases in tenascin-C
and three laminin chains were revealed. Our data suggest novel ECM
mediators of breast cancer progression and demonstrate a protective
influence of ibuprofen on mammary ECM composition.

妊娠后五年内确诊的乳腺癌患者，其肿瘤转移风险升高、生存预后更差。可能介导该不良预后的产后乳腺生物学标志性特征之一，是乳腺退化（mammary gland involution）过程：该过程伴随大量上皮细胞死亡与显著的基质重塑。既往研究表明，从经历产后退化的啮齿类动物乳腺中分离的细胞外基质（extracellular matrix, ECM）具有促肿瘤发生特性。近期研究进一步证实，在乳腺退化阶段给予全身性布洛芬干预，可削弱其促肿瘤活性。本研究采用蛋白质组学方法，对未产大鼠以及经/未予布洛芬干预的产后退化大鼠的乳腺ECM组成进行相对差异分析。凝胶液相色谱-串联质谱（GeLC–MS/MS）实验共鉴定到20327条肽段，对应884个蛋白质，错误发现率低于0.02%。无标记定量分析显示，未产腺体与退化腺体的ECM存在多处差异，这些差异与胶原纤维排布、细胞迁移与黏附以及细胞因子调控相关。已知促肿瘤发生的ECM蛋白骨桥蛋白（osteopontin）、腱生蛋白C（tenascin-C）与层粘连蛋白α1（laminin-α1），以及促炎蛋白信号转导与转录激活因子3（STAT3）和CD68抗原的表达上调，进一步明确了乳腺退化窗口期特异性的乳腺癌进展候选介导因子。经产后布洛芬干预后，腱生蛋白C与3个层粘连蛋白亚基的表达出现下调。本研究数据揭示了新的乳腺癌进展相关ECM介导因子，并证实布洛芬可对乳腺ECM组成产生保护性影响。