Dontu Gabriela, Jackson Kyle W, McNicholas Erin, Kawamura Mari J, Abdallah Wissam M, Wicha Max S
Comprehensive Cancer Center, Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA.
Breast Cancer Res. 2004;6(6):R605-15. doi: 10.1186/bcr920. Epub 2004 Aug 16.
Notch signaling has been implicated in the regulation of cell-fate decisions such as self-renewal of adult stem cells and differentiation of progenitor cells along a particular lineage. Moreover, depending on the cellular and developmental context, the Notch pathway acts as a regulator of cell survival and cell proliferation. Abnormal expression of Notch receptors has been found in different types of epithelial metaplastic lesions and neoplastic lesions, suggesting that Notch may act as a proto-oncogene. The vertebrate Notch1 and Notch4 homologs are involved in normal development of the mammary gland, and mutated forms of these genes are associated with development of mouse mammary tumors.
In order to determine the role of Notch signaling in mammary cell-fate determination, we have utilized a newly described in vitro system in which mammary stem/progenitor cells can be cultured in suspension as nonadherent 'mammospheres'. Notch signaling was activated using exogenous ligands, or was inhibited using previously characterized Notch signaling antagonists.
Utilizing this system, we demonstrate that Notch signaling can act on mammary stem cells to promote self-renewal and on early progenitor cells to promote their proliferation, as demonstrated by a 10-fold increase in secondary mammosphere formation upon addition of a Notch-activating DSL peptide. In addition to acting on stem cells, Notch signaling is also able to act on multipotent progenitor cells, facilitating myoepithelial lineage-specific commitment and proliferation. Stimulation of this pathway also promotes branching morphogenesis in three-dimensional Matrigel cultures. These effects are completely inhibited by a Notch4 blocking antibody or a gamma secretase inhibitor that blocks Notch processing. In contrast to the effects of Notch signaling on mammary stem/progenitor cells, modulation of this pathway has no discernable effect on fully committed, differentiated, mammary epithelial cells.
These studies suggest that Notch signaling plays a critical role in normal human mammary development by acting on both stem cells and progenitor cells, affecting self-renewal and lineage-specific differentiation. Based on these findings we propose that abnormal Notch signaling may contribute to mammary carcinogenesis by deregulating the self-renewal of normal mammary stem cells.
Notch信号通路与细胞命运决定的调控有关，如成体干细胞的自我更新以及祖细胞沿特定谱系的分化。此外，根据细胞和发育环境的不同，Notch信号通路可作为细胞存活和细胞增殖的调节因子。在不同类型的上皮化生病变和肿瘤性病变中均发现了Notch受体的异常表达，提示Notch可能作为一种原癌基因发挥作用。脊椎动物的Notch1和Notch4同源物参与乳腺的正常发育，这些基因的突变形式与小鼠乳腺肿瘤的发生有关。
为了确定Notch信号通路在乳腺细胞命运决定中的作用，我们利用了一种新描述的体外系统，其中乳腺干/祖细胞可以作为非贴壁的“乳腺球”在悬浮液中培养。使用外源性配体激活Notch信号通路，或使用先前鉴定的Notch信号拮抗剂抑制该通路。
利用该系统，我们证明Notch信号通路可作用于乳腺干细胞以促进其自我更新，并作用于早期祖细胞以促进其增殖，添加Notch激活的DSL肽后次级乳腺球形成增加了10倍即证明了这一点。除作用于干细胞外，Notch信号通路还能够作用于多能祖细胞，促进肌上皮谱系特异性定向分化和增殖。刺激该通路还可促进三维基质胶培养中的分支形态发生。这些效应被Notch4阻断抗体或阻断Notch加工的γ-分泌酶抑制剂完全抑制。与Notch信号通路对乳腺干/祖细胞的作用相反，调节该通路对完全定向分化的乳腺上皮细胞没有明显影响。
这些研究表明，Notch信号通路通过作用于干细胞和祖细胞，影响自我更新和谱系特异性分化，在正常人类乳腺发育中起关键作用。基于这些发现，我们提出异常的Notch信号通路可能通过失调正常乳腺干细胞的自我更新而导致乳腺癌发生。
