GPR54 regulates non-small cell lung cancer development via dopa decarboxylase

TL;DR

This study shows that GPR54 promotes non-small cell lung cancer development through its interaction with dopa decarboxylase and specific signaling pathways.

Contribution

The study identifies GPR54's role in NSCLC via dopa decarboxylase and reveals its regulation of glycolysis and signaling pathways.

Findings

GPR54 deletion in mouse models and knockdown in human cell lines caused apoptotic cell death.

GPR54 regulates NSCLC cell proliferation through Gαq/11/AKT and β-arrestin/ERK pathways.

GPR54 influences glycolysis and DDC expression via Gαq/11/PI3K/AKT/mTOR signaling.

Abstract

Non-small cell lung cancer (NSCLC), the most common type of lung cancer, is a leading cause of cancer death. G protein-coupled receptor 54 (GPR54) plays a role in cancer development by interacting with its endogenous ligand kisspeptin encoded by the KISS1 gene. However, the role of GPR54 in NSCLC development is not yet fully understood. Here, we demonstrate that GPR54 regulates NSCLC development via dopa decarboxylase (DDC). A mutant Kras-driven mouse lung cancer model revealed that adenoviral CMV-Cre-mediated Gpr54 deletion attenuated NSCLC development. Both Gpr54 deletion in mouse NSCLC tissues and GPR54 knockdown in human NSCLC cell lines caused apoptotic cell death. In addition, GPR54 regulation of NSCLC cell proliferation involves both the Gαq/11/AKT and β-arrestin/ERK signaling pathways. RNA sequencing revealed that Gpr54 deletion altered a gene set related to glycolysis and genotype-dependently regulated Ddc gene expression. Moreover, the regulation of glycolysis and DDC expression by GPR54 was dependent on the Gαq/11/PI3K/AKT/mTOR signaling pathway. Phosphoprotein arrays further revealed that DDC regulated NF-κB phosphorylation in NSCLC cells. Consistently, DDC regulated both NSCLC cell proliferation in vitro and tumor growth in vivo. Overall, our findings suggest that GPR54 could be a diagnostic marker for NSCLC and that therapeutics targeting GPR54 signaling may be useful for treating NSCLC.

Figures

a–c). Similarly, our TUNEL assays revealed that *Gpr54* deletion resulted in increased numbers of apoptotic cells (Supplementary Fig. 1d and 1e). Taken together, these results provide in vivo evidence that Gpr54 is required for mutant *Kras*-induced NSCLC development.Fig. 1*Gpr54* deletion inhibits growth and induces apoptosis in *Kras*^*G12D*^-driven NSCLC. a A schematic diagram of KG mouse generation and lung tumor induction by intratracheal injection of adenoviral *CMV-Cre*. b Overall survival plot for adenoviral *Cre*-injected KG mice. Statistical significance was determined by the log-ran

a). *Gpr54-IRES-Cre*; *Kras*^*LSL-G12D*^ (*Kras*^*LSL-G12D*^; *GPIC*, herein KGPIC) mice at approximately 40 weeks after birth exhibited labored breathing independently of sex. When we sacrificed KGPIC mice at 40 weeks after birth, the mice developed tumors in the lung (100%, independent of sex), back skin (20% of male KGPIC mice) and mouth (60% of male KGPIC mice) (Supplementary Fig. 2b). When we further examined tumor development at earlier weeks after birth, all KGPIC mice developed pulmonary tumors, mainly beginning at 6 weeks after birth (Supplementary Fig. 2c and 2 d). Thus, our data ind

GPR54 is required for aerobic glycolysis in NSCLC cells. (a) Venn diagram showing the numbers of differentially expressed genes. (b) GSEA/KEGG gene sets were downregulated by oncogenic *Kras* gene mutation in *Gpr54*-null lung tissues compared with wild-type lung tissues. (c) Gene enrichment plot of the KEGG_Glycolysis_Gluconeogenesis gene set from the GSEA/KEGG gene sets. (d-i) Seahorse glycolytic stress test in H358 cells after silencing of *GPR54*, *GNAQ* (*Ga*_*q*_)*, GNA12* (*Ga*_*12*_) or *ARRB2* (*β-arrestin-2*) followed by stimulation with 100 nM KP10. Oxygen consumption rate (OCR) (d)

a). Consistently, *Gpr54* deletion decreased both p-Erk and p-Akt levels in the tumor lysates of the mice at 5 weeks after Ad5-*CMV-Cre* infection (Fig. [2h](#Fig2)). At 10 weeks after Ad5-*CMV-Cre* infection, the p-Erk level significantly decreased in the tumor lysates of KG^fl/+^ mice, and the p-Akt level was not significantly altered in the tumor lysates of KG^fl/fl^ mice (Fig. [2i](#Fig2)). Moreover, both myristoylated AKT (*HA-Myr-AKT*; pcDNA3-*Myr-HA-AKT1*) and ERK2-MEK1 fusion (*myc-MEK-ERK2*; pCMV-*myc-ERK2-LA-MEK1*) blocked the apoptosis of *GPR54*-silenced NSCLC cells (Figs. [2](#Fig

Full text

Introduction

Lung cancer, consisting of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), is the major cause of cancer-related death, and NSCLCs account for ~85% of lung cancer cases.^1^ The NSCLC stage is important for selecting therapeutic approaches.^1^ Surgery is the first option for treating NSCLC. While radiotherapy and chemotherapy are determined by NSCLC stage, targetable mutations are critical for deciding treatment approaches.^1^ Patients with driver mutations receive targeted therapeutics for mutations in epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), Kirsten rat sarcoma virus (KRAS), v-raf murine sarcoma viral oncogene homolog B (BRAF), ROS proto-oncogene 1 (ROS1), rearranged during transfection (RET), c-MET proto-oncogene (MET), human epidermal receptor 2 (HER2) and neuregulin 1 (NRG1).^1^ Patients with no mutations or non-driver mutations are treated with immune checkpoint inhibitors as monotherapy or with platinum-based chemotherapy.^1^ Although recent advances in NSCLC treatment are promising, there are limitations, including drug resistance and side effects. Therefore, understanding the molecular mechanisms involved in NSCLC development is crucial for developing treatment strategies.

G protein-coupled receptors (GPCRs) play broad roles in the body and are frequently selected as drug targets. G protein-coupled receptor 54 (GPR54), also called KISS1 receptor (KISS1R), is known as an endogenous receptor for kisspeptin encoded by the KISS1 gene.^2^ GPR54-mediated signaling activated by kisspeptin regulates various biological mechanisms, including puberty, circadian rhythm, metabolism, fertility, emotion and the innate immune response.^2^ In cancer, GPR54-mediated signaling is known to suppress cancer metastasis.^2,3^ Recently, it has been revealed that mouse Gpr54 heterozygosity delays polyomavirus middle T antigen (PyMT)-induced mammary tumor development^4^ and that GPR54-mediated signaling inhibits tumor angiogenesis.^5^ Moreover, stress-induced Gpr54 signaling in tumor-infiltrating T cells regulates lung cancer development by exacerbating T cell dysfunction and exhaustion.^6^ Therefore, GPR54 appears to play pleiotropic roles in cancer beyond its ability to suppress distant metastases. Moreover, metabolic reprogramming of cancer cells has been well documented as one of the hallmarks of cancer, and major oncogenes have been reported to be master inducers of cancer glycolysis.^7^ GPR54 has been revealed to reprogram breast cancer cell metabolism by decreasing aerobic glycolysis, increasing mitochondrial biogenesis and regulating the levels of the glucose transporter GLUT1, hexokinase 2 (HK2) and glutaminase, which contribute to triggering processes, including the nucleotide synthesis required for tumor growth.^8–11^ Thus, GPR54 signaling appears to play multiple roles in cancer development in addition to its role in metastasis suppression.

