Decreased E‐Cadherin in MCF7 Human Breast Cancer Cells Forming Multicellular Spheroids Exposed to Simulated Microgravity

Abstract

MCF7 human breast cancer cells were cultured under normal gravity (1 g) and on a random positioning machine (RPM) preventing sedimentation. After 2 weeks, adherent 1 g-control and adherent RPM cells (AD) as well as multicellular spheroids (MCS) were harvested. AD and MCS had been exposed to the RPM in the same culture flask. In a subsequent proteome analysis, the majority of the proteins detected showed similar label-free quantification (LFQ) scores in each of the respective subpopulations, but in both AD or MCS cultures, proteins were also found whose LFQs deviated at least twofold from their counterparts in the 1 g-control cells. They included the cell junction protein E-cadherin, which was diminished in MCS cells, where proteins of the E-cadherin autodegradation pathway were enhanced and c-Src (proto-oncogene tyrosine-protein kinase c-Src) was detected. Spheroid formation was prevented by inhibition of c-Src but promoted by antibodies blocking E-cadherin activity. An interaction analysis of the detected proteins that are involved in forming and regulating junctions or adhesion complexes and in E-cadherin autodegradation indicated connections between the two protein groups. This suggests that the balance of proteins that up- or downregulate E-cadherin mediates the tendency of MCF7 cells to form MCS during RPM exposure.

Semantic Analysis of Posttranslational Modification of Proteins Accumulated in Thyroid Cancer Cells Exposed to Simulated Microgravity

“…These proteins were of interest because they were found in spheroid cells but not in control monolayer cells, or were found in spheroid cells at a 1.8-fold higher accumulation than in control cells. In addition, they also were detected by a second independent proteome analysis on MCF-7 breast cancer cells, which grew in a monolayer under normal 1 g laboratory conditions or within spheroids exposed to an RPM for 14 days [23]. Their up-regulation in spheroid cells as compared to control cells was similar to those of the corresponding FTC-133 cells (Table 1).…”

“…[17,23]. Prior to analysis both types of cells had been grown either within a monolayer under normal 1 g laboratory conditions or within spheroids exposed to an RPM [10].…”

“…After harvest, the cells were lysed and subjected to mass spectrometry, obeying the protocols described in refs. [17,23,47,48]. Finally, raw data from the mass spectrometer were processed using MaxQuant (May Planck Society, Munich, Germany) computational proteomics platform (version 1.5.2.22) [49] using the standard parameters.…”

“…It currently is applied in many fields including space research [20,21,22]. Recently, we performed deep proteome analyses on thyroid cancer cells unveiling about 5900 proteins, and on MCF-7 breast cancer cells for comparison, aiming to shed light on the molecular machinery, which enables the cells to move from one kind of growth (monolayer) to another one (spheroid) [17,23]. The studies revealed a number of proteins, whose accumulations were significantly different, when they were found either in monolayer cells of normal 1 g cultures or in spheroids formed under simulated microgravity.…”

“…The studies revealed a number of proteins, whose accumulations were significantly different, when they were found either in monolayer cells of normal 1 g cultures or in spheroids formed under simulated microgravity. Taken together they suggested that there is a relationship between the increment and decrement of distinct proteins and the kind of growth of the cells [17,23]. Furthermore, we observed phosphorylation of profilin-2 and de-phosphorylation of extracellular signal–regulated kinases (ERKs) 1/2 on thyroid cells exposed to microgravity [24,25] and recognized that genes mostly up- and down-regulated during a ten-day space mission coded for enzymes involved in posttranslational modifications (PTM) of target proteins [26].…”