Skip to main content

Advertisement

SpringerLink
Log in

EGFL6 promotes bone metastasis of lung adenocarcinoma by increasing cancer cell malignancy and bone resorption

  • Research Paper
  • Published:
Clinical & Experimental Metastasis Aims and scope Submit manuscript

Abstract

Lung adenocarcinoma is the most common and aggressive type of lung cancer with the highest incidence of bone metastasis. Epidermal growth factor-like domain multiple 6 (EGFL6) is an exocrine protein, and the expression of EGFL6 is correlated with survival of patient with lung adenocarcinoma. However, the association between EGFL6 expression in lung adenocarcinoma and bone metastasis has not been investigated. In this study, we found that EGFL6 levels in lung adenocarcinoma tissues correlate with bone metastasis and TNM stages in surgical patients. In vitro, overexpression of EGFL6 in lung adenocarcinoma cells promoted their proliferation, migration, and invasion ability compared with control by enhancing EMT process and activating Wnt/β-catenin and PI3K/AKT/mTOR pathways. In the nude mouse model, overexpression of EGFL6 enhanced tumor growth and caused greater bone destruction. Moreover, the exocrine EGFL6 of human lung adenocarcinoma cells increased osteoclast differentiation of bone marrow mononuclear macrophages (BMMs) of mice via the NF-κB and c-Fos/NFATc1 signaling pathways. However, exocrine EGFL6 had no effect on osteoblast differentiation of bone marrow mesenchymal stem cells (BMSCs). In conclusion, high expression of EGFL6 in lung adenocarcinomas is associated with bone metastasis in surgical patients. The underlying mechanism may be the increased metastatic properties of lung adenocarcinoma cells with high EGFL6 level and the enhanced osteoclast differentiation and bone resorption by exocrine EGFL6 from tumors. Therefore, EGFL6 is a potential therapeutic target to reduce the ability of lung adenocarcinomas to grow and metastasize and to preserve bone mass in patients with bone metastases from lung adenocarcinomas.

Graphical Abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Abbreviations

BMMs:

Bone marrow mononuclear macrophage

BMSCs:

Bone marrow mesenchymal stem cells

CCK-8:

Cell Counting Kit-8

EGFL6:

Epidermal growth factor-like domain multiple 6

EMT:

Epithelial mesenchymal transformation

IHC:

Immunohistochemistry

M-CSF:

Macrophage colonies stimulating factor

MCP1:

Monocyte chemoattractant protein 1

NF-κB:

Nuclear factor-kappa B

OPG:

Osteoprotegerin

RANKL:

Receptor activator of the NF-κB ligand

References

  1. Siegel RL, Miller KD, Jemal A (2020) Cancer statistics, 2020. CA Cancer J Clin 70(1):7–30

    Article  PubMed  Google Scholar 

  2. Zhang H, Guo L, Chen J (2020) Rationale for Lung Adenocarcinoma Prevention and Drug Development based on Molecular Biology during Carcinogenesis. Onco Targets Ther 13:3085–3091

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Cho YJ, Cho YM, Kim SH, Shin KH, Jung ST, Kim HS (2019) Clinical analysis of patients with skeletal metastasis of lung cancer. BMC Cancer 19(1):303

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bienz M, Saad F (2015) Management of bone metastases in prostate cancer: a review. Curr Opin Support Palliat Care 9(3):261–267

    Article  PubMed  Google Scholar 

  5. Macedo F, Ladeira K, Pinho F, Saraiva N, Bonito N, Pinto L, Goncalves F (2017) Bone Metastases: An Overview Oncol Rev 11(1):321

    PubMed  Google Scholar 

  6. Yang M, Sun Y, Sun J, Wang Z, Zhou Y, Yao G, Gu Y, Zhang H, Zhao H (2018) Differentially expressed and survival-related proteins of lung adenocarcinoma with bone metastasis. Cancer Med 7(4):1081–1092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Yin JJ, Pollock CB, Kelly K (2005) Mechanisms of cancer metastasis to the bone. Cell Res 15(1):57–62

    Article  CAS  PubMed  Google Scholar 

  8. Wood SL, Pernemalm M, Crosbie PA, Whetton AD (2014) The role of the tumor-microenvironment in lung cancer-metastasis and its relationship to potential therapeutic targets. Cancer Treat Rev 40(4):558–566

    Article  CAS  PubMed  Google Scholar 

  9. Papotti M, Kalebic T, Volante M, Chiusa L, Bacillo E, Cappia S, Lausi P, Novello S, Borasio P, Scagliotti GV (2006) Bone sialoprotein is predictive of bone metastases in resectable non-small-cell lung cancer: a retrospective case-control study. J Clin Oncol 24(30):4818–4824