Both GPR54 and kisspeptin were associated with favorable prognostic markers in colorectal, esophageal, thyroid, prostate, ovarian and bladder cancers, whereas poor prognoses were observed in patients with breast and liver cancers.^2^ Thus, the expression patterns of both GPR54 and KISS1 as prognostic markers seem to depend on cancer type and stage. Moreover, the prognostic value of their expression patterns is controversial in lung cancer. The gene expression patterns of both GPR54 and KISS1 were lower in metastatic NSCLC (stage IV) than in locally advanced NSCLC (stage III) or lower in more advanced stages of NSCLC (III-IV) than in low TNM stages of NSCLC (I-II).^12^ However, other studies have shown that the serum kisspeptin level produced from the KISS1 gene is higher in NSCLC patients than in healthy donors or that the serum kisspeptin level is not significantly different between NSCLC patients and healthy donors.^13^ Although a recent study revealed the role of GPR54 in T cells against lung cancer,^6^ the role of GPR54-mediated signaling in lung cancer still needs to be elucidated.

Moreover, DOPA decarboxylase (DDC) catalyzes 3,4-dihydroxyl-L-phenylalanine to dopamine and 5-hydroxytryptophan to serotonin.^14^ DDC activity is detected in neurons of the central nervous system as well as peripheral organs such as the lung, liver, pancreas, kidney, stomach, and endothelial cells of blood vessels.^15–18^ DDC is considered a biomarker for neuroendocrine tumors.^19,20^ For example, high DDC expression levels have been detected in SCLC and neuroblastoma.^21^ Although DDC function has been identified in different cancer types, including SCLC, prostate cancer, breast cancer, gastric cancer, colorectal adenocarcinoma, head and neck squamous cell carcinoma and hepatocellular carcinoma, its role in NSCLC remains unclear.^22–26^ Herein, we investigated the role of GPR54 in NSCLC via both in vitro human lung cancer cell line studies and in vivo mouse tumor models.^27^ Our present studies demonstrate that GPR54 is crucial for NSCLC development and requires DDC-involved signaling.

Results

Loss of Gpr54 inhibits tumor cell growth and induces apoptosis in KrasG12D-driven NSCLC

The intratracheal injection of viral Cre into Kras^LSL-G12D^ knock-in mice results in conditional Kras^G12D^ expression via Cre–lox events and is widely used to understand NSCLC development.^27^ Therefore, to investigate the role of GPR54 in NSCLC development, we intratracheally injected adenoviral CMV-Cre (Ad5-CMV-Cre) into 6-week-old Kras^LSL-G12D^ mice crossed with Gpr54^flox^ mice (Kras^LSL-G12D^;Gpr54^flox^, herein KG) (Fig. 1a). When mouse overall survival was examined, 50% of Kras^G12D^;Gpr54^+/+^ mice injected intratracheally with Ad5-CMV-Cre (herein, KG^+/+^), Kras^G12D^;Kiss1r^flox/+^ with Ad5-CMV-Cre (KG^fl/+^), Kras^G12D^;Kiss1r^flox/flox^ with Ad5-CMV-Cre (KG^fl/fl^) were dead at 123.1, 207.4 and 355.3 days, respectively (Fig. 1b). Moreover, when we measured both the size and number of tumor lesions in the lung tissues at 10 weeks post-infection, Gpr54 deletion reduced both the tumor number and lesion size (Fig. 1c–e). These data suggest that Gpr54 is required for the development of mutant Kras-driven mouse lung cancer. We further found that the tumor lesions of all the genotypes were TTF-1-positive and p63-negative lung adenocarcinomas (Fig. 1f). In addition, Gpr54 deletion caused apoptosis (Fig. 1f–h). Therefore, we additionally examined the histopathology of the mice at 5 weeks after viral infection. IHC data for PCNA and cleaved Caspase-3 levels revealed that Gpr54 deletion caused apoptosis at the early stage of tumor development (Supplementary Fig. 1a–c). Similarly, our TUNEL assays revealed that Gpr54 deletion resulted in increased numbers of apoptotic cells (Supplementary Fig. 1d and 1e). Taken together, these results provide in vivo evidence that Gpr54 is required for mutant Kras-induced NSCLC development.Fig. 1Gpr54 deletion inhibits growth and induces apoptosis in Kras^G12D^-driven NSCLC. a A schematic diagram of KG mouse generation and lung tumor induction by intratracheal injection of adenoviral CMV-Cre. b Overall survival plot for adenoviral Cre-injected KG mice. Statistical significance was determined by the log-rank test. KG^+/+^ (n = 36), KG^fl/+^ (n = 36), and KG^fl/fl^ (n = 30). KG^+/+^ vs. KG^fl/+^ (p = 2.78e-11), KG^+/+^ vs. KG^fl/fl^ (p = 0.0281), KG^fl/+^ vs. KG^fl/fl^ (p = 0.3019). c H&E staining of lung tissues from different genotypes at 10 weeks post-adenoviral CMV-Cre infection (n = 5/group). Top, scale bar, 1,000 μm; Bottom, scale bar, 20 μm. d Tumor numbers in the lung tissues of KG^+/+^, KG^fl/+^, and KG^fl/fl^ mice at 10 weeks after virus injection (n = 5/group). e Percentages of tumor lesion areas in the lung tissues of KG^+/+^, KG^fl/+^, and KG^fl/fl^ mice at 10 weeks after virus injection (n = 5/group). f Representative IHC staining of TTF1, P63, PCNA and cleaved Caspase-3 in the lung tissues of the indicated genotypes (n = 5/group). Scale bar, 20 μm. g PCNA-positive cell percentages in the lung tissues (n = 5/group). h Cleaved Caspase-3-positive cell percentages in the lung tissues (n = 5/group). All data are presented as the mean ± SD. *, p < 0.05; **, p < 0.01; n.s, not significant

To investigate the preference of Gpr54-positive cells (Gpr54^+^) for oncogenic Kras mutation, we crossed Kras^LSL-G12D^ (lox-stop-lox-Kras^G12D^, K) conditional mutant mice with Gpr54-Cre transgenic (GPIC) mice, in which the Gpr54 promoter drives Cre gene expression (Supplementary Fig. 2a). Gpr54-IRES-Cre; Kras^LSL-G12D^ (Kras^LSL-G12D^; GPIC, herein KGPIC) mice at approximately 40 weeks after birth exhibited labored breathing independently of sex. When we sacrificed KGPIC mice at 40 weeks after birth, the mice developed tumors in the lung (100%, independent of sex), back skin (20% of male KGPIC mice) and mouth (60% of male KGPIC mice) (Supplementary Fig. 2b). When we further examined tumor development at earlier weeks after birth, all KGPIC mice developed pulmonary tumors, mainly beginning at 6 weeks after birth (Supplementary Fig. 2c and 2 d). Thus, our data indicate that oncogenic Kras mutations in Gpr54-positive cells (Gpr54^+^) mainly lead to pulmonary tumor development.

