Translocation of Epidermal Growth Factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage

Abstract

Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10 min of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10 min of stimulation with EGF. However, the total volume of EGF clusters increases only after 20 min in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20 min of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study of EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.

Epidermal growth factor (EGF) triggers nuclear calcium signaling through the intranuclear phospholipase Cδ-4 (PLCδ4)

“…Stimulation with EGF induces its RTK EGFR to translocate to the nucleus (18), similar to what has been shown for other RTKs, including the hepatocyte growth factor (HGF) receptor, c-Met, and the insulin receptor (7,8). EGFR translocated to the nucleus in both primary rat hepatocytes and SKHep-1 cells, a human liver cancer cell line (Fig.…”

“…For example, c-Met that is biotinylated at the plasma membrane can be recovered from the nucleus after cells are stimulated with HGF (7). Similarly, fluorescentlylabeled EGF can be tracked from the cell membrane to the cell nucleus over a 10-min period (18). Furthermore, super-resolution imaging can be used to quantify the amount of EGF/EGFR clusters that accumulate in the nucleus (18).…”

“…Similarly, fluorescentlylabeled EGF can be tracked from the cell membrane to the cell nucleus over a 10-min period (18). Furthermore, super-resolution imaging can be used to quantify the amount of EGF/EGFR clusters that accumulate in the nucleus (18). The peak in nuclear translocation of EGFR coincides with the peak in Ca 2ϩ signals (Fig.…”

“…Confocal and super-resolution immunofluorescence imagings were performed as described previously (18). In brief, cells were labeled with rabbit polyclonal anti-EGFR (1:250, Santa Cruz Biotechnology, catalog no.…”

“…Nuclear EGFR function has been the subject of extensive investigation, as has been nuclear targets of EGFR. EGFR can shuttle from the cell surface to the nucleus (17,18) where it acts as a transcriptional regulator (19,20), transmits signals (21,22), and is involved in processes such as cell proliferation, tumor progression, and DNA repair and replication (23). However, it is unknown whether EGFR is linked to nuclear calcium release.…”