Neuronal-driven glioma growth requires Gαi1 and Gαi3

Abstract

Neuroligin-3 (NLGN3) is necessary and sufficient to promote glioma cell growth. The recruitment of Gαi1/3 to the ligand-activated receptor tyrosine kinases (RTKs) is essential for mediating oncogenic signaling. Methods: Various genetic strategies were utilized to examine the requirement of Gαi1/3 in NLGN3-driven glioma cell growth. Results: NLGN3-induced Akt-mTORC1 and Erk activation was inhibited by decreasing Gαi1/3 expression. In contrast ectopic Gαi1/3 overexpression enhanced NLGN3-induced signaling. In glioma cells, NLGN3-induced cell growth, proliferation and migration were attenuated by Gαi1/3 depletion with shRNA, but facilitated with Gαi1/3 overexpression. Significantly, Gαi1/3 silencing inhibited orthotopic growth of patient-derived glioma xenografts in mouse brain, whereas forced Gαi1/3-overexpression in primary glioma xenografts significantly enhanced growth. The growth of brain-metastatic human lung cancer cells in mouse brain was largely inhibited with Gαi1/3 silencing. It was however expedited with ectopic Gαi1/3 overexpression. In human glioma Gαi3 upregulation was detected, correlating with poor prognosis. Conclusion: Gαi1/3 mediation of NLGN3-induced signaling is essential for neuronal-driven glioma growth.

Keywords: Gαi1/3; NLGN3; Neuron-glioma communication; Signaling.

Figure 1

Gαi1 and Gαi3 double knockout largely inhibits NLGN3-induced Akt, Erk and mTORC1 activation in MEFs. Wild-type (WT), Gαi1 and Gαi3 double knockout (DKO) (A-B), Gαi1, or Gαi3 single knockout (SKO) (C) mouse embryonic fibroblasts (MEFs), or WT MEFs with Gαi1 plus Gαi3 CRISPR/Cas9 KO constructs (“CRISPR-Gαi1/3-DKO”, C), were treated with NLGN3 (50 ng/mL) for applied time, tested by Western blotting of listed proteins in total cell lysates. “MW” stands for molecular weight (Same for all Figures). Data were expressed as mean ± standard deviation (SD, same for all Figures). Quantifications were from five replicate blot data. *P < 0.05.

Figure 2

Gαi1 and Gαi3 are required for NLGN3-induced Akt, Erk and mTORC1 activation in MEFs. WT MEFs with the scramble control shRNA (“scr-shRNA”), the lentiviral Gαi1 shRNA, the lentiviral Gαi3 shRNA, or both (“Gαi1/3 DshRNA”), were treated with NLGN3 (50 ng/mL) for applied time periods, and tested by Western blotting of listed proteins in total cell lysates (A). Gαi1/3 DKO MEFs (B) or WT MEFs (C) were transfected with the adenovirus Gαi1 construct (“Ad-Gαi1”), the adenovirus Gαi3 construct (“Ad-Gαi3”) or the empty vector (“Ad-Vec”), treated with NLGN3 (50 ng/mL) for applied time periods, and tested of listed proteins. Quantifications were from five replicate blot data. *P < 0.05 vs. “scr-shRNA” (A) or WT MEFs (B). ^#P < 0.05. “n.s.” stands for P > 0.05 (no statistical differences, C).

Figure 3

Gαi1 and Gαi3 are essential for NLGN3 signaling in glioma cells. The primary human glioma cells (“P1”) or U251MG cells, with Gαi1 shRNA plus Gαi3 shRNA (“Gαi1/3 DshRNA”) or the scramble control shRNA (“scr-shRNA”), were treated with NLGN3 (50 ng/mL) for applied time periods, listed proteins in total cell lysates or endosomal fractions were tested by Western blotting assays (A-C). P1 glioma cells or U251MG cells were transfected with the adenovirus Gαi1 construct plus the adenovirus Gαi3 construct (“OE-Gαi1/3”, two lines: “sL1/sL2”) or the empty vector (“Ad-Vec”), treated with NLGN3 (50 ng/mL) for 10 min, and tested by Western blotting assays of listed proteins (D). Quantifications were from five replicate blot data. ^#P < 0.05 (A).* P < 0.05 vs. “Ad-Vec” cells (D). “n.s.” stands for P > 0.05 (no statistical differences, C).

Figure 4

Gab1 is a key adaptor protein of NLGN3 signaling. The primary human glioma cells (“P1”) (A) or U251MG cells (B), with Gab1 shRNA or the scramble control shRNA (“scr-shRNA”), were treated with or without NLGN3 (50 ng/mL) for applied time periods, listed proteins in total cell lysates were tested by Western blotting. Wild-type (WT) and Gab1 knockout (Gab1-KO) MEFs were treated with NLGN3 (50 ng/mL) for applied time, tested by Western blotting of listed proteins in total cell lysates (C). U251MG cells were treated with NLGN3 (50 ng/mL) for 5 min, association of Gαi1/3-Gab1-SHP2-p85 was tested by co-IP assays (D). WT, Gαi1/3 DKO (E), Gαi1 or Gαi3 SKO (F), as well as WT MEFs with Gαi1 plus Gαi3 CRISPR/Cas9 DKO constructs (F) or Gαi1 shRNA plus Gαi3 shRNA (“Gαi1/3 DshRNA”) (G) were treated with NLGN3 (50 ng/mL) for applied time, total- and p-Gab1 were tested. Gαi1/3 DKO MEFs were transfected with the adenovirus Gαi1 construct (“Ad-Gαi1”), the adenovirus Gαi3 construct (“Ad-Gαi3”) or the empty vector (“Ad-Vec”), treated with NLGN3 (50 ng/mL) for applied time periods, total- and p-Gab1 were tested (H). Quantifications were from five replicate blot data. ^#P < 0.05.

