Membrane progesterone receptor α (mPRα) enhances hypoxia-induced vascular endothelial growth factor secretion and angiogenesis in lung adenocarcinoma through STAT3 signaling

Abstract

Lung cancer remains a huge challenge to public health because of its high incidence and mortality, and lung adenocarcinoma (LUAD) is the main subtype of lung cancer. Hypoxia-induced vascular endothelial growth factor (VEGF) release and angiogenesis have been regarded as critical events in LUAD carcinogenesis. In the present study, membrane progesterone receptor α (mPRα) is deregulated within LUAD tissue samples; increased mPRα contributes to a higher microvessel density (MVD) in LUAD tissues. mPRα knockdown in A549 and PC-9 cells significantly inhibited STAT3 phosphorylation, as well as HIF1α and VEGF protein levels, decreasing cancer cell migration and invasion. The in vivo xenograft model further confirmed that mPRα enhanced the aggressiveness of LUAD cells. Furthermore, mPRα knockdown significantly inhibited hypoxia-induced upregulation in HIF1α and VEGF levels, as well as LUAD cell migration and invasion. Under the hypoxic condition, conditioned medium (CM) derived from mPRα knockdown A549 cells, namely si-mPRα-CM, significantly inhibited HUVEC migration and tube formation and decreased VEGF level in the culture medium. In contrast, CM derived from mPRα-overexpressing A549 cells, namely mPRα-CM, further enhanced HUVEC migration and tube formation and increased VEGF level under hypoxia, which was partially reversed by STAT3 inhibitor Stattic. In conclusion, in LUAD cells, highly expressed mPRα enhances the activation of cAMP/JAK/STAT3 signaling and increases HIF1α-induced VEGF secretion into the tumor microenvironment, promoting HUVEC migration and tube formation under hypoxia.

Keywords: Angiogenesis; Hypoxia; Lung adenocarcinoma; Membrane progesterone receptor α (mPRα); STAT3 signaling; Vascular endothelial growth factor (VEGF).

Fig. 1

Expression of mPRα in tissue samples. A The mRNA expression of mPRα in 483 lung adenocarcinoma (LUAD) tissue samples and 347 noncancerous tissue samples according to TCGA database. B Cases from the TCGA-LUAD database were divided into two groups using the best cutoff of mPRα expression. The correlation between mPRα expression and the overall survival in patients with LUAD was analyzed by KMPLOT (https://kmplot.com/analysis/). C The mRNA expression of mPRα was determined in 20 paired LUAD and adjacent noncancerous tissue samples using real-time PCR. D The protein content and distribution of mPRα were determined in LUAD and adjacent noncancerous tissue samples using immunohistochemical (IHC) staining. E The protein content and distribution of mPRα were determined in LUAD and adjacent noncancerous tissue samples using Immunoblotting. F The CD31-MVD was determined in LUAD tissues samples with a high or low mPRα level. **P < 0.01, ***P < 0.005

Fig. 2

mPRα activates cAMP/JAK/STAT3 signaling pathway in LUAD cells A549 and PC-9 cells were transfected with si-mPRα or mPRα and examined for (A) cAMP concentrations using a cAMP Direct Immunoassay kit; B VEGF concentrations using ELISA; C the protein levels of p-STAT3, STAT3, VEGF, and HIF1α. Next, A549 and PC-9 cells were transfected with mPRα in the presence or absence of STAT3 inhibitor Stattic (5 μM for 24 h) and examined for (D) VEGF concentrations using ELISA and E the protein levels of p-STAT3, STAT3, VEGF, and HIF1α. *P < 0.05, **P < 0.01, compared to si-NC; #P < 0.05, ##P < 0.01, ###P < 0.005 compared to NC group; $$ P < 0.01, compared to mPRα

Fig. 3

Specific effects of mPRα on LUAD cell migration and invasion Next, A549 and PC-9 cells were transfected with si-mPRα or mPRα and examined for A, B cell migration and invasion by Transwell assays and C cell migration by wound healing assay. *P < 0.05, **P < 0.01, compared to si-NC; #P < 0.05, ##P < 0.01, ###P < 0.005 compared to NC group

Fig. 4

mPRα promoted LUAD cell growth in the xenograft model. A–C xenograft mouse tumor model was established in BALB/C nude mice accordingly. MPRα knockdown or overexpression lentivirus infected A549 cells were injected under the skin of the left flank of nude mice. The tumor volume B was measured every 3 days for 21 days; on day 21, mice were sacrificed, and the tumor weight was measured (C); D the protein levels of mPRα, ki67, and PCNA in tumor tissues were examined using IHC staining. E The protein levels of mPRα, PCNA, p-STAT3, STAT3, VEGF, and HIF1α in tumor tissues were examined using Immunoblotting. **P < 0.01, compared to si-NC. ## P < 0.01, ### P < 0.005, compared to NC

Fig. 5

mPRα knockdown decreases hypoxia-induced HIF1α/VEGF content and LUAD cell aggressiveness A549 and PC-9 cells were transfected with si-mPRα or si-NC, exposed to hypoxia (1% O₂) or normoxia (20% O₂), and examined for A the protein levels of HIF1α and VEGF by Immunoblotting; B VEGF concentrations by ELISA; C, D cell migration and invasion by Transwell assays; E cell migration by wound healing assay. *P < 0.05, **P < 0.01, compared to 20% O₂ si-NC; ##P < 0.01, compared to 20% O₂ + si-mPRα; &&P < 0.01, compared to 1% O₂ si-NC group

Fig. 6

mPRα enhances HUVEC tube formation and migration under hypoxia through HIF1α-induced VEGF release, and STAT3 signaling A549 cells were transfected with si-mPRα or si-NC (negative control). The culture medium was collected for HUVEC culture (marked as si-mPRα-CM/si-NC-CM). HUVECs were cultured in different conditioned mediums under 1% O₂ and examined for A cell migration by wound healing assay, B tube formation by Tube formation assay, and C VEGF content in the culture medium by ELISA. Next, A549 cells were transfected with NC (negative control) or mPRα-overexpressing vector, and the culture medium was collected for HUVEC culture (marked as NC-CM or mPRα-CM). HUVECs were cultured in different conditioned mediums in the presence or absence of STAT3 inhibitor Stattic under hypoxia (1% O₂) and examined for D cell migration by wound healing assay, E tube formation by Tube formation assay, and F VEGF content in the culture medium by ELISA. *P < 0.05, **P < 0.01, compared to NC or si-NC-CM; #P < 0.05, ##P < 0.01 compared to mPRα-CM