Meta-analysis data from a web portal (https://lce.biohpc.swmed.edu/lungcancer/) supportively revealed that the GPR54 expression level is higher in NSCLC tumor tissues than in normal tissues (Supplementary Fig. 3a and 3b) and that its expression level is correlated with the KRAS expression level (Supplementary Fig. 3c). Moreover, high GPR54 mRNA expression was correlated with poor overall survival (especially stages I and II), first progression survival and post progression survival according to the Kaplan‒Meier plotter (kmplot.com) (Supplementary Fig. 3d). These data indicate that GPR54 is crucial for NSCLC development.

GPR54 is required for NSCLC cell proliferation

To elucidate whether the effect of GPR54 on NSCLC development is limited to KRAS mutation or independent of subtypes and mutations, we examined GPR54 expression in various NSCLC cell lines.

GPR54 expression was higher in NSCLC cell lines (A549, H358, HCC827, H1975, H460, H1299, HCC95, and HCC1588) than in human airway epithelial cells (HPSAEpiC) and pulmonary alveolar epithelial cells (HPAEpiC) (Fig. 2a). Accordingly, we conducted mouse tumor xenograft assays with NSCLC cells in which either GPR54 was silenced or not silenced. GPR54 silencing delayed tumor growth in vivo (Fig. 2b and 2c, Supplementary Fig. 4a). Consistently, the TUNEL assays revealed that GPR54 silencing increased the number of apoptotic cells in the tumor burden (Fig. 2d, Supplementary Fig. 4b). Similarly, our western blot assays revealed that GPR54 silencing caused apoptosis independently of subtype and mutation (Fig. 2c, Supplementary Fig. 4a). Cell proliferation, colony formation and Annexin V assays confirmed that GPR54 silencing retarded NSCLC cell proliferation with increasing apoptosis (Fig. 2e–g, Supplementary Fig. 4c and 5).Fig. 2GPR54 silencing results in the apoptosis of NSCLC cells. a Western blot analysis of GPR54 expression in normal lung epithelial cells and NSCLC cells. b Xenograft tumor growth curves comparing siControl and siGPR54 groups in NSCLC cell lines (n = 7/group). Scrambled control siRNAs (siControl), GPR54 siRNA (siGPR54). c Heatmap showing the western blot data. d Quantification of apoptotic cell numbers was measured via TUNEL assays in NSCLC (shown as fold change relative to siControl). e A retardation of NSCLC cell proliferation by GPR54 gene silencing. f Colony formation by GPR54 silencing. g Annexin V apoptosis analysis after GPR54 silencing. Western blot analysis of phosphorylation levels of both Erk and Akt in mouse lung tissues at 5 weeks (h) or at 10 weeks (i) after Ad5-CMV-Cre infection. The bar graphs show the relative levels of p-Erk and p-Akt in the mouse lung tissues. j Western blot analysis of apoptotic effect in GPR54-silenced H358 and HCC1588 cells with HA-Myr-AKT1 overexpression. k Western blot analysis of apoptotic effect in GPR54-silenced H358 and HCC1588 cells with myc-MEK-ERK2 overexpression. All data present as the mean ± SD. *, p < 0.05; n.s, not significant

GPR54 appears to regulate multifarious intracellular signaling pathways, including the AKT and MAPK signaling pathways, which are crucial for cell proliferation.^2^ Thus, we investigated whether GPR54 deletion affects MAPK and AKT signaling in NSCLC cells. In NSCLC cell lines, GPR54 silencing reduced the levels of phosphorylated ERK and AKT (Fig. 2c and Supplementary Fig. 4a). Consistently, Gpr54 deletion decreased both p-Erk and p-Akt levels in the tumor lysates of the mice at 5 weeks after Ad5-CMV-Cre infection (Fig. 2h). At 10 weeks after Ad5-CMV-Cre infection, the p-Erk level significantly decreased in the tumor lysates of KG^fl/+^ mice, and the p-Akt level was not significantly altered in the tumor lysates of KG^fl/fl^ mice (Fig. 2i). Moreover, both myristoylated AKT (HA-Myr-AKT; pcDNA3-Myr-HA-AKT1) and ERK2-MEK1 fusion (myc-MEK-ERK2; pCMV-myc-ERK2-LA-MEK1) blocked the apoptosis of GPR54-silenced NSCLC cells (Figs. 2j and 2k, Supplementary Fig. 6a and 6b). Therefore, our data suggest that GPR54 regulates proliferation via ERK- and AKT-involved signaling pathways in NSCLC cells.

GPR54 regulates glycolysis in NSCLC cells

To investigate the underlying mechanisms by which GPR54 regulates NSCLC development, we performed mRNA sequencing to analyze gene expression patterns in KG^+/+^, KG^fl/+^ and KG^fl/fl^ mice at 5 weeks after Ad5-CMV-Cre infection. Gpr54 deletion altered the gene expression patterns, as shown in the Venn diagram (Fig. 3a). Gene set enrichment analysis (GSEA) revealed that 8 KEGG signatures, including ‘starch and sucrose metabolism’, ‘olfactory transduction’, ‘ascorbate and aldarate metabolism’, ‘glycolysis gluconeogenesis’, ‘pentose and glucuronate interconversions’, “drug metabolism cytochrome P450”, “gap junction” and “neuroactive ligand receptor interaction”, were downregulated in the lung tissues of KG^fl/+^ and KG^fl/fl^ mice (Fig. 3b). GPR54-mediated signaling is known to alter aerobic glycolysis in cancer cells.^8–11^ Accordingly, Gpr54 appeared to be associated with gene sets related to glycolysis and gluconeogenesis in NSCLC (Fig. 3c).Fig. 3GPR54 is required for aerobic glycolysis in NSCLC cells. (a) Venn diagram showing the numbers of differentially expressed genes. (b) GSEA/KEGG gene sets were downregulated by oncogenic Kras gene mutation in Gpr54-null lung tissues compared with wild-type lung tissues. (c) Gene enrichment plot of the KEGG_Glycolysis_Gluconeogenesis gene set from the GSEA/KEGG gene sets. (d-i) Seahorse glycolytic stress test in H358 cells after silencing of GPR54, GNAQ (Gaq), GNA12 (Ga12) or ARRB2 (β-arrestin-2) followed by stimulation with 100 nM KP10. Oxygen consumption rate (OCR) (d), extracellular acidification rate (ECAR) (e), proton efflux rate (PER) (f), mOCR (g), basal GlycoPER (h), and compensatory GlycoPER (i). j–o Glycolysis stress test in H358 cells pre-treated for 10 minutes with 100 nM KP234 (GPR54 antagonist), 1 μM FR900359 (Gα_q/11_ inhibitor), 25μM LY294002 (PI3K inhibitor), 1 μM rapamycin (mTOR inhibitor), 10 μM barbadin (β-arrestin inhibitor), 25 mM LiCl (GSK-3β inhibitor), and 25 μM PD98059 (MEK inhibitor), followed by stimulation with 100 nM KP10. OCR (j), ECAR (k), PER (l), mOCR (m), basal GlycoPER (n), and compensatory GlycoPER (o). p Relative glucose consumption levels after GPR54, GNAQ, GNA12 and ARRB2 silencing, followed by 100 nM KP10 in H358 cells. q Relative lactate production levels after GPR54, GNAQ, GNA12 and ARRB2 silencing, followed by 100 nM KP10 in H358 cells. r Relative percentages of apoptotic H358 cells, as determined by Annexin V assays, following silencing of GPR54, GNAQ, GNA12, or ARRB2. s Western blots for cleaved Caspase-3 and PARP by GNAQ and ARRB2 silencing in H358 and HCC1588 cells. t Relative glucose consumption levels of H358 cells treated with 100 nM KP234, 1 μM FR900359, 25 μM LY294002, 1 μM rapamycin, 10 μM barbadin, 25 mM LiCl and 25 μM PD98059, followed by 100 nM KP10 10 min later. u Relative lactate production levels in H358 cells treated with 100 nM KP234, 1 μM FR900359, 25 μM LY294002, 1 μM rapamycin, 10 μM barbadin, 25 mM LiCl and 25 μM PD98059, followed by 100 nM KP10 10 min later. v Relative percentages of apoptotic H358 cells, as determined by Annexin V assays, following treatment with 100 nM KP234, 1 μM FR900359, 25 μM LY294002, 1 μM rapamycin, 10 μM barbadin, 25 mM LiCl and 25 μM PD98059. w Western blots for cleaved Caspase-3 and PARP by 100 nM KP234, 1 μM FR900359, 25 μM LY294002, 1 μM rapamycin and 25 μM PD98059 in H358 and HCC1588 cells. All data present the mean ± SD. *, p < 0.05

