Graves Marcia L, Cipollone Jane A, Austin Pamela, Bell Erin M, Nielsen Julie S, Gilks C Blake, McNagny Kelly M, Roskelley Calvin D
Department of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada.
Deeley Cancer Research Center, 2410 Lee Avenue, Victoria, V8R 6V5, BC, Canada.
Breast Cancer Res. 2016 Jan 22;18(1):11. doi: 10.1186/s13058-015-0670-4.
Overexpression of the transmembrane sialomucin podocalyxin, which is known to play a role in lumen formation during polarized epithelial morphogenesis, is an independent indicator of poor prognosis in a number of epithelial cancers, including those that arise in the breast. Therefore, we set out to determine if podocalyxin plays a functional role in breast tumor progression.
MCF-7 breast cancer cells, which express little endogenous podocalyxin, were stably transfected with wild type podocalyxin for forced overexpression. 4T1 mammary tumor cells, which express considerable endogenous podocalyxin, were retrovirally transduced with a short hairpin ribonucleic acid (shRNA) targeting podocalyxin for stable knockdown. In vitro, the effects of podocalyxin on collective cellular migration and invasion were assessed in two-dimensional monolayer and three-dimensional basement membrane/collagen gel culture, respectively. In vivo, local invasion was assessed after orthotopic transplantation in immunocompromised mice.
Forced overexpression of podocalyxin caused cohesive clusters of epithelial MCF-7 breast tumor cells to bud off from the primary tumor and collectively invade the stroma of the mouse mammary gland in vivo. This budding was not associated with any obvious changes in histoarchitecture, matrix deposition or proliferation in the primary tumour. In vitro, podocalyxin overexpression induced a collective migration of MCF-7 tumor cells in two-dimensional (2-D) monolayer culture that was dependent on the activity of the actin scaffolding protein ezrin, a cytoplasmic binding partner of podocalyxin. In three-dimensional (3-D) culture, podocalyxin overexpression induced a collective budding and invasion that was dependent on actomyosin contractility. Interestingly, the collectively invasive cell aggregates often contained expanded microlumens that were also observed in vivo. Conversely, when endogenous podocalyxin was removed from highly metastatic, but cohesive, 4T1 mammary tumor cells there was a decrease in collective invasion in three-dimensional culture.
Podocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.
跨膜唾液酸黏蛋白足突细胞黏蛋白的过表达在极化上皮形态发生过程中的管腔形成中起作用，是包括乳腺癌在内的多种上皮癌预后不良的独立指标。因此，我们着手确定足突细胞黏蛋白在乳腺肿瘤进展中是否发挥功能作用。
内源性足突细胞黏蛋白表达很少的MCF-7乳腺癌细胞用野生型足突细胞黏蛋白进行稳定转染以实现强制过表达。内源性足突细胞黏蛋白表达相当多的4T1乳腺肿瘤细胞用靶向足突细胞黏蛋白的短发夹核糖核酸（shRNA）进行逆转录病毒转导以实现稳定敲低。在体外，分别在二维单层和三维基底膜/胶原凝胶培养中评估足突细胞黏蛋白对集体细胞迁移和侵袭的影响。在体内，在免疫受损小鼠原位移植后评估局部侵袭情况。
足突细胞黏蛋白的强制过表达导致上皮性MCF-7乳腺肿瘤细胞的黏附簇从原发性肿瘤上脱落，并在体内集体侵袭小鼠乳腺的基质。这种出芽与原发性肿瘤的组织结构、基质沉积或增殖的任何明显变化无关。在体外，足突细胞黏蛋白过表达在二维（2-D）单层培养中诱导MCF-7肿瘤细胞的集体迁移，这依赖于肌动蛋白支架蛋白埃兹蛋白的活性，埃兹蛋白是足突细胞黏蛋白的细胞质结合伴侣。在三维（3-D）培养中，足突细胞黏蛋白过表达诱导集体出芽和侵袭，这依赖于肌动球蛋白收缩性。有趣的是，集体侵袭的细胞聚集体通常含有扩大的微管腔，在体内也观察到了这种微管腔。相反，当从高转移性但黏附性的4T1乳腺肿瘤细胞中去除内源性足突细胞黏蛋白时，三维培养中的集体侵袭减少。
足突细胞黏蛋白是实验性集体肿瘤细胞侵袭和肿瘤出芽的肿瘤细胞内在调节因子。
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