ADRB2 Regulates the Proliferation and Metastasis of Gastrointestinal Stromal Tumor Cells by Enhancing the ETV1-c-KIT Signaling

Abstract

Background: Gastrointestinal stromal tumor (GIST) originates from a pacemaker cell, the Cajal cell. However, little is known about the cancer neuroscience in GIST. In this study, we aimed to elucidate the clinical and biological roles of adrenoceptor beta 2 (ADRB2) in GIST.

Methods: Immunohistochemistry was used to evaluate the expression of ADRB2 in GIST tissues. The biological effects of ADRB2 on GIST cell proliferation, migration, invasion, and apoptosis were explored using Cell Counting Kit -8, plate colony formation assay, transwell invasion assay, and flow cytometry. We also explored the growth and metastasis of xenograft tumors in nude mice. Western blotting was used to quantify protein expression and phosphorylation.

Results: ADRB2 is generally highly expressed in GIST. High ADRB2 expression was significantly associated with risk level, tumor size, mitotic count, and metastasis. Overexpression of ADRB2 promoted GIST cell proliferation, migration, invasion, and apoptosis, while silencing ADRB2 expression showed the opposite effects. Furthermore, we found that silencing endogenous ADRB2 inhibited GIST progression and metastasis in nude mice. ADRB2-induced ETV1 upregulation enhanced the activation of c-KIT.

Conclusion: ADRB2 plays an important role in the proliferation and metastasis of GIST and is expected to be a potential target for the treatment of GIST.

Figure 1

High ADRB2 expression is associated with prognosis in patients with gastrointestinal stromal tumors. (a) Kaplan–Meier disease-free survival curves for 122 patients with gastrointestinal stromal tumor stratified by high and low expression of ADRB2. (b) Real-time PCR was used to detect the mRNA levels of ADRB2 in high immunoreactivity-score group compared to low immunoreactivity-score group. (c–f) The mRNA levels of ADRB2 correlate with risk level, tumor size, mitotic count and liver metastasis. (g) Representative staining of ADRB2 and c-KIT in GIST tissues. Magnification: 400×; ^∗∗∗P < 0.001.

Figure 2

ADRB2 regulates tumor proliferation in gastrointestinal stromal tumors. (a) Western blot and real-time PCR confirmed overexpression and knockdown of ADRB2 in GIST-882 and GIST-T1 cells. (b, c) Cell proliferation abilities were detected by CCK-8 assay and colony formation assay. Data are presented as the mean ± SD. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 3

ADRB2 regulates GIST cell migration, invasion, and apoptosis. (a) Cell migration ability was determined by the wound healing assay; (b) transwell assay was used to determine cell invasion ability; (c) cell apoptosis was examined by staining with Annexin V/PI. Data are presented as the mean ± SD. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 4

ADRB2 promotes the proliferation and metastasis of gastrointestinal stromal tumors in animal experiments. (a) Images of excised tumors from nude mice. (b) Tumor growth curves were used to evaluate the growth of xenograft tumors. (c) Representative images of gastric orthotopic tumors. (d) H&E staining of gastric orthotopic tumors. (e) Tumor volume of gastric orthotopic tumors. (f) Representative images of live metastasis form gastric orthotopic tumors. (g) H&E staining of liver metastasis. (h) Number of metastatic nodes. (i, j) In vivo image system was used to show the metastasis in intravenous injection model. (k) Ki67 expression was detected by IHC. Data are presented as the mean ± SD. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 5

ADRB2 enhances the ETV1-c-KIT signaling. (a) Western blotting was used to detect the expression levels of ETV1, c-KIT, and p-ERK (Y204). (b) GIST cells were treated with or without imatinib mesylate, and ETV1, c-KIT, and p-ERK (Y204) expression levels were detected by western blotting.

Figure 6

Schematic representation shows ADRB2 enhancing ETV1-c-KIT signaling by inducing ETV1.