To validate our mRNA sequencing data, we examined the mRNA expression levels of glycolysis-related genes, including Hif-1α, G6pd, Slc1a5, Hk2, Pkm2 and Glut1. The Hif-1α, G6pd, Slc1a5 and Hk2 mRNA expression levels decreased significantly in the lung tissues of both the KG^fl/+^ and KG^fl/fl^ mice at 5 weeks post-infection (Supplementary Fig. 7a), and the Hif-1α and Hk2 mRNA expression levels decreased markedly in the lung tissue of the KG^fl/fl^ mice at 10 weeks post-infection (Supplementary Fig. 7b). Moreover, Gpr54 deletion reduced Hif1α protein level (Supplementary Fig. 7c). Thus, we further investigated whether GPR54 silencing regulates the protein levels of HIF-1α, c-MYC, p-mTOR and mTOR, which are master regulators of glycolysis. GPR54 silencing commonly decreased HIF-1α, c-MYC, and p-mTOR levels in NSCLC cell lines (Supplementary Fig. 7d). In addition, the PI3K inhibitor LY294002 decreased HIF-1α expression (Supplementary Fig. 7e) and both glucose consumption and lactate production in NSCLC cells (Supplementary Fig. 7f). These results suggest that GPR54, via the AKT-involved intracellular signaling pathway in NSCLC, might regulate mainly HIF-1α expression and glycolytic metabolism, including glucose consumption and lactate production.

To decipher the role of GPR54 signaling in glycolysis in NSCLC cells, we measured the oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and proton efflux rate (PER). In the cells treated with kisspeptin-10 (KP10), both GPR54 and GNAQ (encoding Gaq) silencing, but not GNA12 (Ga12) or ARRB2 (β-arrestin-2) silencing, blocked the OCR, ECAR, and PER (Fig. 3d–i, Supplementary Fig. 8a–f). Similarly, kisspeptin-234 (KP234, GPR54 antagonist), FR900359 (Gα_q/11_ inhibitor), LY294002 (PI3K inhibitor), and rapamycin (mTOR inhibitor), but not barbadin (β-arrestin inhibitor), LiCl (GSK-3β inhibitor), or PD98059 (MEK inhibitor), blocked the kisspeptin-induced changes in the OCR, ECAR, and PER (Fig. 3j–o, Supplementary Fig. 8g–l). Consistently, GPR54 and GNAQ silencing altered glucose consumption and lactate production (Figs. 3p and 3q, Supplementary Fig. 9a and 9b). Accordingly, we further found that KP234, FR900359, LY294002, and rapamycin reduced both glucose consumption and lactate production in NSCLC cells (Fig. 3t and 3u, Supplementary Fig. 11a and 11b). However, the silencing of genes we tested resulted in apoptosis (Figs. 3r and 3s, Supplementary Fig. 10). Consistently, all the inhibitors we tested caused apoptosis (Figs. 3v and 3w, Supplementary Fig. 12). Therefore, our data suggest that GPR54 may regulate glycolysis via Gα_q/11_-AKT-mTOR signaling, whereas GPR54 requires both the AKT- and ERK-involved pathways for NSCLC cell proliferation.