    Article  CAS  PubMed  Google Scholar 

  10. Zhang L, Hou X, Lu S, Rao H, Hou J, Luo R, Huang H, Zhao H, Jian H, Chen Z, Liao M, Wang X (2010) Predictive significance of bone sialoprotein and osteopontin for bone metastases in resected chinese non-small-cell lung cancer patients: a large cohort retrospective study. Lung Cancer 67(1):114–119

    Article  PubMed  Google Scholar 

  11. Peng X, Guo W, Ren T, Lou Z, Lu X, Zhang S, Lu Q, Sun Y (2013) Differential expression of the RANKL/RANK/OPG system is associated with bone metastasis in human non-small cell lung cancer. PLoS ONE 8(3):e58361

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nakamura ES, Koizumi K, Kobayashi M, Saitoh Y, Arita Y, Nakayama T, Sakurai H, Yoshie O, Saiki I (2006) RANKL-induced CCL22/macrophage-derived chemokine produced from osteoclasts potentially promotes the bone metastasis of lung cancer expressing its receptor CCR4. Clin Exp Metastasis 23(1):9–18

    Article  CAS  PubMed  Google Scholar 

  13. Chim SM, Qin A, Tickner J, Pavlos N, Davey T, Wang H, Guo Y, Zheng MH, Xu J (2011) EGFL6 promotes endothelial cell migration and angiogenesis through the activation of extracellular signal-regulated kinase. J Biol Chem 286(25):22035–22046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chang CC, Sung WW, Hsu HT, Yeh CM, Lee CH, Chen YL, Liu TC, Yeh KT (2018) Validation of EGFL6 expression as a prognostic marker in patients with lung adenocarcinoma in Taiwan: a retrospective study. BMJ Open 8(6):e021385

    Article  PubMed  PubMed Central  Google Scholar 

  15. An J, Du Y, Fan X, Wang Y, Ivan C, Zhang XG, Sood AK, An Z, Zhang N (2019) EGFL6 promotes breast cancer by simultaneously enhancing cancer cell metastasis and stimulating tumor angiogenesis Oncogene 38(12):2123–2134

    CAS  PubMed  Google Scholar 

  16. Huo FC, Zhu WT, Liu X, Zhou Y, Zhang LS, Mou J (2021) Epidermal growth factor-like domain multiple 6 (EGFL6) promotes the migration and invasion of gastric cancer cells by inducing epithelial-mesenchymal transition. Invest New Drugs 39(2):304–316

    Article  CAS  PubMed  Google Scholar 

  17. Zhu Z, Ni H, You B, Shi S, Shan Y, Bao L, Duan B, You Y (2018) Elevated EGFL6 modulates cell metastasis and growth via AKT pathway in nasopharyngeal carcinoma. Cancer Med 7(12):6281–6289

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yeung G, Mulero JJ, Berntsen RP, Loeb DB, Drmanac R, Ford JE (1999) Cloning of a novel epidermal growth factor repeat containing gene EGFL6: expressed in tumor and fetal tissues. Genomics 62(2):304–307

    Article  CAS  PubMed  Google Scholar 

  19. Kang J, Wang J, Tian J, Shi R, Jia H, Wang Y (2020) The emerging role of EGFL6 in angiogenesis and tumor progression. Int J Med Sci 17(10):1320–1326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rami-Porta R, Asamura H, Travis WD, Rusch VW (2017) Lung cancer - major changes in the american Joint Committee on Cancer eighth edition cancer staging manual. CA Cancer J Clin 67(2):138–155

    Article  PubMed  Google Scholar 

  21. Salvador F, Llorente A, Gomis RR (2019) From latency to overt bone metastasis in breast cancer: potential for treatment and prevention. J Pathol 249(1):6–18

    Article  PubMed  PubMed Central  Google Scholar 

  22. Popper HH (2016) Progression and metastasis of lung cancer. Cancer Metastasis Rev 35(1):75–91

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhang X (2019) Interactions between cancer cells and bone microenvironment promote bone metastasis in prostate cancer. Cancer Commun (Lond) 39(1):76

    Article  PubMed  Google Scholar 

  24. Hirsch FR, Scagliotti GV, Mulshine JL, Kwon R, Curran WJ Jr, Wu YL, Paz-Ares L (2017) Lung cancer: current therapies and new targeted treatments. Lancet 389(10066):299–311

