Carcinoembryonic antigen potentiates non-small cell lung cancer progression via PKA-PGC1ɑ axis

doi:10.21203/rs.3.rs-3212042/v1

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Carcinoembryonic antigen potentiates non-small cell lung cancer progression via PKA-PGC1ɑ axis

https://doi.org/10.21203/rs.3.rs-3212042/v1

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Background

Carcinoembryonic antigen (CEA) is a tumor-associated antigen primarily produced by tumor cells. CEA has been reported to be involved in various biological processes, including cell adhesion, proliferation, differentiation, and metastasis. However, the impact of CEA on the tumor immune escape remains largely unknown.

Methods

The A549 tumor-bearing mouse models were established to investigate the role of CEA during tumor progression. CCK-8 assay, Colony formation assay, ELISA, immunohistochemistry and flow cytometry analysis were used to assess the role of CEA on fatty acid metabolism and proliferation of A549 and H1299 cells.

Results

In this study, we demonstrate that CEA promotes the proliferation and migration of non-small cell lung cancer (NSCLC). Our findings indicate that CEA enhances NSCLC proliferation and migration through the activation of fatty acid metabolism-related genes and PGC1 expression. Mechanistically, CEA promotes PGC1 expression via the PKA-PGC1ɑ signaling pathway. Inhibition of the PKA-PGC1ɑ signaling pathway attenuates CEA-induced proliferation and migration of NSCLC. Interestingly, we also observed that inhibition of PGC1α downregulates the expression of PD-L1 in NSCLC.

Conclusion

These results reveal the involvement of CEA in fatty acid metabolism during lung cancer development and highlight its impact on immunotherapy of NSCLC.

Carcinoembryonic antigen

lung cancer

PKA-PGC1ɑ

fatty acid metabolism

Lung cancer is a leading cause of cancer-related deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of all lung cancer cases (Chen et al. 2014). Unfortunately, most lung cancer patients are diagnosed at an advanced stage, leaving only a small percentage eligible for curative surgery or drug treatments, while the majority can only receive palliative chemotherapy and have a life expectancy of 1–2 years (Wang et al. 2021). Therefore, early detection of lung cancer plays a crucial role in its treatment. Currently, imaging examinations such as chest X-ray, CT scan, and PET-CT are commonly used diagnostic methods for lung cancer (Restrepo et al. 2023). Tumor markers, which are substances produced by tumor cells or the body's response to tumor cells during tumor development and growth, have also been utilized in clinical practice to assist in lung cancer diagnosis (J and Boyle 2022).

Carcinoembryonic antigen (CEA) is a well-known tumor marker that was identified over 30 years ago (Beauchemin and Arabzadeh 2013). This protein is released by many solid tumors and plays a role in cancer cell survival and differentiation through an autocrine function (Kelleher et al. 2019). The concentration of CEA in the serum of lung cancer patients is significantly higher compared to healthy individuals, making it a valuable diagnostic marker (Grunnet and Sorensen 2012). High expression of CEA has been found to be associated with poor prognosis in patients with non-small cell lung cancer (Facchinetti et al. 2015), and its levels are elevated in embryonic or tumor tissues. Apart from its clinical use as a tumor marker, recent studies suggest that CEA has diverse biological functions. These include participation in intercellular adhesion, transduction of regulatory signals, regulation of cell proliferation, differentiation, apoptosis, angiogenesis, and immune response (Hao et al. 2019). However, the precise biological role of CEA in the tumor immune escape is not yet fully understood.

The tumor microenvironment can subject cancer cells to various stresses, such as nutrient and oxygen deprivation, as well as acidification of the extracellular space (Anderson and Simon 2020). For tumor progression to occur, cancer cells need to adapt to these stresses and develop survival mechanisms within this microenvironment. This adaptation may involve the oncogene-driven expression of nutrient transporters (Nwosu et al. 2023), the ability to derive energy from different nutrient sources (including scavenged proteins, recycled organelles, and necrotic debris), and metabolic cooperation (Faubert et al. 2020), all of which contribute to the tumor cells' ability to thrive in the challenging tumor microenvironment. In this study, we aimed to investigate the role of soluble CEA on the proliferation and migration in NSCLC and elucidate the underlying mechanism involved in regulating the tumor metabolism and immunity.