GPR54 regulates DDC expression in NSCLC cells

Next, we investigated genes whose expression patterns corresponded to the Gpr54 genotype. According to our transcriptomic analysis setting a threshold fold change (FC > 2.0 or <0.5), the data for the differentially expressed genes (DEGs) revealed that Ddc gene expression significantly decreased in a Gpr54 genotype-dependent manner and that the expression of 12 genes (Sowaha, Wfdc18, Hspa1a, H2-M2, Obp2a, Lcn11, Lcn4, Mup4, Mup5, Gm14744, 5430402E10Rik and Gm14743) inversely increased (Fig. 4a). Crosstalk between GPR54 and DDC in the brain seems to be plausible on the basis of recent brain studies,^28–31^ whereas their functional relationship has not been revealed in cancer. Moreover, the roles of both GPR54 and DDC in NSCLC have yet to be clearly defined, while DDC is thought to play a particular role in SCLC.^19,32^ Thus, we focused on DDC in NSCLC. Using the DAVID tool, we found that upregulated DEGs in KG^fl/fl^ mice compared with those in KG^+/+^ mice were significantly enriched in ‘cell body (GO:0044297)’, ‘extracellular region part (GO:0044421)’, ‘extracellular regions (GO:0005576)’, and ‘extracellular space (GO:0005615)’ in the cellular component (CC) category. Biological process enrichment suggested that the upregulated DEGs in KG^fl/fl^ were significantly enriched in the ‘inflammatory response (GO:0006954)’. The molecular function terms ‘ion binding (GO:0043167)’, ‘cation binding (GO:0043169)’, ‘metal ion binding (GO:0046872)’, and ‘carboxylic acid binding (GO:0031406)’ were enriched among the DEGs whose expression was downregulated in KG^fl/fl^ compared with that in KG^+/+^ mice. Ddc was included in the gene set for its ‘carboxylic acid binding’ function (Fig. 4b). We confirmed that Ddc protein levels decreased in a Gpr54 genotype-dependent manner in lung tissues at 10 weeks post-infection (Fig. 4c). Similarly, the DDC expression level was higher in tumor tissues than in normal adjacent tissues (Supplementary Fig. 13a), which was consistent with DDC protein levels in normal lung epithelial cells and NSCLC cells (Supplementary Fig. 13b). Moreover, high DDC mRNA expression was correlated with poor overall survival (especially stage III) according to the Kaplan‒Meier plotter (kmplot.com) (Supplementary Fig. 13c). Moreover, our results revealed that the serum dopamine level, but not the serotonin level, was higher in KG^+/+^ mice than in KG^fl/fl^ mice (Fig. 4d). However, both dopamine and serotonin levels were not significantly altered in the lung tissues (Supplementary Fig. 14a), whereas Gpr54 and Ddc mRNA levels were altered in a Gpr54 genotype-dependent manner (Supplementary Fig. 14b). Moreover, both dopamine and serotonin failed to affect the viability of A549, H460 and HCC827 cells (Supplementary Fig. 14c).Fig. 4GPR54 regulates DDC expression. a Heatmap showing genes whose expression was significantly downregulated or upregulated in a Gpr54 genotype-dependent manner. The data are presented as log_2_ values. The Gpr54 genotype-dependent Ddc expression pattern is marked by the red arrow. b GO enrichment analysis via the DAVID web tool. The red arrow indicates that GO:0031406~ carboxylic acid binding contains Ddc. c Ddc expression levels in lung tissues from the mice at 10 weeks after Ad5-CMV-Cre infection. Scale bar, 20 μm. d Serum dopamine and serotonin levels in mice at 10 weeks post Ad5-CMV-Cre infection. Western blot analysis of DDC expression and AKT/mTOR phosphorylation status in H358 and HCC1588 cells pre-treated with 100 nM KP234 (e), 1μM FR900359 (f), 25μM LY294002 (g), 1μM rapamycin (h), 25 mM LiCl (i) and 25μM PD98059 (j) followed by 10 minutes of 100 nM KP10 treatment. Western blot analysis of DDC expression and AKT/mTOR phosphorylation status in H358 and HCC1588 cells silenced by GPR54 (k), GNAQ (l), GNA12 (m) or ARRB2 (n) cells followed by 100 nM KP10 treatment. Effects of HA-Myr-AKT1 overexpression on DDC and HIF-1α protein levels in GPR54-silenced (o) or GNAQ-silenced (p) cells. Effect of HA-Myr-AKT1 overexpression on glucose consumption in GPR54- (q) or GNAQ-silenced (r) cells. Effects of HA-Myr-AKT1 overexpression on lactate production in GPR54-silenced (s) or GNAQ-silenced (t) cells. All data present the mean ± SD. *, p < 0.05

To decipher GPR54-mediated signaling for the regulation of DDC expression, NSCLC cells were pretreated with different inhibitors (KP234, FR900359, LY294002, rapamycin, LiCl, and PD98059) and treated with KP10. DDC protein levels were altered by KP234, FR900359, LY294002, and rapamycin (Fig. 4e–j), suggesting that GPR54 activation by kisspeptin involves Gα_q/11_-AKT-mTOR to regulate DDC expression. Kisspeptin-activated GPR54 also regulated AKT phosphorylation via Gα_q/11_, as FR900359 blocked AKT phosphorylation even in cells treated with KP10 (Fig. 4f). Consistently, GPR54 and GNAQ silencing but not GNA12 or ARRB2 silencing in the cells blocked kisspeptin-induced AKT phosphorylation and DDC expression (Fig. 4k and 4n). In addition, Myr-AKT1 overexpression in both GPR54- and GNAQ-silenced cells rescued both DDC and HIF-1α protein levels (Figs. 4o and 4p) and glucose consumption and lactate production (Fig. 4q–t). Thus, the GPR54-Gα_q/11_-AKT-mTOR pathway seems to be crucial for DDC expression in NSCLC cells. Moreover, DDC promoter analysis revealed NF-κB binding regions between −250bp and −500bp (Supplementary Fig. 15a). Consistently, NF-κB luciferase assays revealed that the kisspeptin-activated GPR54-Gα_q/11_-AKT-mTOR pathway activated NF-κB transcriptional activity (Supplementary Fig. 15b and 15c), indicating that kisspeptin-activated GPR54 regulates NF-κB-mediated DDC expression via the Gα_q/11_-AKT-mTOR pathway.

GPR54-mediated DDC expression regulates NSCLC cell proliferation

To investigate DDC-mediated downstream signaling, we examined the effect of DDC gene silencing on protein phosphorylation profiles in A549 cells (Fig. 5a). Gene Ontology (GO) analysis of the top 10 genes associated with upregulated and downregulated protein phosphorylation status via DAVID revealed that DDC gene silencing altered protein phosphorylation profiles, including ‘negative regulation of extrinsic apoptotic signaling pathway (GO:2001234)’, ‘positive regulation of NF-kappaB transcription factor activity (GO0051092)’, ‘regulation of extrinsic apoptotic signaling pathway (GO:2001236)’, ‘negative regulation of signal transduction (GO:0009968)’, ‘response to ketone (GO:1901654)’, ‘extrinsic apoptotic signaling pathway (GO:0097191)’, ‘negative regulation of cell communication (GO:0010648)’, ‘negative regulation of signaling (GO:0023057)’ and ‘negative regulation of apoptotic signaling pathway (GO:2001234)’ (Fig. 5b). DDC silencing altered the gene ontology ‘response to ketone (GO:1901654)’, which appears to support our finding that GPR54-mediated DDC expression regulates glycolytic metabolism. However, our data revealed that the main pathways altered by DDC silencing were related to apoptotic cell death. Our tumor xenograft study confirmed that DDC silencing decreased tumor growth in vivo (Fig. 5c). Our TUNEL assay data also revealed that DDC gene silencing increased the number of apoptotic cells (Fig. 5d, Supplementary Fig. 16a). Consistently, DDC silencing reduced in vitro cell proliferation (Fig. 5e) and colony formation (Fig. 5f, Supplementary Fig. 16b), thereby resulting in apoptosis (Figs. 5g and 5h, Supplementary Fig. 16c and 16d). Thus, our data indicate that DDC is required for NSCLC development.Fig. 5GPR54-DDC pathway is required for NSCLC cell proliferation. a Differences in protein phosphorylation levels between scrambled control siRNA (siControl) and DDC siRNA (siDDC) knockdown A549 cells. b GO analysis of genes significantly altered via DAVID. c Comparison of tumor volume between the siControl group and the siDDC group in a xenograft assay using NSCLC cell lines (each group n = 7). d Relative number of apoptotic cells in tumor tissues from nude mice xenografted with DDC knockdown NSCLC cells was determined via a TUNEL assay. e Growth curves for 96 hours after DDC knockdown in NSCLC cells. f Effects of DDC knockdown on colony formation in NSCLC cells. g Heatmap showing protein levels after DDC silencing in NSCLC cells. h Relative apoptotic cell numbers after DDC knockdown in NSCLC (H358, HCC827, H460 and HCC1588) cells from Annexin V assays. i Bar plot of the levels of phosphorylated proteins most significantly downregulated by DDC knockdown, with a cutoff of a fold change ≤ 0.5 (log₂ ≤ –1) and p < 0.05. Glycolysis stress test in H358 and HCC1588 cells after DDC silencing. OCR (j), ECAR (k), PER (l), mOCR (m), basal GlycoPER (n), and compensatory GlycoPER (o) in H358 cells after DDC silencing, followed by 100 nM KP10 in H358 cells. OCR (p), ECAR (q), PER (r), mOCR (s), basal GlycoPER (t), and compensatory GlycoPER (u) after DDC silencing, followed by 100 nM KP10 in HCC1588 cells. All data present the mean ± SD. *, p < 0.05

