Advertisement

Journal of Cancer Research and Clinical Oncology

, Volume 145, Issue 11, pp 2675–2687 | Cite as

Inhibition of bone morphogenetic protein signaling reduces viability, growth and migratory potential of non-small cell lung carcinoma cells

  • Jelena Mihajlović
  • Laura A. M. Diehl
  • Andreas Hochhaus
  • Joachim H. ClementEmail author
Original Article – Cancer Research
  • 127 Downloads

Abstract

Purpose

BMP signaling has an oncogenic and tumor-suppressing activity in lung cancer that makes the prospective therapeutic utility of BMP signaling in lung cancer treatment complex. A more in-depth analysis of lung cancer subtypes is needed to identify BMP-related therapeutic targets. We sought to examine the influence of BMP signaling on the viability, growth and migration properties of the cell line LCLC-103H, which originates from a large cell lung carcinoma with giant cells and an extended aneuploidy.

Methods

We used BMP-4 and LDN-214117 as agonist/antagonist system for the BMP receptor type I signaling. Using flow cytometry, wound healing assay, trans-well assay and spheroid culture, we examined the influence of BMP signaling on cell viability, growth and migration. Molecular mechanisms underlying observed changes in cell migration were investigated via gene expression analysis of epithelial–mesenchymal transition (EMT) markers.

Results

BMP signaling inhibition resulted in LCLC-103H cell apoptosis and necrosis 72 h after LDN-214117 treatment. Cell growth and proliferation are markedly affected by BMP signaling inhibition. Chemotactic motility and migratory ability of LCLC-103H cells were clearly hampered by LDN-214117 treatment. Cell migration changes after BMP signaling inhibition were shown to be coupled with considerable down-regulation of transcription factors involved in EMT, especially Snail.

Conclusions

BMP signaling inhibition in LCLC-103H cells leads to reduced growth and proliferation, hindered migration and accelerated cell death. The findings contribute to the pool of evidence on BMP signaling in lung cancer with a possibility of introducing BMP signaling inhibition as a novel therapeutic approach for the disease.

Keywords

BMP signaling BMP-4 Large cell lung carcinoma cell line Live cell imaging Snail 

Notes

Acknowledgements

The technical assistance of Cornelia Jörke is greatly acknowledged. We highly appreciate Dr. Mike Fischer for helpful instructions with flow cytometry and Dr. Katrin Hoffmann for support with statistical analysis. We also would like to thank Dr. Christine Gräfe for her helpful support in preparing and paraphrasing this manuscript. JM received a scholarship from the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) Jena. This work was supported in part by Europäische Fonds für regionale Entwicklung-Europa für Thüringen (EFRE, FKZ 2016 FGI 0006).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Human and animal participants

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

432_2019_3026_MOESM1_ESM.docx (483 kb)
Supplementary material 1 (DOCX 482 kb)

