The role of NF\u03baB in spheroid formation of human breast cancer cells cultured on the Random Positioning Machine

Sascha Kopp,Jayashree Sahana,Lasse Slumstrup,Markus Wehland,Asbjørn Graver Petersen,Kathrin Saar,Thomas J Corydon,Johann Bauer,Ronald Luetzenberg,Stefan Riwaldt,Daniela Grimm,Tawhidul Islam,Manfred Infanger,Herbert Schulz,Norbert Huebner

doi:10.1038/s41598-017-18556-8

Abstract

Human MCF-7 breast cancer cells were exposed to a Random Positioning Machine (RPM). After 24 hours (h) the cells grew either adherently within a monolayer (AD) or within multicellular spheroids (MCS). AD and MCS populations were separately harvested, their cellular differences were determined performing qPCR on genes, which were differently expressed in AD and MCS cells. Gene array technology was applied to detect RPM-sensitive genes in MCF-7 cells after 24 h. Furthermore, the capability to form multicellular spheroids in vitro was compared with the intracellular distribution of NF-kappaB (NFκB) p65. NFκB was equally distributed in static control cells, but predominantly localized in the cytoplasm in AD cells and nucleus in MCS cells exposed to the RPM. Gene array analyses revealed a more than 2-fold change of only 23 genes including some whose products are affected by oxygen levels or regulate glycolysis. Significant upregulations of the mRNAs of enzymes degrading heme, of ANXA1, ANXA2, CTGF, CAV2 and ICAM1, as well as of FAS, Casp8, BAX, p53, CYC1 and PARP1 were observed in MCS cells as compared with 1g-control and AD cells. An interaction analysis of 47 investigated genes suggested that HMOX-1 and NFκB variants are activated, when multicellular spheroids are formed.

Highlights

With 3D aggregation, microgravity-induced apoptosis was detected in breast cancer cells[6] like it has been observed in other types of cells[11,12,13]
The principal aim of this paper was to investigate the early phases of Random Positioning Machine (RPM)-exposure (24 h) of Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and to test whether there is a link between enhancement of apoptosis, changes in NFκB expression and spheroid formation
After MCF-7 human breast adenocarcinoma cells had been cultured on the RPM for 24 h, we detected two different phenotypes: Cells growing adherently within a 2D monolayer (AD) and cells growing in form of 3D aggregates exhibiting no glandular structures after this short-term exposure

Summary

Introduction

With 3D aggregation, microgravity-induced apoptosis was detected in breast cancer cells[6] like it has been observed in other types of cells[11,12,13]. In a recent deep proteome analysis the translocation inhibitor IκBKB showed up in AD cells after culturing FTC-133 cells on the RPM, but could not be detected in MCS cells of the same culture flask[17,18]. These observations created the idea that a link between spheroid formation, initiation of apoptosis and NFκB expression may exist[14]. The principal aim of this paper was to investigate the early phases of RPM-exposure (24 h) of MCF-7 breast cancer cells and to test whether there is a link between enhancement of apoptosis, changes in NFκB expression and spheroid formation. We investigated the impact of the poly ADP ribose polymerase (PARP) inhibitor olaparib, the effect of dexamethasone (DEX) and the phosphodiesterase-4 (PDE-4) inhibitor rolipram on spheroid formation

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