Hypoxic Conditions Induce a Cancer-Like Phenotype in Human Breast Epithelial Cells

IntroductionSolid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis.
MethodsNormal human primary breast epithelial cells and immortalized non-malignant mammary epithelial MCF-10A cells were grown in a three-dimensional overlay culture on laminin-rich extracellular matrix for up to 21 days at normoxic or hypoxic conditions. Acinar morphogenesis and expression of markers of epithelial differentiation and cell polarization were analyzed by immunofluorescence, immunohistochemistry, qPCR and immunoblot.
ResultsIn large ductal carcinoma in situ patient-specimens, we find that epithelial cells with high HIF-1α levels and multiple cell layers away from the vasculature are immature compared to well-oxygenated cells. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. Normoxic cultures formed polarized acini-like spheres with the anticipated distribution of marker proteins associated with mammary epithelial polarization e.g. α6-integrin, laminin 5 and Human Milk Fat Globule/MUC1. At hypoxia, cells were not polarized and the sub-cellular distribution pattern of the marker proteins rather resembled that reported in vivo in breast cancer. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state.

【引言】实体瘤的氧合程度低于其起源组织。肿瘤内低氧水平与乳腺癌不良预后、转移潜能增强及未成熟表型相关。本团队此前已报道肿瘤缺氧与乳腺癌低分化状态相关。目前对于缺氧对非恶性细胞的影响尚所知甚少。本研究旨在探讨缺氧是否会影响非恶性乳腺上皮细胞的分化阶段，进而可能与肿瘤发生的早期阶段相关。【方法】将正常人类原代乳腺上皮细胞与永生化非恶性乳腺上皮MCF-10A细胞，在富含层粘连蛋白的细胞外基质上进行三维覆盖培养，于常氧或缺氧条件下培养至多21天。通过免疫荧光、免疫组织化学、定量聚合酶链式反应(qPCR)及免疫印迹法，分析腺泡形态发生情况以及上皮分化、细胞极化相关标志物的表达。【结果】在大型导管原位癌患者标本中，我们发现缺氧诱导因子-1α(HIF-1α)高表达且远离脉管系统多层细胞的上皮细胞，相较于氧合良好的细胞呈现未成熟表型。我们证实，在富含层粘连蛋白的基质上离体培养时，缺氧条件会损害原代及永生化乳腺上皮细胞的腺泡形态发生。常氧培养物可形成极化的腺泡样球体，其标志物蛋白分布符合乳腺上皮极化的预期特征，例如整合素α6(α6-integrin)、层粘连蛋白5(laminin 5)及人乳脂肪球蛋白/MUC1(Human Milk Fat Globule/MUC1)。而在缺氧条件下，细胞无法发生极化，标志物蛋白的亚细胞分布模式与体内乳腺癌中报道的特征相似。缺氧细胞维持在有丝分裂状态，而常氧条件下腺泡形态发生时细胞增殖停止。我们发现，未分化的缺氧MCF-10A细胞结构中，分化抑制因子ID1的表达被诱导上调。腺泡形态发生伴随整体组蛋白去乙酰化，而缺氧乳腺上皮细胞则表现出持续的整体组蛋白乙酰化——这一状态通常与活跃转录及未分化的增殖状态相关。