Cell lines and treatment

Human lung carcinoma A549 and H1299 cells were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) in 2015. All cell lines were examined Mycoplasma-free using MycAwayTM-Color One-Step Mycoplasma Detection Kit (Yeasen Bio-technol) and these cells were authenticated and certified by ChengDu Nuohe Biotech co., LTD (Sichuan, China). Cells were cultured in DMEM medium containing high glucose supplemented with 10% FBS and 100 U/mL penicillin/streptomycin.

Animals and tumor models

Nude mice were purchased from the Animal Institute of the Academy of Medical Science (Beijing, China). These mice were kept in specific pathogen-free conditions under a 12 hr light cycle, and were given a regular chow diet at Chongqing University Cancer Hospital. A549 cells (5 × 10⁶) were implanted subcutaneously into male nude mice. Tumor growth was measured using calipers every 3 days. Tumor volume was calculated as follows: V= (length × width²) × 0.5. For SR18292 (SR, 45 mg/kg, Cat No. HY-101491, MCE) and H89 (10 mg/kg, Cat No. HY-15979A, MCE) treatment studies, treatment was given intraperitoneally every 3 days once tumors approximately 100 mm³ until the mice were sacrificed. All animal experiments were approved by the ethics Committee of the Chongqing University Cancer Hospital, Chongqing, China. All animal studies were conducted in accordance with the national and international Guidelines for the Care and Use of Laboratory, and with the approval of the Animal Care and Use Committee (IACUC) of Chongqing University Cancer Hospital and complied with the Declaration of Helsinski.

Flow Cytometry (FCM)

The A549 and H1299 cells were prepared and blocked with rat IgG (10 µg/mL; Sigma) for at least 20 minutes on ice. After that, they were washed with staining buffer (PBS containing 2% FBS). Then, the cells were labeled with the indicated antibodies (1:100) for 30 minutes at 4°C. Dead cells were excluded using a Fixable Viability Dye Efluor 780 (1:1000; Cat No. 65-0865-14, eBioscience). PD-L1 (Cat No. 329708) was purchased from Biolegend. Bodipy558/568 (Cat No. D3835) was obtained from Thermo Fisher Scientific. Mito-Tracker Green probes (Cat No. C1048) were from Beyotime (Shanghai, China). Flow cytometry (FCM) was performed on BD FACS Canto II platforms, and the results were analyzed with FlowJo software version 10.0.7 (TreeStar). Sorting was performed on a BD FACSAria II instrument (BD Biosciences).

Colony formation assay

The A549 and H1299 cells were adjusted to 1 × 10³ cells/mL. Cell suspension was supplemented with culture medium and seeded in the 6-well plate. Then, these cells were uniformly dispersed and cultured for 2–3 weeks. Cell growth was terminated and the culture medium was discarded. The cells were treated by methanol for 15 min and dyed with crystal violet staining solution for 10 min. The colony number visible to naked eyes was counted, and the colony rate = (colony number/seeded cell number) × 100%.

CCK-8 assay

The A549 and H1299 single cell suspension was diluted and adjusted to 1 × 10⁴ cells/mL. 200 µL of the diluted cell suspension was absorbed and seeded in the 96-well plate. The experiment was completed by following the instructions of CCK-8 kit (Dojindo, Tokyo, Japan).

Apoptosis assay

The A549 cells were treated with different concentrations of CEA, DDP (Cat No. ST1164, Beyotime) or combined for 24 h. Apoptotic ratio was measured by Annexin V-FITC/PI double staining kit (Cat No. FXP018, Beijing 4A Biotech) according to the manufacturer’s protocol.

Transfection of shRNAs in A549 cells

Three pLKD-CMV-EGFP-2A lentivectors containing shRNAs targeting PPARGC1A or scrambled shRNA lentivector were obtained from Genechem (Shanghai, China). A549 cells were plated at 1× 10⁶ cells per ml in 6-well plates and transduced with lentiviral particles (at MOI of 20) with 5µg/ml Polybrene (GenePharma). Cells were harvested and screened for further experiments 3 days after transfection.

