Function and biomedical implications of myoglobin in breast cancer

肌红蛋白在骨骼和心肌细胞中被称为O2结合呼吸蛋白，在过去10年中也在各种肿瘤实体中被检测到，主要是上皮起源的。例如，在管腔性乳腺癌中，珠蛋白的表达与总体生存率的增加显著相关，这表明MB具有肿瘤抑制作用。我们已经报道过，癌细胞中的MB转录是由另一种基因调控机制维持的，这种机制不适用于肌细胞。然而，MB在肿瘤中的生理作用仍然不明确。我们的初步工作表明，珠蛋白在肿瘤细胞内的细胞供氧中并不发挥其经典功能。相反，我们能够通过全面的RNA-Seq分析和验证Western Blot实验表明，RNAi介导的MB击倒的细胞反应严重影响主要转录因子HIF1a和p53的目标基因的差异表达。我们的基本工作模型表明，MB通过其在缺氧条件下产生NO或在常氧条件下清除NO的酶功能来调节这两种转录因子的活性，这些功能已经在MB的平滑肌肉中描述过了。没有分子，也可能是MB产生的ROS，可以显著诱导野生型P53、突变P53和HIF1a的活性。我们的初步数据进一步表明，MB的存在/缺失也会影响癌细胞的表型，例如导致凋亡细胞数量和迁移/侵袭能力的改变。因此，珠蛋白似乎在肿瘤细胞新陈代谢中起着关键作用。然而，有趣的是，观察到的影响与细胞的p53基因不同。在这个项目提案中，我们打算根据我们的工作假设，研究MB在癌细胞中的作用。在不同的生理O2和NO条件下，针对不同的P53基因类型，我们将首先验证PHD2/HIF1a和P53在有无MB存在的乳腺癌细胞中的活性。我们将进一步研究转录因子目标基因的表达，以及由此产生的细胞代谢、细胞周期进展和细胞迁移能力的适应。然后，在细胞模型中描述的MB依赖的蛋白质相互作用和细胞级联将在体内研究MB表达或以MB基因敲除基因为特征的小鼠乳腺肿瘤模型。我们进一步计划继续研究MB、P53和HIF1a靶基因在人类乳腺癌活检组织中的表达与其P53基因型和其他临床病理参数的相关性。我们的项目有望获得关于MB在癌细胞中的肿瘤抑制作用的基础知识。此外，对MB在癌细胞中的作用的理解将可能为开发新的诊断和治疗方法铺平道路。

Myoglobin, known as an O2-binding respiratory protein in skeletal and cardiac myocytes, has also been detected in the past 10 years in various tumor entities, mostly of epithelial origin. For example, the globin expression is significantly correlated to increased overall survival rates in luminal breast cancers, suggesting a tumor-suppressive role of MB. We already reported that MB transcription in cancer cells is maintained by an alternative gene regulatory machinery, which is not used in myocytes. The physiological role of MB in tumors, however, remains ambiguous. Our preliminary work suggests that the globin does not exert its classic function in cellular O2-supply within tumor cells. Instead, we were able to show by comprehensive RNA-Seq analyses and verifying Western Blot experiments that the cellular response of an RNAi-mediated MB-knockdown severely impacts the differential expression of target genes of the master transcription factors HIF1a und p53. Our fundamental working model implies that MB modulates the activity of both transcription factors via its enzymatic functions in producing NO under hypoxia or scavenging NO under normoxia, functions which have already been described for MB e.g. in the context of smooth muscles. NO molecules, and possibly also MB-produced ROS, were previously reported to significantly induce the activity of wildtype p53, mutated p53 and HIF1a. Our preliminary data further imply that the presence/absence of MB also impacts the cancer cell phenotype, e.g. resulting in altered amounts of apoptotic cells and migration/invasion capacities. The globin thus appears to play a key role in tumor cell metabolism. Interestingly, the observed effects however differ with regard to the p53 genotype of the cell.In this project proposal, we intend to study the role of MB in cancer cells, as based on our working hypotheses. At different physiological O2 and NO conditions and with respect to different p53 genotypes, we will first validate the activity of PHD2/HIF1a and p53 in the presence or absence of MB in breast cancer cells. We will further examine the expression of the transcription factors target genes and resulting adaptions in cellular metabolism, cell-cycle progression and cell migration capacities. MB-dependent protein interactions and cellular cascades described in cell models will then be investigated in vivo in mouse breast tumor models who are either MB-expressing or feature an MB-knockout genotype. We further plan to continue studying the expression correlations between MB, p53 and HIF1a target genes in human breast cancer biopsies with respect to their p53-genotype and additional clinicopathological parameters.Our project promises gaining fundamental knowledge on the tumor-suppressing role of MB in cancer cells. Furthermore, the achieved understanding of the role of MB in cancer cells will potentially pave the way for developing novel diagnostic and therapeutic approaches.