    Article  CAS  PubMed  Google Scholar 

  25. Kuchuk M, Kuchuk I, Sabri E, Hutton B, Clemons M, Wheatley-Price P (2015) The incidence and clinical impact of bone metastases in non-small cell lung cancer. Lung Cancer 89(2):197–202

    Article  PubMed  Google Scholar 

  26. Zhang W, Bado IL, Hu J, Wan YW, Wu L, Wang H, Gao Y, Jeong HH, Xu Z, Hao X, Lege BM, Al-Ouran R, Li L, Li J, Yu L, Singh S, Lo HC, Niu M, Liu J, Jiang W, Li Y, Wong STC, Cheng C, Liu Z, Zhang XH (2021) The bone microenvironment invigorates metastatic seeds for further dissemination Cell, 184(9): p. 2471–2486.e20

  27. Wang X, Yuan W, Wang X, Qi J, Qin Y, Shi Y, Zhang J, Gong J, Dong Z, Liu X, Sun C, Chai R, Le Noble F, Liu D (2016) The somite-secreted factor Maeg promotes zebrafish embryonic angiogenesis. Oncotarget 7(47):77749–77763

    Article  PubMed  PubMed Central  Google Scholar 

  28. Zhang G, Chen L, Khan AA, Li B, Gu B, Lin F, Su X, Yan J (2018) miRNA-124-3p/neuropilin-1(NRP-1) axis plays an important role in mediating glioblastoma growth and angiogenesis Int J Cancer, 143(3): p. 635–644

  29. Park JH, Lee NK, Lee SY (2017) Current understanding of RANK Signaling in Osteoclast differentiation and maturation. Mol Cells 40(10):706–713

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Macian F (2005) NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol 5(6):472–484

    Article  CAS  PubMed  Google Scholar 

  31. Zhang L, Feng M, Li Z, Zhu M, Fan Y, Chu B, Yuan C, Chen L, Lv H, Hong Z, Hong D (2018) Bulleyaconitine A prevents Ti particle-induced osteolysis via suppressing NF-kappaB signal pathway during osteoclastogenesis and osteoblastogenesis. J Cell Physiol 233(9):7067–7079

    Article  CAS  PubMed  Google Scholar 

  32. Brabletz T, Kalluri R, Nieto MA, Weinberg RA (2018) EMT in cancer Nat Rev Cancer 18(2):128–134

    Article  CAS  PubMed  Google Scholar 

  33. Zhang Y, Wang X (2020) Targeting the Wnt/beta-catenin signaling pathway in cancer. J Hematol Oncol 13(1):165

    Article  PubMed  PubMed Central  Google Scholar 

  34. Martini M, De Santis MC, Braccini L, Gulluni F, Hirsch E (2014) PI3K/AKT signaling pathway and cancer: an updated review. Ann Med 46(6):372–383

    Article  CAS  PubMed  Google Scholar 

  35. Peluso I, Yarla NS, Ambra R, Pastore G, Perry G (2019) MAPK signalling pathway in cancers: olive products as cancer preventive and therapeutic agents. Semin Cancer Biol 56:185–195

    Article  CAS  PubMed  Google Scholar 

  36. Meurette O, Mehlen P (2018) Notch Signaling in the Tumor Microenvironment. Cancer Cell 34(4):536–548

    Article  CAS  PubMed  Google Scholar 

  37. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD (2001) Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol 17:615–675

    Article  CAS  PubMed  Google Scholar 

  38. Guerrero-Zotano A, Mayer IA, Arteaga CL (2016) PI3K/AKT/mTOR: role in breast cancer progression, drug resistance, and treatment. Cancer Metastasis Rev 35(4):515–524

    Article  CAS  PubMed  Google Scholar 

  39. Fattahi S, Amjadi-Moheb F, Tabaripour R, Ashrafi GH, Akhavan-Niaki H (2020) PI3K/AKT/mTOR signaling in gastric cancer: epigenetics and beyond. Life Sci 262:118513

    Article  CAS  PubMed  Google Scholar 

  40. Ediriweera MK, Tennekoon KH, Samarakoon SR (2019) Role of the PI3K/AKT/mTOR signaling pathway in ovarian cancer: Biological and therapeutic significance. Semin Cancer Biol 59:147–160

    Article  CAS  PubMed  Google Scholar 

  41. Chen H, Zhou L, Wu X, Li R, Wen J, Sha J, Wen X (2016) The PI3K/AKT pathway in the pathogenesis of prostate cancer. Front Biosci (Landmark Ed) 21:1084–1091

    Article  CAS  PubMed  Google Scholar 

  42. Iksen S, Pothongsrisit, Pongrakhananon V (2021) Targeting the PI3K/AKT/mTOR signaling pathway in Lung Cancer: an update regarding potential drugs and Natural Products. Molecules, 26(13)