ELISA

Intracellular cAMP expression in A549 cells were measured using a cAMP ELISA kit (Cat No. ab138880, Abcam) according to the manufacturer's instruction.

Immunohistochemistry

Formalin-fixed paraffin-embedded tumor samples slides were incubated in 0.3% H₂O₂ in methanol for 30 min to block endogenous peroxidase activity. Antigens were retrieved with 10 mmol L-1 sodium citrate (pH 6) for 5 min in a pressure cooker. The slides were then incubated with the antibodies (anti-PCNA (1:100, Cat. No. 2586S, Cell Signaling Technology), P-PKA (1:100, Cat. No. 9621S, Cell Signaling Technology), PGC1ɑ (1:100, Cat. No. 66369-1-Ig, Proteintech) and PD-L1 antibody (1:100, Cat. No. 13684S, Cell Signaling Technology) at 4℃ overnight. The slides without treatment with primary antibody served as negative controls. Then, the sections were imaged on a phase contrast microscopy (Leica, Wetzlar, Germany).

Quantitative real-time PCR

Total RNA was extracted using RNAiso Plus (TAKARA, Japan) and the RNA concentration was measured using NanoDrop 2000 (Thermo Scientific). 1 µg total RNA was converted to complementary DNA (cDNA) using the PrimeScript RT-PCR Kit (RR014A, Takara) according to the manufacturers’ instructions. qPCR was performed using TB Green Fast qPCR Mix Kit (RR430A, Takara) on a CFX384 system (BIO-RAD), and the relative quantification (2^−ΔΔCt) method was used to analyze gene expression. β-actin mRNA was used as a reference for mRNA quantification. All qPCR experiments were repeated at least three times.

RNA sequencing library construction

Total RNA was extracted from A549 cells after CEA or vehicle treatment. The RNA-seq library for these RNA samples was constructed according to the strand-specific RNA sequencing library preparation protocol. mRNA transcripts were enriched by two rounds of poly-(A+) selection with Dynabeads oligo-(dT) 25 (Invitrogen) before library construction. The prepared libraries were sequenced on an Illumina Novaseq 6000 platform.

Western blotting

Cells were lysed by RIPA lysis buffer, and the cell lysates were incubated on ice for 30 min and centrifuged at 13,000g, 4°C for 15 min before the supernatant was collected. Western blot analysis was performed as previously described (Yan et al. 2018). The primary antibodies included anti-PGC1-α (1:1,000; Cat No. 2178S, Cell Signaling Technology) anti-p-PKA (1:1,000; Cat No. 4781S, Cell Signaling Technology) and anti-PD-L1 (1:1,000; Cat No. 13684S, Cell Signaling Technology).

Statistical analysis

Statistical parameters and n values are indicated in figure legends. All results were confirmed in at least three independent experiments and were expressed as means ± SD. Student’s t-test and one- or two-way ANOVA were used for calculation statistical significance with GraphPad Prism software (version 8.0). For survival curves, the Kaplan-Meier method was used, and the differences in survival curves were analyzed using the log-rank test. P < 0.05 was considered statistically significant.

CEA promotes proliferation, migration and invasion of NSCLC cells

To evaluate the oncogenic role of CEA in non-small cell lung cancer (NSCLC) in vivo, we subcutaneously injected A549 cells into nude mice. After 25 days of CEA treatment, the mice were sacrificed and the tumors were isolated. The CEA treatment significantly caused growth in A549 xenografts (Fig. 1A). Moreover, the A549 tumor volume and weight were noticeably increased (Fig. 1B and C). PCNA staining also indicated a remarkable increase in A549 cell proliferation upon CEA treatment (Fig. 1D). CEA treatment significantly promoted cell growth in both A549 and H1299 cells, as determined by CCK8 assays (Fig. 1E). Furthermore, cell cycle analysis revealed a significant increase in proliferation of A549 and H1299 cells upon CEA treatment (Fig. 1F). Colony-formation assays demonstrated that CEA treatment enhanced the clonogenic capacity of both A549 and H1299 cells (Fig. 1G). Additionally, cell migration and invasion assays demonstrated that CEA promoted the migration and invasion abilities of A549 and H1299 cells (Fig. 1H). Cisplatin (DDP) is an anti-cancer agent known to induce tumor cell apoptosis through multiple mechanisms (Sun et al. 2020). DDP significantly induced apoptosis in A549 and H1299 cells, and this effect was reversed by CEA (Fig. 1I). These results suggest that CEA plays crucial roles in the proliferation, migration, and invasion of NSCLC, both in vitro and in vivo.

