Transcription profiling of mouse WT and Cav-3 knockouts on FVB/N genetic background mammary glands reveals loss of caveolin-3 induces the development of a lactogenic microenvironment

Here, we show that functional loss of a single gene is sufficient to confer constitutive milk protein production and protection against mammary tumor formation. Caveolin-3 (Cav-3), a muscle-specific caveolin-related gene, is highly expressed in striated and smooth muscle cells. We demonstrate that Cav-3 is also expressed in myoepithelial cells within the mammary gland. To determine if genetic ablation of Cav-3 expression affects adult mammary gland development, we next studied the phenotype(s) of Cav-3 (-/-) null mice. Interestingly, detailed analysis of Cav-3 (-/-) virgin mammary glands shows dramatic increases in ductal thickness, side-branching, and the development of extensive lobulo-alveolar hyperplasia, akin to the changes normally observed during pregnancy and lactation. Analysis by genome-wide expression profiling reveals the upregulation of gene transcripts associated with pregnancy/lactation, mammary stem cells, and human breast cancers, consistent with a constitutive lactogenic phenotype. The expression levels of three key transcriptional regulators of lactation, namely Elf5, Stat5a, and c-Myc are also significantly elevated. Experiments with pregnant mice directly show that Cav-3 (-/-) mice undergo precocious lactation. Finally, using orthotopic implantation of a transformed mammary cell line (known as Met-1), we demonstrate that virgin Cav-3 (-/-) mice are dramatically protected against mammary tumor formation. Interestingly, Cav-3 (+/-) mice also show similar protection, indicating that even reductions in Cav-3 levels are sufficient to render these mice resistant to tumorigenesis. Thus, Cav-3 (-/-) mice are a novel preclinical model to study the protective effects of a constitutive lactogenic microenviroment on mammary tumor onset and progression. Our current studies have broad implications for using the lactogenic micro-environment as a paradigm to discover new therapies for the prevention and/or treatment of human breast cancers. Most importantly, a lactation-based therapeutic strategy would provide a more natural and nontoxic approach to the development of novel anti-cancer therapies. Experiment Overall Design: All WT and Cav-3 knockout (KO) mice used in this study were in the FVB/N genetic background. 4-month old virgin female mice were utilized in a micro array study between 3 wild type and 3 Caveolin-3 knock-out mammary glands.本研究证实，单个基因的功能缺失足以使机体获得持续性乳蛋白合成能力，并可抵御乳腺肿瘤发生。小窝蛋白3（Caveolin-3, Cav-3）作为一种肌肉特异性小窝相关基因，在横纹肌与平滑肌细胞中呈高表达。本研究发现，Cav-3在乳腺的肌上皮细胞中同样存在表达。 为探究Cav-3表达的基因敲除是否会影响成年乳腺发育，我们后续对Cav-3敲除纯合（Cav-3⁻/⁻）小鼠的表型展开了研究。值得注意的是，对未交配雌性Cav-3⁻/⁻小鼠乳腺的详细分析显示，其导管厚度、侧支分支以及广泛的腺泡-肺泡增生均出现显著增加，这与妊娠及泌乳期的正常生理变化高度相似。 全基因组表达谱分析结果表明，与妊娠/泌乳、乳腺干细胞及人类乳腺癌相关的基因转录本均发生上调，这与持续性泌乳表型的特征一致。此外，三种关键泌乳转录调控因子——Elf5、Stat5a及c-Myc的表达水平也显著升高。 对妊娠小鼠的直接实验证实，Cav-3⁻/⁻小鼠会出现泌乳提前的现象。最后，通过原位移植转化乳腺细胞系Met-1的实验，我们发现未交配的Cav-3⁻/⁻小鼠可显著抵御乳腺肿瘤的发生。有趣的是，Cav-3杂合（Cav-3⁺/⁻）小鼠同样表现出类似的肿瘤抵抗效果，这表明即便仅降低Cav-3的表达水平，也足以使小鼠对肿瘤发生产生抗性。 因此，Cav-3⁻/⁻小鼠是一种新型临床前模型，可用于研究持续性泌乳微环境对乳腺肿瘤发生及进展的保护作用。本研究的成果为以泌乳微环境为范式开发人类乳腺癌预防与治疗新疗法提供了广泛的理论支撑。尤为重要的是，基于泌乳的治疗策略将为新型抗癌疗法的开发提供一种更天然、无毒的途径。 实验总体设计：本研究中使用的所有野生型（Wild Type, WT）及Cav-3敲除（Knockout, KO）小鼠均为FVB/N遗传背景。我们选取4月龄未交配雌性小鼠，对3只野生型与3只Cav-3敲除小鼠的乳腺组织进行了基因芯片分析。