Figure 5

Gαi1 and Gαi3 mediate NLGN3-induced glioma cell progression in vitro. The P1 primary glioma cells or U251MG cells, with Gαi1 shRNA plus Gαi3 shRNA (“Gαi1/3 DshRNA”) or the scramble control shRNA (“scr-shRNA”), were treated with or without NLGN3 (50 ng/mL) for applied time periods, cell proliferation (A and E), growth (B), and migration (C and E) were examined by the assays mentioned in the text, with Gαi1 and Gαi3 expression tested as well (D, for P1 glioma cells). P1 glioma cells, with the adenovirus Gαi1 construct plus the adenovirus Gαi3 construct (“OE-Gαi1/3”, two lines: “sL1/sL2”) (F-I), were treated with or without NLGN3 (50 ng/mL) for applied time period, cell proliferation (F), growth (G), migration (H) and Gαi1/3 expression (I) were tested, with results quantified. For the EdU staining assay, ten random views were included to calculate EdU/DAPI ratios. For the “Transwell” assays, ten random views of each condition were included to calculate the average number of migrated cells. For all functional assays exact same number of viable cells of different genetic treatments were initially seeded into each well/dish (at 0h). “C” stands for medium control. Blotting data was repeated five times (D and I). *P < 0.05 (A-E). *P < 0.05 vs. “Ad-Vec” cells (F-H). “n.s.” stands for non-statistical difference (A-C). Scale bar=100 μm (A and C).

Figure 6

Gαi1 and Gαi3 are required for orthotopic growth of primary glioma xenografts in mouse brain. Brain MRI images of a primary glioma patient providing primary human glioma cells (“P1” cells, A). The exact same amount of glioma cells (5 × 10⁵ cells of each mouse), expressing Gαi1 shRNA plus Gαi3 shRNA (“Gαi1/3 DshRNA”), scramble non-sense shRNA (“scr-shRNA”), the adenovirus Gαi1 plus Gαi3 constructs (“OE-Gαi1/3”), or the empty vector (“Ad-Vec”), were intracranially injected to brains of nude mice (5-6 week old), after 26/20 days, representative MRI images of orthotopic glioma xenografts were presented (B and G); Animals were decapitated and tumors were isolated by surgery, tumor volumes (C and H) and mice body weights (D and I) were recorded. Tumor tissue lysates were tested by Western blot assay of listed proteins (E and J). Immunohistochemistry (IHC) images of p-Akt and p-S6K were shown (F). Western blotting quantifications were from five replicate blot data. IHC experiments were repeated in three pairs of tissues. ^#P < 0.05. “n.s.” stands for non-statistical difference (D and I). Scale bar=100 μm (F).

Figure 7

Orthotopic growth of brain-metastatic human lung cancer cells requires Gαi1 and Gαi3. Brain MRI images of a primary lung cancer patient with brain metastasis (A) providing the brain-metastatic human lung cancer cells (“bmLCs”). bmLCs (5 × 10⁵ cells of each mouse), expressing Gαi1 shRNA plus Gαi3 shRNA (“Gαi1/3 DshRNA”), scramble non-sense shRNA (“scr-shRNA”), the adenovirus Gαi1 plus Gαi3 constructs (“OE-Gαi1/3”), or the empty vector (“Ad-Vec”), were intracranially injected to brains of nude mice (5-6 week old); After 25/17 days, representative MRI images of orthotopic bmLCs xenografts were presented (B and G); Animals were decapitated and tumors were isolated by surgery, tumor volumes (C and H) and mice body weights (D and I) were recorded. The orthotopic bmLCs xenograft tissue lysates were tested by Western blot assay of listed proteins (E and J). Immunohistochemistry (IHC) images of p-Akt and p-S6K were shown (F). Western blotting quantifications were from five replicate blot data. IHC experiments were repeated in three pairs of tissues.^#P < 0.05. “n.s.” stands for non-statistical difference (D and I). Scale bar=100 μm (F).

Figure 8

Gαi3 upregulation in human glioma tissues. TCGA database shows Gαi3 and NLGN3 expression (RNASeq, RSEM) in glioma tissues and normal brain tissues (A and F). Kaplan Meier Survival analyses of Gαi3-low and Gαi3-high glioma (LGG and GBM) patients (B). Human glioma tissues (“T”, 16 high-grade and 16 low-grade) and the paired normal brain tissues (“N”) were homogenized and dissolved in the tissue lysis buffer, Gαi3 mRNA and protein levels were tested by qPCR (C) and Western blotting (D), respectively, with results quantified. NLGN3 mRNA was tested by qPCR (E). * P < 0.05 vs.“N” group. ^#P < 0.05.