CEA accelerates NSCLC cells growth by reprogramming fatty acid metabolism

To investigate the role of CEA in NSCLC development, we conducted RNA sequencing (RNA-seq) analysis on A549 cells treated with CEA and control cells. The analysis revealed that CEA treatment globally altered the expression of 1,786 genes, with 633 genes up-regulated and 1082 genes down-regulated (Fig. 2A). Further analysis of these genes identified several enriched pathways, including TRAF-mediated signal transduction, fatty acid metabolism, regulation of metanephros size, and metal ion transmembrane transporter activity (Fig. 2B). Fatty acid metabolism is known to play a crucial role in NSCLC proliferation, migration, and invasion within the tumor microenvironment (Koundouros and Poulogiannis 2020). Notably, our analysis demonstrated significant up or down regulation of genes related to fatty acid metabolism following CEA treatment (Fig. 2C). Consistent with our expectations, we observed increased mitochondria numbers and accumulation of lipid droplets in A549 and H1299 cells treated with CEA (Fig. 2D, E). These findings suggest that CEA promotes the proliferation and metastasis of NSCLC cells through its impact on fatty acid metabolism.

CEA enhances PGC1α and PD-L1 expression via activating PKA signaling.

PPARγ coactivator-1α (PGC-1α) is a crucial regulator of mitochondrial biosynthesis and a coactivator of nuclear transcription. It plays a significant role in various energy metabolism processes, including mitochondrial biosynthesis, hepatic gluconeogenesis, and fatty acid β-oxidation (Fontecha-Barriuso et al. 2020; Rius-Perez et al. 2020). In light of the fact that CEA treatment led to an increase in the number of mitochondria and affected lipid metabolism, we were curious to investigate whether the promotion of NSCLC cell proliferation and metastasis by CEA was regulated by PGC1α. Our findings indicated that PGC1α expression was up-regulated after CEA treatment in A549 and H1299 cells. Additionally, genes related to fatty acid metabolism, such as CYP2J2 and PLA2G10, exhibited significant up-regulation or down-regulation (Fig. 3A). Interestingly, CEA treatment also resulted in the promotion of PD-L1 expression in NSCLC cells (Fig. 3A).

As a glycophosphatidylinositol-anchored protein lacking a cytoplasmic domain, CEA relies on a transmembrane interaction partner to facilitate intracellular signal transduction (Beauchemin and Arabzadeh 2013). Previous studies have observed the co-localization of CEA and integrins in lipid rafts, which play a role in regulating integrin-dependent signaling pathways like phosphoinositide 3-kinase (PI3K) and AKT, which are integrin-linked kinases (Camacho-Leal et al. 2007; Ordonez et al. 2007). Additionally, previous studies have shown that CEA expression can be selectively enhanced by cAMP (Guadagni et al. 1991). In our study, we observed an upregulation of cAMP levels in lung cancer cells treated with CEA (Fig. 3B). This suggests that CEA may increase intracellular cAMP concentration, leading to the activation of PKA and promoting the expression of PGC1α. Indeed, we found that CEA treatment resulted in increased levels of PGC1α and p-PKA in A549 and H1299 cells (Fig. 3C). Furthermore, CEA treatment also promoted the protein expression of PD-L1 in NSCLC cells (Fig. 3D, E). Moreover, inhibiting PGC1α with SR18292 or inhibiting PKA with H89 down-regulated the expression of PD-L1 and p-PKA in NSCLC (Fig. 3F, G). These findings suggest that CEA can activate the expression of PGC-1α and PD-L1 through the cAMP-PKA signaling pathway.

Knockdown of PPARGC1A attenuates CEA-mediated NSCLC cell proliferation in vitro.