DDC silencing significantly reduced the phosphorylation levels of the androgen receptor (AR), RELA (NF-κB-p65), NF-κB1 (NF-κB-p105/p50), ATF2, DAB1, Cortactin, and ERBB2 according to our protein phosphorylation profiling (Fig. 5i). Thus, DDC is assumed to regulate the canonical NF-κB pathway. We confirmed that DDC silencing reduced the phosphorylation levels of IKK, Iκ,B and NF-κB in NSCLC cell lines (Fig. 5g, Supplementary Fig. 16c). Consistently, carbidopa increased the levels of the cleaved forms of both Caspase-3 and PARP but decreased the phosphorylation levels of IKK, IκB, and NF-κB (Supplementary Fig. 17a). Furthermore, carbidopa reduced colony formation and caused apoptosis (Supplementary Fig. 17b and 17c). Thus, our data suggest that DDC, via the NF-κB pathway, regulates NSCLC cell proliferation. Next, we examined the DDC requirement for the activation of the NF-κB pathway and proliferation in GPR54-silenced cells to test GPR54 dependency. DDC overexpression (pcDNA3.1-DDC) in GPR54-silenced cells rescued the activation of the NF-κB pathway (Supplementary Fig. 17d) and cell proliferation (Supplementary Fig. 17e). Considering that GPR54 regulates the NF-κB pathway for DDC expression and that DDC is involved in the activation of the NF-κB pathway, it is plausible that DDC may sustain the activation of the NF-κB pathway for GPR54-mediated NSCLC cell proliferation (Fig. 6).Fig. 6. Scheme of GPR54-DDC pathway signaling. GPR54 is stimulated by Kisspeptin signals to G_αq_/G_11_ and G_β_/G_γ_. The G_αq_/G_11_ pathway activates the PI3K/AKT/mTOR/NF-κB pathway. GPR54-mediated regulation of HK2 expression is crucial for glycolytic function, thereby regulating glucose consumption and lactate production. The G_β_/G_γ_ pathway activates KRAS/MEK/ERK signaling and/or regulates β-arrestin2-associated ERK signaling to control proliferation. GPR54-dependent NF-κB activation regulates DDC expression, resulting in a positive feedback loop that maintains proliferation and glycolysis. In summary, GPR54 signaling via DDC is crucial for maintaining NSCLC cell proliferation and metabolic reprogramming. BioRender (www.biorender.com) was used for the image production

Furthermore, DDC silencing reduced HIF-1α protein levels (Supplementary Fig. 18a) and reduced both glucose consumption and lactate production (Supplementary Fig. 18b and 18c). Similarly, carbidopa reduced glucose consumption and lactate production (Supplementary Fig. 18d and 18e). Accordingly, DDC silencing and carbidopa altered the OCR, ECAR and PER (Fig. 5j-u, Supplementary Fig. 19a–l). Taken together, our data suggest that the GPR54-mediated Gα_q/11_-AKT-NF-κB signaling pathway may regulate DDC expression, resulting in the regulation of HIF-1α expression and glycolytic metabolism.

We further hypothesized that targeting pathways involving GPR54 and DDC might be applicable to NSCLC treatment. Thus, we examined the synergistic effect of KP234 and carbidopa on NSCLC cell viability. When NSCLC cells were treated with KP234 and carbidopa, the combinatorial treatment reduced NSCLC cell viability and had a synergistic effect, independently of mutation and subtype (Supplementary Figs. 20 and 21). Our western blot and Annexin V assay data confirmed the synergistic effect (Supplementary Fig. 22). Furthermore, our in vivo xenograft experiment in which NSCLC cells were orthotopically injected into the lungs of BALB/C nude mice revealed the combined treatment with KP234 and carbidopa reduced tumor growth in vivo, as co-treatment significantly inhibited tumor weight and volume compared with those of the control and single treatment with either KP234 or carbidopa, with no effect on body weight (Supplementary Fig. 23a). Consistently, the combined treatment increased the number of apoptotic cells when the tumor tissues were stained with cleaved Caspase-3 and PCNA (Supplementary Fig. 23b). Thus, our results showed that blocking GPR54-mediated signaling with KP234 and carbidopa suppressed NSCLC growth more effectively.

We further examined the effects of these agents with targeted therapies, including gefitinib, RMC-6236 (also called daraxonrasib), and sotorasib. Neither gefitinib-sensitive HCC827 nor gefitinib-resistant H1975 cells exhibited synergistic effects of gefitinib with KP234 and carbidopa (Supplementary Fig. 24a–d). KRAS^G12C^-targeting sotorasib shows synergism with KP234 and carbidopa in H358 cells with the KRAS^G12C^ mutation (Supplementary Fig. 25a–d). RMC-6236 targeting pan-RAS mutations showed synergistic effects with KP234 and carbidopa in A549, H460, H358, and H1299 cells that are known to have RAS mutations (Supplementary Figs. 26, 27 and 28a, b). Thus, our data indicate that blockade of the GPR54-mediated signaling pathway with targeted therapies against driver mutations would be beneficial for treating NSCLC development.

Discussion

Emerging evidence suggests that GPR54 signaling plays important roles in cancer.^2^ Although it is focused on cancer metastases, its role in earlier stages of cancer development is suggested by clinical data where GPR54 and/or KISS1 is highly expressed either in tumor tissues compared with normal tissues or in metastatic tumor tissues compared with non-metastatic tumor tissues.^3^ A recent study with PyMT mouse model revealed that GPR54 is required for breast cancer development.^4^ Moreover, chronic stress facilitates lung cancer progression via GPR54 signaling in T cells.^6^ Thus, recent findings from GEMM studies support that GPR54 signaling is required for cancer development. Our present study revealed that Gpr54 deletion attenuated Kras^G12D^-driven NSCLC. In our subsequent GEMM studies, oncogenic Kras mutations in Gpr54^+^ cells resulted mostly in the development of tumors in the lung. Our data obtained from GEMM may explain why the GPR54 expression level in clinical data is higher either in tumor tissues than in normal tissues or in metastatic cancer tissues than in non-metastatic cancer tissues.^2,3,12,13^ Moreover, our in vitro and in vivo studies suggest that the role of GPR54 is crucial for NSCLC development. Although the loss of chromosome 19p was suggested to be linked to lung tumorigenesis or metastasis,^33^ the frequency of homologous GPR54 deletion in NSCLC was not over 1.5% in cBioPortal (cbioportal.org). On the contrary, GPR54 levels are higher in NSCLC tissues than in normal tissues but are not correlated with survival.^34^ Similarly, the kisspeptin level in the serum increased in NSCLC patients compared to healthy donors, and kisspeptin sensitized cisplatin-induced apoptosis.^35^ Thus, GPR54 and genes involved in GPR54-mediated signaling may work as biomarkers and therapeutic targets. In our present data, GPR54 was required for both metabolic reprogramming and proliferation in NSCLC cells, thereby regulating tumor growth. Our finding that Gpr54-regulated expression patterns of genes involved in glycolytic metabolism is partly in line with recent reports, while those findings showed that GPR54 signaling reversed the Warburg effect.^8–11^ Although recent studies have stimulated GPR54 signaling exogenously, we focused on endogenous GPR54 signaling.^9–11^ Thus, different experimental approaches seem to provide opposite results. We could suggest that GPR54 signaling may positively affect earlier stages of cancer development but inhibit metastatic progression by altering glycolytic metabolism. Another possibility is that GPR54 signaling may need to reach a net independently of time (or disease stage).^36^

