The cell surface mucin podocalyxin regulates collective breast tumor budding.

Marcia L Graves,Kelly M Mcnagny,Calvin D Roskelley,Pamela Austin,Erin M Bell,C Blake Gilks,Jane A Cipollone,Julie S Nielsen

doi:10.1186/s13058-015-0670-4

Marcia L Graves, Kelly M Mcnagny + Show 6 more

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https://doi.org/10.1186/s13058-015-0670-4

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Abstract

BackgroundOverexpression 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.MethodsMCF-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.ResultsForced 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.ConclusionsPodocalyxin is a tumor cell-intrinsic regulator of experimental collective tumor cell invasion and tumor budding.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0670-4) contains supplementary material, which is available to authorized users.

Highlights

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
Podocalyxin overexpression expands apical membrane domains on the free surface of epithelial breast cancer cells that are maintained in monolayer culture where it alters the subcellular localization of two associated actin-binding scaffolding proteins, NHERF-1 and ezrin [34], both of which have been implicated in breast tumor progression when they are mistargeted [35,36,37]
Casey and colleagues demonstrated that a podocalyxin–ezrin complex increases breast tumor motility [38]. These findings, coupled with the recent observation that podocalyxin overexpression is positively correlated with lymphovascular invasion (LVI) in breast cancer [31], which itself is well correlated with tumor budding [8], led us to ask whether podocalyxin overexpression plays a functional role in collective breast tumor cell invasion

Summary

Introduction

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. Mostov and colleagues recently demonstrated that podocalyxin can cause normal epithelial cells to become collectively invasive in three-dimensional (3-D) culture when polarity cues are actively disrupted such that apical membrane domains and lumens do not form efficiently [19]. Casey and colleagues demonstrated that a podocalyxin–ezrin complex increases breast tumor motility [38] These findings, coupled with the recent observation that podocalyxin overexpression is positively correlated with lymphovascular invasion (LVI) in breast cancer [31], which itself is well correlated with tumor budding [8], led us to ask whether podocalyxin overexpression plays a functional role in collective breast tumor cell invasion

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