To investigate the role of PGC1α in fatty acid metabolism and cell proliferation of NSCLC cells, we utilized three shRNAs to knock down PPARGC1A in A549 cells (Fig. 4A). The knockdown of PPARGC1A significantly hindered the growth of A549 cells, as evidenced by CCK8 assays (Fig. 4B). Moreover, the migration and invasion abilities of A549 cells were notably reduced upon silencing of PPARGC1A (Fig. 4C). The colony-formation assays also demonstrated that clonogenic capacity was suppressed following PPARGC1A knockdown (Fig. 4D). Additionally, PPARGC1A deficiency impaired the ability of CEA to increase the number of mitochondria and accumulate lipid droplets (Fig. 4E, F). These findings suggest that CEA promotes the proliferation and metastasis of NSCLC cells by activating PGC-1α.

Blocking PKA-PGC1ɑ axis blunts CEA-mediated NSCLC growth in vivo.

To evaluate the oncogenic role of PGC1ɑ in NSCLC in vivo, we injected PGC-1α deficient A549 cells and control cells subcutaneously into nude mice. We observed that the PGC1ɑ-deficient A549 cells were noticeably smaller than those in the control group (Fig. 5A). Furthermore, the tumor volume and weight were reduced after PGC1ɑ knockdown compared to the control group (Fig. 5B and C). In addition, the PGC1ɑ-knockdown tumors showed decreased levels of PCNA, P-PKA, PGC1ɑ, and PD-L1 signals compared to the control tumor (Fig. 5D). We also found that the treatment with SR18292 and H89 inhibited the growth of A549 tumors in vivo (Fig. E-G). Moreover, the tumors treated with SR18292 and H89 exhibited decreased levels of PCNA, P-PKA, PGC1ɑ, and PD-L1 signals. Taken together, these results demonstrate that CEA promotes NSCLC growth by regulating the PKA-PGC1ɑ axis.

CEA, a glycoprotein primarily used as a tumor marker, is of significant interest in understanding its function and relevance to malignant transformation, especially in colorectal cancer (Beauchemin and Arabzadeh 2013). In this study, we demonstrate that CEA plays a crucial role in promoting NSCLC growth and metastasis both in vivo and in vitro. Specifically, CEA facilitates fatty acid metabolism in lung cancer cells, leading to enhanced cell proliferation. Through RNA-seq analysis, we found that CEA reprograms lipid metabolism in NSCLC, which is dependent on the PKA-PGC1α signaling pathways. Inhibition of PKA or PGC1α resulted in suppressed NSCLC growth and downregulation of PD-L1 expression in tumor cells. Overall, our findings reveal a novel function of CEA in the development of NSCLC.

As our understanding of tumor biology deepens, we are also gaining insights into the intricate nature of tumor metabolism. Metabolic reprogramming is a crucial characteristic of tumor cells (Hanahan 2022). While the alteration of glycolytic metabolism, known as the Warburg effect, is considered a pivotal aspect of tumor metabolism, tumor cells must also effectively coordinate energy production and biosynthetic pathways to promote cell proliferation (Martinez-Reyes and Chandel 2021). Recent studies have highlighted the significance of lipid metabolism, particularly increased de novo lipogenesis, as a new hallmark of many aggressive cancers (Feitelson et al. 2023). Additionally, cancer cells exhibit heightened levels of de novo adipogenesis due to enhanced expression of various lipogenic enzymes like ACLY (ATP citrate lyase) and FASN (fatty acid synthase) (Reina-Campos et al. 2017). In our study, we discovered that the CEA-elevated proliferation and metastasis of NSCLC cells were dependent on fatty acid metabolism. Treatment of A549 and H1299 cells with CEA resulted in an increase in the number of mitochondria and accumulation of lipid droplets. The upregulation of PGC1α by CEA was found to be crucial for NSCLC fatty acid metabolism and proliferation. Knockdown of PGC1α reduced CEA-mediated NSCLC cell proliferation both in vitro and in vivo. PGC1α is a transcription coactivator that plays a central role in regulating mitochondrial biogenesis and cellular energy metabolism (Fontecha-Barriuso et al. 2020). PGC-1α has been reported to be involved in obesity, diabetes, and cancers. In our present study, we observed a significant upregulation of the PGC-1α signaling pathway in CEA-treated NSCLC cells, suggesting that CEA may regulate cell proliferation and lipid metabolism of NSCLC cells through the PGC-1α signaling.