DDC plays a key role in the synthesis of neurotransmitters such as dopamine and serotonin.^14^ Thus, the DDC inhibitor carbidopa is usually used in combination with levodopa for the treatment of Parkinson’s disease.^37^ Our transcriptomic analysis revealed that Ddc expression was associated with the Gpr54 genotype. High DDC expression levels are often found in malignant tissues, including SCLC, neuroblastoma, and colorectal cancer.^22,25,38^ In the case of NSCLC, DDC activity was detected at very low levels in an adenocarcinoma cell line (ADLC-5M2) but not in large cell carcinoma, squamous cell carcinoma, and biphasic mesothelioma cell lines.^32^ The ADLC-5M2 cell line was originally obtained from a 67-year-old male patient with adenocarcinoma but is now considered problematic because it is contaminated by the cervical adenocarcinoma cell line HeLa.^39^ Therefore, the role of DDC in NSCLC remains unclear, while it has been known that stress increases plasma dopamine levels in NSCLC patients.^40^ In this study, we revealed that GPR54 regulated DDC expression in NSCLC cell lines via the Gα_q/11_-AKT-mTOR-NF-κB-mediated signaling pathway (Fig. 6). This finding is consistent with a recent finding that the PI3K/AKT pathway regulates DDC expression.^41^ In addition, DDC silencing and carbidopa treatment inhibited glycolysis via HIF-1α expression. Thus, our findings suggest that GPR54-DDC signaling may manage metabolic reprogramming and proliferation in NSCLC cells (Fig. 6). Carbidopa appears to affect glycolysis in mouse skeletal muscles.^42^ However, the mechanisms by which carbidopa regulates glycolysis are yet deciphered, even in cancer. Thus, it remains to decipher the mechanisms by which carbidopa affects glycolysis.

Moreover, DDC silencing reduced the activation of the canonical NF-κB pathway. As the NF-κB pathway is crucial for cancer development,^43^ DDC seems to sustain GPR54-induced NF-κB signaling for NSCLC development. Furthermore, considering our recent innovative finding,^6^ GPR54-DDC signaling in NSCLC cells may affect the stress-induced increase in plasma dopamine level and CD8^+^ T-cell exhaustion. Therefore, it is worth investigating how multicellular GPR54 signaling in the tumor microenvironment, including in NSCLC cells and T cells, regulates NSCLC development. Moreover, the Gpr54 gene dosage was inversely correlated with the expression levels of genes, including Sowaha, Obp2a, and Hspa1a. Human orthologs of Sowaha, Obp2,a and Hspa1a appear to be negatively regulated by NF-κB transcriptional activity, while we need to study how GPR54 signaling governs NF-κB transcriptional regulation of global gene expression. SOWAHA seems to be important to determine the immunotherapy against colorectal cancer and oral squamous cell carcinoma.^44–46^ OBP2A, a member of the lipocalin superfamily, is known to promote prostate cancer castration resistance by enhancing the infiltration of myeloid-derived suppressor cells.^47^ Upregulation of HSPA1A, a member of the HSP70 family, in BAG-1-silenced A549 cells increased the degree of sensitization to cisplatin-induced apoptosis. Moreover, HSPA1A was downregulated in the T cells of non-responders,^48^ suggesting its prognostic contribution to immunotherapy. Accordingly, these genes may be commonly involved in immune cell infiltration into the tumor microenvironment, albeit with contrary roles in cancer progression, which is consistent with the role of GPR54 recently revealed.^6^ Considering that GPR54 signaling occurs in tumor cells, one scenario is that genes inversely expressed by Gpr54 deletion may work as a window for tumor cell crisis by the loss of GPR54 signaling. Otherwise, the expression of these genes may affect the heterogeneity in the tumor microenvironment as well as the tumor cell population. As experimental data for these genes in lung cancer are not enough, it remains to investigate their roles in NSCLC.

Our data showed that the role of GPR54 was not restricted to adenocarcinomas with KRAS mutations but was needed in various NSCLC cells, independently of mutation status and subtype. Moreover, the synergistic effect of KP234 and carbidopa was broadly found in different NSCLC cell lines. Although our data further suggest their usage with targeted therapeutic agents, there are limitations to conclude both the efficacy and safety of those agents. Thus, our ongoing studies for non-GLP safety and efficacy, as well as GLP dose range findings, will support the new therapeutic approach. Recent research has suggested the great value of GPR54 inhibition in CAR-T cell immunotherapy.^6^ Immunotherapy-based approaches are applied for the treatment of NSCLC patients with non-driver mutations.^1^ Thus, it remains to investigate if the combination of those agents is also likely to be effective in NSCLC cells with non-driver mutations. GEMMs are frequently used in NSCLC research, as the tumor microenvironment with the immune system and NSCLC development in GEMM models mimic those of human NSCLC.^49^ However, the biological characteristics of GEMMs do not completely reflect those of human NSCLC. For example, the genetic composition of GEMM tumors is different from that of human NSCLC tumors.^49^ Tumor xenograft assays also have disadvantages in human NSCLC research.^49^ Thus, additional researches with various models, including patient-derived xenograft (PDX) and organoid models, is required to clearly define the role of GPR54 in heterogeneous tumor microenvironments. Moreover, based on the recent findings and our present data,^6^ we suppose that the PDX model may be appropriate for understanding GPR54-dependent crosstalk between tumor cells and immune cells, to reveal the role of GPR54 in the tumors of patients with non-driver mutations, and to investigate GPR54 signaling as a target for immunotherapies. Nonetheless, our study revealed that GPR54 regulates NSCLC development via DDC. This finding broadens the understanding of GPR54 signaling in cancer development and suggests that targeting this signaling pathway may be applicable for treating NSCLC. In addition, deciphering whether this signaling pathway could be applied to SCLC is intriguing. Thus, this would be one of our ongoing works.

