Bombesin Receptor Subtype-3 Regulates Tumor Growth by HER2 Tyrosine Phosphorylation in a Reactive Oxygen Species-Dependent Manner in Lung Cancer Cells

Abstract

Bombesin receptor subtype-3 (BRS-3) is a type 1 G-protein-coupled receptor. BRS-3 is an orphan GPCR which is structurally related to the neuromedin B and gastrin-releasing peptide receptors. When activated, BRS-3 causes phosphatidylinositol turnover in lung cancer cells. BRS-3 stimulates tyrosine phosphorylation of the epidermal growth-factor receptor (ErbB1), however it is unknown if it transactivates ErbB2/HER2. Adding the nonpeptide BRS-3 allosteric-agonist, MK-5046 or the peptide agonist, BA1 to the lung cancer cell line, NCI-H727 or BRS-3-transfected NCI-H1299 lung cancer cells, increased tyrosine phosphorylation of HER2/ERK2. This increase was antagonized by the BRS-3 peptide antagonist, Bantag-1 and the small molecule BRS-3 antagonist, ML-18. The increase in HER2/ERK phosphorylation caused by MK-5046 was inhibited by ROS inhibitors, N-acetylcysteine and Tiron (superoxide-scavenger). Adding MK-5046 to lung cancer cells increased reactive-oxygen species which was inhibited by NAC or Tiron. MK-5024 and BA1 increased NSCLC colony formation whereas, Bantag-1/ML-18 inhibited proliferation. These results indicate that in lung cancer cells activation of BRS-3 regulates HER2-transactivation in ROS-dependent manner which can mediate tumor growth. These results raise the possibility that the use of HER2-inhibiting compounds alone or in combination with other agents, could be a novel approach in the treatment of these tumors.

Keywords: BRS-3; HER2; ROS; lung cancer; proliferation μ; transactivation.

Fig. 1.. Western blot

The presence of BRS-3, EGFR, HER2 and tubulin was determined in 5 NSCLC cell lines. This experiment is representative of 3 others.

Fig. 2.. Effects of BRS-3 agonists.

(A) The presence of PY¹²⁴⁸-HER2, P-ERK and tubulin were determined using 0.01, 0.1 and 1 μM MK-5046 dose and BRS-3-H1299 cells. (B, C). The PY¹²⁴⁸-HER2, P-ERK and tubulin are shown 5, 10 of 20 min after addition of 0.1 μM MK-5046 to BRS-3-H1299 cells. (D). The presence of PY¹²⁴⁸-HER2 (E) and P-ERK (F) was determined as a function of time and the mean value ± S.D. of 3 determinations is indicated; p < 0.05, *; p < 0.01, **; relative to the control by ANOVA.

Fig. 3.. Effects of BRS-3 ligands.

Results from both immunoprecipitation studies (A-C) and direct Western blotting (D-F) are shown. In (A), extracts from BRS-3-H1299 cells were immuno-precipitated with anti- and probed with anti-PY¹²⁴⁸-HER2 (Top Layer) or immune-precipitated with anti-HER2 and probed with anti-PY¹⁰⁶⁸-EGFR (Bottom Layer). In panels B and C cells were stimulated with BA1 (100 nM) (Lane 2) or MK-5046 (0.1 μM) (Lane 3). Western blotting with PY¹²⁴⁸-HER2 (B) or PY¹⁰⁶⁸-EGFR (C) was done after immunoprecipitation with anti-EGFR or anti-HER2, respectively. Panel D shows Western blotting results of PY¹²⁴⁸-HER2 and P-ERK after incubating BRS-3-H1299 cells with 0.1 μM MK-5046. Panels E and F shows Western blotting results of PY¹²⁴⁸-HER2 and P-ERK, respectively, after incubating BRS-3-H1299 cells with 0.1 μM MK-5046 and inhibiting with Bantag-1 (Lane 3) or ML-18 (Lane 4). The mean value ± S.D. of 3 determinations is indicated; p < 0.05 *; p < 0.01**; relative to the control by ANOVA.

Fig. 4.. ROS inhibitors.

Adding 100 nM MK-5046 to BRS-3-H1299 cells increased PY¹²⁴⁸-HER2 and P-ERK (A). NAC (10 mM) or Tiron (5 mM) impaired the ability of MK-5046 to increase PY¹²⁴⁸-HER2 (B) or P-ERK (C). The mean value ± S.D. of 3 determinations is indicated; p < 0.05, *; p < 0.01 **; relative to the control by ANOVA.

Fig. 5.. MTT growth assay.

The effects of varying doses of Bantag-1 (● ) and ML-18 (○) on the proliferation of NCI-H727 (A) and BRS-3-H1299 (B) cells were investigated. The mean value ± S.D. of 8 determinations is indicated; p < 0.05 *; p < 0.01 **; relative to the control by ANOVA.