The cell surface mucin podocalyxin regulates collective breast tumor budding

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.

Interactive dynamics of matrix adhesion and reaction-diffusion predict diverse multiscale strategies of cancer cell invasion

“…Our previously published experiments, which were crucial to the development of the computational model we have explored here, described multiscale invasion, which is phenomenologically similar to tumor budding. Indeed, early investigations by others confirm our empirical and theoretical findings that surface proteins and ECM play key roles contributing to multiscale invasion (Graves et al, 2016;Masaki et al, 2003).…”

A novel 3D in vitro metastasis model elucidates differential invasive strategies during and after breaching basement membrane

“…This highlights the importance of understanding the cellular and molecular underpinnings of collective cancer cell invasion and the need for physiologically relevant in vitro models supporting this crucial mode of invasion. To date, in vitro settings for the study of collective cancer cell migration have relied primarily on 2D scratch/wound assays or on assays using spheroids or organoids embedded in 3D matrices, typically composed of collagen I or BME [48][49][50][51][52]. We and others have reported differential invasive behavior for cancerous cells in fibrillar (collagen I) vs. non-fibrillar (BME) 3D matrices, with collagen I typically being more supportive of invasion than is non-fibrillar BME, which did not lead to invasion in either spheroids or organoids of known tumorigenic breast, ovarian and prostate cancer cells [27,[52][53][54].…”

Emerging Role of Podocalyxin in the Progression of Mature B-Cell Non-Hodgkin Lymphoma

“…In MCF-7 breast cancer cells, forced expression of PODXL perturbed cell-cell junctions, a process which could facilitate breast carcinoma invasion [22]. Additionally, PODXL has been shown to induce collective tumor migration and invasion, as well as tumor budding of MCF-7 cells both in vitro and in vivo [84]. Furthermore, in MCF-7 breast cancer and P3C prostate cancer cell lines, PODXL enhanced cell migration and invasion, matrix metalloproteinase 1 and 9 expression, and activation of MAPK and PI3K activity through its interaction with ezrin in in vitro assays [85].…”