  43. Mortensen DS, Fultz KE, Xu S, Xu W, Packard G, Khambatta G, Gamez JC, Leisten J, Zhao J, Apuy J, Ghoreishi K, Hickman M, Narla RK, Bissonette R, Richardson S, Peng SX, Perrin-Ninkovic S, Tran T, Shi T, Yang WQ, Tong Z, Cathers BE, Moghaddam MF, Canan SS, Worland P, Sankar S, Raymon HK (2015) CC-223, a potent and selective inhibitor of mTOR kinase: in Vitro and in vivo characterization. Mol Cancer Ther 14(6):1295–1305

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We highly appreciated the patient samples provided by the Biological Resource Center of Enze Medical Center, Taizhou Hospital, and the support of Taizhou Discipline Group Fund.

Funding

This work was supported by the Medical and Health Science and Technology Program Project of Zhejiang Province for Dun Hong, Grant/Award Number: 2020PY030; the Basic and Public Research Project of Zhejiang Province for Mingxuan Ming, Grant/Award Number: LGF19H070003.

Author information

Authors and Affiliations

  1. Department of Orthopedics, Taizhou Hospital of Zhejiang Province Affiliated to Wenzhou Medical University, No.150 Ximen Road, Linhai, 317000, Zhejiang, China

    Xiaoting Song, Xu Cheng, Shengyu Ruan, Xianquan Xu, Xinhui Wu, Fangying Lu, Liwei Zhang, Haixiao Chen, Zhenghua Hong & Dun Hong

  2. Bone Metabolism and Development Research Center, Taizhou Hospital affiliated to Wenzhou Medical University, Linhai, China

    Xiaoting Song, Xu Cheng, Shengyu Ruan, Xianquan Xu, Xinhui Wu, Fangying Lu, Liwei Zhang, Haixiao Chen, Zhenghua Hong & Dun Hong

  3. Wenzhou Medical University, Wenzhou, China

    Xiaoting Song, Xu Cheng, Shengyu Ruan, Xianquan Xu, Xinhui Wu, Fangying Lu & Renshan Ge

  4. Taizhou Hospital of Zhejiang Province, Zhejiang University, Linhai, China

    Xiangang Jin & Feng Lu

  5. Department of Orthopedics, Taizhou Central Hospital affiliated to Taizhou College, Taizhou, China

    Mingxuan Feng

Authors
  1. Xiaoting Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xu Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiangang Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shengyu Ruan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xianquan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Feng Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xinhui Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fangying Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mingxuan Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Liwei Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Renshan Ge
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Haixiao Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Zhenghua Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Dun Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Dun Hong and Zhenghua Hong designed the study. The material preparation was performed by Xu Cheng, Xianquan Xu and Shengyu Ruan. Xinhui Wu, Feng Lu and Fangying Lu collected and analyzed the data. The animal experiment was conducted by Xiangang Jin and Mingxuan Feng. The first draft was written by Xiaoting Song. Review and editing were performed by Dun Hong, Zhenghua Hong, Renshan Ge and Haixiao Chen. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Zhenghua Hong or Dun Hong.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics approval

This research was approved by the Medical Ethics Committee of the Taizhou Hospital and obtained the informed consent of all patients. All experiments of animal followed the instruction of the Institutional Animal Ethics Committee of Taizhou Hospital.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10585_2023_10219_MOESM1_ESM.jpg

Supplementary Material 1: Fig. S1 Detection of the siRNAs targeting EGFL6 and LV-EGFL6-RNAi transfection efficiency of A549 cells. (a) Western blot analysis is utilized to detect the levels of EGFL6 in blank control, normal control and three kinds of siRNA transfection. (b) RT-PCR shows that the result of EGFL6 knockdown efficiency was consistent with WB. (c,d) Fluorescence microscopy shows lentivirus transfection efficiency. (e,g) Western blot analysis shows that EGFL6 knockdown group and EGFL6 overexpression group were constructed successfully. This sequence 899 has been shown to have a greater ability to knock down EGFL6 in A549 cells, so we used it directly for EGFL6 knockdown in NCI-H292 cells. (f,h) RT-PCR verifies the result of WB

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Song, X., Cheng, X., Jin, X. et al. EGFL6 promotes bone metastasis of lung adenocarcinoma by increasing cancer cell malignancy and bone resorption. Clin Exp Metastasis 40, 357–371 (2023). https://doi.org/10.1007/s10585-023-10219-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10585-023-10219-5

Keywords

Search

Navigation