Singleminded-2s (Sim2s) promotes delayed Involution of the Mouse Mammary Gland through suppression of Stat3 and NFkB

Post-lactational involution of the mammary gland provides a unique model to study breast cancer susceptibility and metastasis. We have shown that the short isoform of Singleminded-2 (Sim2s), a bHLH/PAS transcription factor, plays a role in promoting lactogenic differentiation, as well as in maintaining mammary epithelial differentiation and malignancy. Sim2s is dynamically expressed during mammary gland development, with expression peaking during lactation, and decreasing in early involution. To determine the role of Sim2s in involution, we used transgenic mice expressing Sim2s under the mouse mammary tumor virus (MMTV-Sim2s) promoter. Over-expression of Sim2s in the mouse mammary gland resulted in delayed involution, indicated by a lower proportion of cleaved caspase-3 positive cells and slower re-establishment of the mammary fat pad. Immunohistochemical and quantitative RNA analysis showed a decrease in apoptotic markers and inflammatory response genes, and an increase in anti-apoptotic genes, which were accompanied by inhibition of signal transducer and activator of transcription 3 (Stat3) activity. Microarray analysis confirmed that genes in the Stat3 signaling pathway were repressed by Sim2s expression, along with NFκB and other key pathways involved in mammary gland development. Multiparous MMTV-Sim2s females displayed a more differentiated phenotype compared to wild-type controls, characterized by enhanced β-casein expression and alveolar structures. Together, these results suggest a role for Sim2s in the normal involuting gland, and identify potential down-stream pathways regulated by Sim2s. Gene expression was measured in 6 mammary gland samples using CodeLink Mouse Whole Genome microarrays. 3 samples were from wild-type mice harvested at 72 hours post weaning and 3 samples were MMTV-Sim2s mice harvested at 72 hours post weaning.

泌乳后乳腺复旧是研究乳腺癌易感性与肿瘤转移的独特实验模型。本研究团队此前证实，单minded-2（Singleminded-2）的短异构体（Sim2s）——一种bHLH/PAS转录因子（bHLH/PAS transcription factor）——可促进泌乳相关分化，并维持乳腺上皮分化状态与恶性表型。Sim2s在乳腺发育过程中呈动态表达模式：泌乳期表达量达峰值，复旧早期则表达水平下降。为明确Sim2s在乳腺复旧中的功能，我们构建了以小鼠乳腺肿瘤病毒（mouse mammary tumor virus, MMTV）为启动子表达Sim2s的转基因小鼠（MMTV-Sim2s小鼠）。在小鼠乳腺中过表达Sim2s可导致乳腺复旧延迟，具体表现为裂解型半胱天冬酶-3（cleaved caspase-3）阳性细胞比例降低、乳腺脂肪垫重建速度减慢。免疫组化与定量RNA分析显示，凋亡标志物与炎症应答基因的表达水平下调，抗凋亡基因表达上调，同时伴随信号转导与转录激活因子3（signal transducer and activator of transcription 3, Stat3）活性受抑制。基因芯片分析证实，Sim2s的表达会抑制Stat3信号通路相关基因的转录，同时也下调核因子κB（NFκB）及其他参与乳腺发育的关键信号通路。经产MMTV-Sim2s雌性小鼠相较于野生型对照，呈现更为成熟的分化表型，具体为β-酪蛋白（β-casein）表达增强与肺泡结构发育更完善。综上，上述结果证实了Sim2s在正常乳腺复旧过程中的功能，并明确了其调控的潜在下游信号通路。本研究采用CodeLink小鼠全基因组基因芯片（CodeLink Mouse Whole Genome microarrays）检测了6份乳腺样本的基因表达水平：其中3份取自断奶后72小时的野生型小鼠，剩余3份取自断奶后72小时的MMTV-Sim2s转基因小鼠。