Materials and methods

KrasG12D lung cancer model with Gpr54 deletion

Kras^LSL-G12D^ (lox-stop-lox-Kras^G12D^) mice were purchased from Jackson Laboratory (JAX stock #008179, allele symbol Kras^tm4Tyj^).^27^ Gpr54^flox^ mice received from Dr. Novaira (Johns Hopkins University, Baltimore, MD, USA) were crossed with Kras^LSL-G12D^ mice to produce Kras^LSL-G12D^; Gpr54^flox^ mice.^50^ Kras^LSL-G12D^; Gpr54^+/+^ (KG^+/+^), Kras^LSL-G12D^; Gpr54^fl/+^ (KG^fl/+^), and Kras^LSL-G12D^; Gpr54^fl/fl^ (KG^fl/fl^) mice were euthanized at 5 and 10 weeks after virus infection. All care and treatment of the experimental animals were monitored according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) and were approved by the Kyung Hee University Animal Care Center (approval number: KHUASP (S)-21-168). The mice were genotyped via PCR using genomic DNA samples from mouse tails. The Ad5-CMV-Cre-expressing virus was purchased from the Viral Vector Core Facility at the University of Iowa (VVC-U of Iowa-5, vectorcore.medicine.uiowa.edu). At 6 to 7 weeks after birth, the mice were given 1 × 10^8^ pfu of Ad5-CMV-Cre via intratracheal infection. Materials and methods for histological analyses, cell-based experiments, and others are described in the Supplementary Materials and Methods (Supplementary Information).

Histology and Immunohistochemistry

The lungs were fixed in 4% paraformaldehyde and processed for paraffin embedding. Tissues were sectioned at 5μm thickness. After deparaffinization and rehydration, slides were stained with hematoxylin and eosin. For immunohistochemistry, tissues were boiled in a pH 6.0 sodium citrate solution, followed by endogenous peroxidase blocking. Blocking was performed for 1 hour with 5% normal goat serum. Tissues were incubated in primary antibodies overnight at 4 °C. The next day, tissues were washed with PBS and incubated with secondary antibodies and with ABC reagent (30 minutes, RT), followed by DAB detection (NC9276270, Vector Laboratories, Newark, CA, USA). Sections were counterstained with hematoxylin. For TUNEL assays, tissue slides were deparaffinized and permeabilized with 1% Triton X-100 in PBS. Blocking was conducted in 3% BSA solution. Tissues were incubated with the primary antibody. The next day, slides were washed, and cell death was detected by TUNEL assay kit - BrdU-Red (ab66110, Abcam) according to the instructions.

Cell culture

NSCLC cell lines (A549, H460, H1299, H1975, HCC95, HCC1588, H358, and HCC827) were purchased from the Korean Cell Line Bank (Seoul, Korea). Human small airway epithelial cells (HPSAEpiC, Cat. No. 3250, ScienCell, Carlsbad, CA, USA) and human pulmonary alveolar epithelial cells (HPAEpiC, Cat. No. 3200, ScienCell, Carlsbad, CA, USA) were purchased from ScienCell. H1975 cells were cultured in DMEM with 10% fetal bovine serum and 1% penicillin/streptomycin. A549, H1299, HCC95, HCC1588, H358, and H460 cells were cultured in RPMI-1640 with 10% fetal bovine serum and 1% penicillin/streptomycin. HCC827 cells were cultured in RPMI-1640 with 10% fetal bovine serum, 1% penicillin/streptomycin, and 5% HEPES.

RNA sequencing

Total RNA was isolated using Trizol reagent (Invitrogen). RNA quality and quantity were assessed using an Agilent TapeStation 4000 system (Agilent Technologies, Amstelveen, The Netherlands) and an ND-2000 spectrophotometer (Thermo Inc., DE, USA), respectively. Poly(A)-selected mRNA libraries were prepared using an Illumina-compatible protocol and sequenced on an Illumina platform. High-throughput sequencing was performed on a NextSeq 500 platform (Illumina, Inc., USA) to generate single-end 75 bp reads for QuantSeq 3’ mRNA-seq. Reads were aligned to the reference genome/transcriptome (GRCh38/hg38) using Bowtie2, and gene-level read counts were obtained using Bedtools. Count data were normalized using the TMM + CPM method, and differential expression analysis was performed with edgeR in R/Bioconductor. Data mining and graphic visualization were performed using ExDEGA (https://www.e-biogen.com). The heatmap was generated by ggplot2 in R software. The RNA-seq dataset is accessible through the GEO series accession number GSE212538.

Phosphoprotein array

50μg of protein lysates were biotin-labeled using biotin/DMF and labeling buffer, and the reaction was stopped with stop reagent. Phosphorylation profiles were measured using the Phospho Explorer Antibody Microarray (Full Moon Biosystems, Sunnyvale, CA), followed by Cy3-streptavidin detection (GE Healthcare, Chalfont St. Giles, UK). Arrays were scanned with a GenePix 4100A scanner and quantified using GenePix 7.0 software (Axon Instrument, USA). Proteins were annotated using the UniProt database, and downstream analysis/visualization was performed using ExDEGA (https://www.e-biogen.com). The raw data were uploaded as “Supplementary Data.xlsx” in the Supplementary Information.

Glycolysis stress assay

Glycolytic activity of the NSCLC cell lines H358 (1 × 10^4^ cells/well) and HCC1588 (1.5 × 10^4^ cells/well) was measured using the XFe96 Extracellular Flux Analyzer (Agilent Seahorse Bioscience, Billerica, USA). Cells were seeded in Seahorse XF96 microplates 1 day before the assay, and the sensor cartridge was hydrated overnight with Seahorse XF Calibrant (Agilent, Cat. No. 100840-000). For experiments requiring transfection, siControl, siGPR54, siGNAQ, siGNA12, siARRB2, or siDDC were introduced at a final concentration for 48 hours. On the day of the assay, XF Base Medium (Agilent, Cat. No. 103334-100) supplemented with 4mM L-glutamine (Gibco, Cat. No. 250303-081) was used as the assay medium, and cells were equilibrated in a CO_2_-free incubator at 37 °C. During the assay, compounds were sequentially injected into the designated ports as follows: port A, KP10; port B, rotenone and antimycin A; port C, 2-deoxy-D-glucose. Measurements were acquired in mix-wait-measure cycles, and oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and glycolytic proton efflux rate (GlycoPER) were recorded. To confirm GPR54-related signaling pathway experiments, cells were pretreated for 5 minutes with inhibitors. Following pretreatment, the medium was replaced with XF assay medium, equilibrated, and analyzed under the same injection sequence and measurement conditions as described above. For data analysis, mitochondrial OCR (mOCR) was calculated as (Pre-stim-Residual), basal GlycoPER as (Pre-stim-NGA), and compensatory GlycoPER (Post R/A – NGA). NGA is non-glycolytic acidification.

Statistical analysis

All experiments were performed at least three times, as indicated in the figure legends. Data are presented as the means ± standard deviations (SD). Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Prism Software Inc.). Comparisons of three or more groups were assessed with the Kruskal-Wallis non-parametric test with Dunn’s post-hoc test, and comparisons between two groups were assessed with an unpaired two-tailed Student’s t-test. Kaplan–Meier survival curves with 95% confidence intervals were compared using the log-rank test. GO enrichment analysis was performed using DAVID v6.8, and terms with Benjamini–Hochberg adjusted p < 0.05 are shown. Gene set enrichment analysis (GSEA) was conducted on all expressed genes on the GSEA portal (http://www.broad.it.edu/GSEA/) with phenotype labels (rest vs control), 1,000 permutations, and gene set permutation. A p-value < 0.05 was considered statistically significant. Plots were generated using R and/or GraphPad Prism. *, p < 0.05, **, p < 0.01; ns, not significant.

Supplementary information

Supplementary Information Supplementary Data