DRG Neurons Promote Perineural Invasion of Endometrial Cancer via GluR2

Abstract

Background: Perineural invasion (PNI) is correlated with negative prognosis in multiple cancers, but its role in endometrial cancer (EC) is still largely unknown; thus, targeted treatment for nerve infiltration is lacking as well. Methods: The interaction between nerve and EC cells were investigated by in vitro neural invasion assay and transwell coculture system. Then the nerve-related receptor gene glutamate ionotropic receptor AMPA type subunit 2 (GRIA2) was detected in EC tissues and cells using PCR array, western blotting, and immunohistochemistry. The role of GluR2 (gene name GRIA2) on EC proliferation, migration and invasion was evaluated by a GluR2 antagonist and shRNA. At the same time, the neurotransmitter effect on GluR2 (glutamate) from the cocultured conditional medium was measured using high-performance liquid chromatography (HPLC). Results: EC cell line Ishikawa (ISK) showed the ability to migrate along neurites in vitro and the numbers of migrated/invaded EC cells in the DRG neuron coculture group were significantly increased. The expression of GluR2 in EC tissue was found to be higher than that in para-carcinoma tissue. After GluR2 antagonist and GluR2 shRNA treatment, the proliferation, migration and invasion of ISK cells was markedly inhibited. Moreover, the ability of DRG neurons to promote the migration and invasion of ISK cells could also be attenuated by downregulation of GluR2, and the concentration of the neurotransmitter glutamate was notably increased in the coculture conditional medium compared to that in the DRG neuron or ISK cells alone. Conclusions: DRG neurons promote metastasis of EC cells via GluR2, which might be a risk factor for PNI in EC. Moreover, the perineural system may promote tumor invasion and metastasis under certain circumstances.

Keywords: DRG neurons; GluR2; endometrial cancer; metastasis; perineural invasion.

Figure 1

DRG neurons promote the migration and invasion of EC cells. (A) EC tissue with or without PNI was stained in Hematoxylin and eosin (HE). The nerve area was marked by dashed lines. N = nerves; C = cancer cells. (B-C) DRG was placed in the center of Matrigel. The interaction of EC cell line and DRG was confirmed in bright field (black arrow) and immunofluorescence staining (yellow arrow). ISK cells were stained by pan-cytokeratin (pan-CK), green, scale bar, 100 μm. (D) Representative bright field image of DRG neurons after culture for 3 days (black arrow); scale bar, 50μm. (E) Immunofluorescence staining of DRG neurons. The dissociated DRG neurons were defined as DAPI+/MAP2+ (red arrow: DRG neuron, yellow arrow: nerve fiber); scale bar, 50μm. (F) Transwell coculture systems were involved to evaluate the migration/invasion effect of DRG neurons on EC cells; scale bar, 100μm. (G) The migrated/invaded cells were then counted in five random fields and statistically analyzed. Data are presented as the mean±SD; **P<0.01.

Figure 2

Expression of nerve-related receptor genes in EC tissue and cell lines. (A) Clustered heat map of differentially expressed nerve-related genes in endometrial cancer and para-cancer normal tissue (C1-C5 represents cancer tissues, N1-N3 represents normal tissues); 45 genes displayed a ≥2-fold increase or decrease in expression level. Each color patch represents the expression level of related genes with a continuum of expression levels from blue (lowest) to red (highest). (B-G) Expression of interested nerve-related receptor genes in EC cells lines (Ishikawa, HEC-1A and KLE) detected at the mRNA level; *P<0.05.

Figure 3

GluR2 is expressed in EC cell lines and tissues. (A-D) IHC analysis of GluR2 in normal endometrium (N), atypical hyperplasia endometrium (AH), EC and stage I-III EC tissue. The mean level of GluR2 (encoded by GRIA2 gene) was significantly higher in AH than in normal and EC groups, whereas no significant difference was detected in different EC stages. Semiquantitative analysis of IHC staining was practiced among the three groups; scale bar, 50μm. (E, F) The expression of GluR2 at the protein level in EC cell lines was detected by western blotting, and the results were further quantified by densitometry three times. (G) Representative image of neuro-filament light chain (NF-L) in AH tissues (the long and linear fiber stained in green, red arrow); scale bar, 50μm. (H) The number of nerve fibers in normal, AH and EC tissues were counted in 10 random fields/slide and statistically analyzed; scale bar, 50μm. Data are presented as the mean ± SD; *P< 0.05.

Figure 4

GluR2 antagonist decreases proliferation, migration and invasion of EC cells. (A-B) Ishikawa cells were treated with GluR2 agonist from 10nM to 100μM or antagonist from 0.1μM to 10μM for 0-96 h. CCK-8 assay was used to test the cell proliferation ability. (C-D) ISK cells treated with GluR2 agonist/antagonist (both at 10μM) and its control were subjected to wound-healing migration assay. Representative images of wounds at different time points (0, 24, 48 h) were displayed, and the percentage of wound closure was measured (percentage of wound healing: 0-24 h width of wound/0 h width of wound or 0-48 h width of wound/0 h width of wound). (E-G) Transwell migration/invasion assays were applied to evaluate the ISK cells while treated with GluR2 agonist or antagonist; scale bar, 50μm. Data are presented as the mean ± SD; *P< 0.05.

Figure 5

Knockdown of GluR2 decreases proliferation, migration and invasion of endometrial cancer cells. (A-B) Ishikawa cells were transfected with negative control shRNA or shRNA-GRIA2 (50, 51, 52). Downregulation of GRIA2 was verified in whole cell lysates from these cells at both the RNA and protein level, especially in the shRNA-50 group. (C) After infection with GRIA2 shRNA, stable colonies were selected using 1μg/mL of puromycin, and the proliferation rate of cells (both control and infected cells) were detected using the CCK-8 assay at different time points (0, 24, 48, 72, and 96 h). (D) Transwell assay was used to test the migration and invasion ability of EC cells (including control ISK cells, ISK+DRG neurons, ISK+DRG neurons+GluR2 antagonist at 10μM and ISK-shRNA + DRG neurons); scale bar, 50μm. (E- F) The migrated/invaded cells were counted in five random fields and statistically analyzed. Data are presented as the mean ± SD. (G- H) The concentration of glutamic acid in four different groups were tested by HPLC; *P < 0.05, **P < 0.01.

Figure 6

DRG neurons promote perineural invasion (PNI) of EC cells via GluR2. DRG neurons could release more glutamic acid when cocultured with EC cells. Then, the high-concentration glutamic acid activates tumor-expressed GluR2. The GluR2 activation promotes the migration and invasion of EC cells and contributes to PNI in EC as well. Although both endogenous and exogenous knockdown of GluR2 inhibited the ability of metastasis in EC cells, targeting GluR2 might block PNI by disrupting nerve-cancer crosstalk.