A significant amount of clinical research data has demonstrated that the immune evasion of tumor cells plays a crucial role in tumor progression. Understanding the molecular mechanism behind this phenomenon has become a key focus in the field of tumor immunotherapy (Hu et al. 2021). Programmed death ligand-1 (PD-L1) is a pivotal molecule that is highly expressed in various types of cancers (Jiang et al. 2021). PD-L1 interacts with the PD-1 receptor on activated T cells, resulting in T cell exhaustion and facilitating tumor immune evasion (Yu et al. 2016). In our study, we discovered that CEA can activate the expression of PD-L1 by phosphorylating PKA. However, the precise mechanisms through which CEA influences PD-L1 expression in tumors remain unclear. Therefore, a comprehensive investigation into the molecular mechanism by which CEA regulates PD-L1 expression in lung cancer cells will offer novel insights for the clinical treatment of lung cancer.

In summary, the present study demonstrates that CEA induces proliferation and migration in NSCLC. Furthermore, CEA promotes the expression of fatty acid metabolism-related genes and PGC1α in lung cancer cells. The study also reveals that CEA-induced NSCLC proliferation relies on the PKA-PGC1α signaling pathway. Additionally, inhibiting PGC1α leads to a decrease in the expression of PD-L1 in tumor cells. These findings provide evidence that CEA regulates fatty acid metabolism and the proliferation of NSCLC cells. Consequently, CEA could be considered as a potential target for immunotherapy against cancer.

Availability of data and materials

The data and code generated or analyzed in this study are available from the corresponding authors upon reasonable request.

Acknowledgements

The authors wish to thank Yu Zhou for their assistance in Western blot analysis and Immunohistochemistry procedure.

Funding

This work was supported by the Major International (Regional) Joint Research Program of the National Natural Science Foundation of China (No. 81920108027), Natural Science Foundation of Chongqing (cstc2020jcyj-msxmX0516), Chongqing Outstanding Youth Foundation, and Funding for Chongqing Youth Talents (No. CQYC202003006).

Author information

Authors and Affiliations

Department of Medical Oncology, Chongqing University Cancer Hospital, Chongqing 400030, China

Juan Lei, Lei Wu, Jiangang Zhang, Dairong Li and Yongsheng Li

School of Medicine, Chongqing University, Chongqing 400030, China

Nan Zhang, Xudong Liu, Liwen Kuang, Jiongming Chen, Yijiao Chen,

Contributions

Y. Li and D. Li conceived of the study and wrote the manuscript. J. Lei, L. Wu, N. Zhang, X. Liu, and J. Zhang performed cells and animal experiments; J. Zhang and J. Chen performed WB and RT-qPCR experiments; N. Zhang, X. Liu, L. Kuang performed bioinformatics analysis; L. Wu, J. Zhang, Y. Chen performed Flow cytometry experiments. Y. Li and L. Wu analyzed the data. All authors reviewed and edited the manuscript.

Corresponding author

Correspondence to Yongsheng Li ([email protected])

Ethics declarations

Ethics approval and consent to participate

All animal experiments were approved by the ethics Committee of the Chongqing University Cancer Hospital, Chongqing, China. All animal studies were conducted in accordance with the national and international Guidelines for the Care and Use of Laboratory, and with the approval of the Animal Care and Use Committee (IACUC) of Chongqing University Cancer Hospital and complied with the Declaration of Helsinski.

Consent for publication

Not applicable.

Competing interests

The authors have declared that no competing interest exists.

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Carcinoembryonic antigen potentiates non-small cell lung cancer progression via PKA-PGC1ɑ axis

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Abstract

Figures

Background

Materials and methods

Cell lines and treatment

Animals and tumor models

Flow Cytometry (FCM)

Colony formation assay

CCK-8 assay

Apoptosis assay

Transfection of shRNAs in A549 cells

ELISA

Immunohistochemistry

Quantitative real-time PCR

RNA sequencing library construction

Western blotting

Statistical analysis

Results

CEA promotes proliferation, migration and invasion of NSCLC cells

CEA accelerates NSCLC cells growth by reprogramming fatty acid metabolism

Discussion

Conclusions

Declarations

Consent for publication

References

Status:

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