References

  1. Alarmo EL, Kallioniemi A (2010) Bone morphogenetic proteins in breast cancer: dual role in tumourigenesis? Endocr Relat Cancer 17:R123–R139PubMedCrossRefGoogle Scholar
  2. Alarmo EL, Huhtala H, Korhonen T, Pylkkänen L, Holli K, Kuukasjärvi T, Parkkila S, Kallioniemi A (2013) Bone morphogenetic protein 4 expression in multiple normal and tumor tissues reveals its importance beyond development. Mod Pathol 26:10–21PubMedCrossRefGoogle Scholar
  3. Ampuja M, Jokimäki R, Juuti-Uusitalo K, Rodriguez-Martinez A, Alarmo EL, Kallioniemi A (2013) BMP4 inhibits the proliferation of breast cancer cells and induces an MMP-dependent migratory phenotype in MDA-MB-231 cells in 3D environment. BMC Cancer 13:429–442PubMedPubMedCentralCrossRefGoogle Scholar
  4. Ampuja M, Alarmo EL, Owens P, Havunen R, Gorska AE, Moses HL, Kallioniemi A (2016) The impact of bone morphogenetic protein 4 (BMP4) on breast cancer metastasis in a mouse xenograft model. Cancer Lett 375:238–244PubMedCrossRefGoogle Scholar
  5. Bellusci S, Henderson R, Winnier G, Oikawa T, Hogan BL (1996) Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development 122:1693–1702PubMedGoogle Scholar
  6. Bénazet JD, Bischofberger M, Tiecke E, Gonçalves A, Martin JF, Zuniga A, Naef F, Zeller R (2009) A self-regulatory system of interlinked signaling feedback loops controls mouse limb patterning. Science 323:1050–1053PubMedCrossRefGoogle Scholar
  7. Bepler G, Koehler A, Kiefer P, Havemann K, Beisenherz K, Jaques G, Gropp C, Haeder M (1988) Characterization of the state of differentiation of six newly established human non-small-cell lung cancer cell lines. Differentiation 37:158–171PubMedCrossRefGoogle Scholar
  8. Brandao GDA, Brega EF, Spatz A (2012) The role of molecular pathology in non-small-cell lung carcinoma-now and in the future. Curr Oncol 19:S24–S832PubMedPubMedCentralCrossRefGoogle Scholar
  9. Buckley S, Shi W, Driscoll B, Ferrario A, Anderson K, Warburton D (2004) BMP4 signaling induces senescence and modulates the oncogenic phenotype of A549 lung adenocarcinoma cells. Am J Physiol Lung Cell Mol Physiol 286:L81–L86PubMedCrossRefGoogle Scholar
  10. Clement JH, Marr N, Meissner A, Schwalbe M, Sebald W, Kliche KO, Höffken K, Wölfl S (2000) BMP-2 induces sequential changes of ID gene expression in the breast cancer cell line MCF-7. J Cancer Res Clin Oncol 126:271–279PubMedCrossRefGoogle Scholar
  11. Danesh SM, Villasenor A, Chong D, Soukup C, Cleaver O (2009) BMP and BMP receptor expression during murine organogenesis. Gene Expr Patterns 9:255–265PubMedPubMedCentralCrossRefGoogle Scholar
  12. Deng H, Ravikumar TSS, Yang WL (2009) Overexpression of bone morphogenetic protein 4 enhances the invasiveness of Smad4-deficient human colorectal cancer cells. Cancer Lett 281:220–231PubMedCrossRefGoogle Scholar
  13. Fang WT, Fan CC, Li SM et al (2014) Downregulation of a putative tumor suppressor BMP4 by SOX2 promotes growth of lung squamous cell carcinoma. Int J Cancer 135:809–819PubMedCrossRefGoogle Scholar
  14. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F (2015) Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 136:E359–E386PubMedCrossRefGoogle Scholar
  15. Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Piñeros M, Znaor A, Soerjomataram I, Bray F (2018). Global cancer observatory: cancer today. International Agency for Research on Cancer, Lyon. https://gco.iarc.fr/today. Accessed 14 Feb 2019
  16. Fotinos A, Nagarajan N, Martins AS, Fritz DT, Garsetti D, Lee AT, Hong CC, Rogers MB (2014) Bone morphogenetic protein-focused strategies to induce cytotoxicity in lung cancer cells. Anticancer Res 34:2095–2104PubMedPubMedCentralGoogle Scholar
  17. Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA (2009) Spheroid-based drug screen: considerations and practical approach. Nature Protoc 4:309–324CrossRefGoogle Scholar
  18. Gonzalez DM, Medici D (2014) Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal 7(344):re8.  https://doi.org/10.1126/scisignal.2005189 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Goswami M, Uzgare AR, Sater AK (2001) Regulation of MAP kinase by the BMP-4/TAK1 pathway in Xenopus ectoderm. Dev Biol 236:259–270PubMedCrossRefGoogle Scholar
  20. Guo D, Huang J, Gong J (2012) Bone morphogenetic protein 4 (BMP4) is required for migration and invasion of breast cancer. Mol Cell Biochem 363:179–190PubMedCrossRefGoogle Scholar
  21. Hao J, Lee R, Chang A, Fan J, Labib C, Parsa C, Orlando R, Andresen B, Huang Y (2014) DMH1, a small molecule inhibitor of BMP type I receptors, suppresses growth and invasion of lung cancer. PLoS One 9:e90748.  https://doi.org/10.1371/journal.pone.0090748 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Haramis APG, Begthel H, van den Born M, van Es J, Jonkheer S, Offerhaus GJA, Clevers H (2004) De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303:1684–1686PubMedCrossRefGoogle Scholar
  23. Haubold M, Weise A, Stephan H, Dünker N (2010) Bone morphogenetic protein 4 (BMP4) signaling in retinoblastoma cells. Int J Biol Sci 6:700–715PubMedPubMedCentralCrossRefGoogle Scholar
  24. Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148:3–15PubMedPubMedCentralCrossRefGoogle Scholar
  25. Hollnagel A, Oehlmann V, Heymer J, Rüther U, Nordheim A (1999) Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells. J Biol Chem 274:19838–19845PubMedCrossRefGoogle Scholar
  26. Kallioniemi A (2012) Bone morphogenetic protein 4-a fascinating regulator of cancer cell behavior. Cancer Genet 205:267–277PubMedCrossRefGoogle Scholar
  27. Kim JS, Kurie JM, Ahn YH (2015) BMP4 depletion by miR-200 inhibits tumorigenesis and metastasis of lung adenocarcinoma cells. Mol Cancer 14:173–183PubMedPubMedCentralCrossRefGoogle Scholar
  28. Kretzschmar M, Doody J, Massagué J (1997) Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1. Nature 389:618–622PubMedCrossRefGoogle Scholar
  29. Langenfeld E, Hong CC, Lanke G, Langenfeld J (2013) Bone morphogenetic protein type I receptor antagonists decrease growth and induce cell death of lung cancer cell lines. PLoS One 8:e61256.  https://doi.org/10.1371/journal.pone.0061256 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Lubbe SJ, Pittman AM, Olver B, Lloyd A, Vijayakrishnan J, Naranjo S, Dobbins S, Broderick P, Gómez-Skarmeta JL, Houlston RS (2012) The 14q22.2 colorectal cancer variant rs4444235 shows cis-acting regulation of BMP4. Oncogene 31:3777–3784PubMedCrossRefGoogle Scholar
  31. Maegdefrau U, Amann T, Winklmeier A et al (2009) Bone morphogenetic protein 4 is induced in hepatocellular carcinoma by hypoxia and promotes tumour progression. J Pathol 218:520–529PubMedCrossRefGoogle Scholar
  32. McCormack N, Molloy EL, O’Dea S (2013) Bone morphogenetic proteins enhance an epithelial-mesenchymal transition in normal airway epithelial cells during restitution of a disrupted epithelium. Respir Res 14:36.  https://doi.org/10.1186/1465-9921-14-36 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Miyazaki Y, Oshima K, Fogo A, Ichikawa I (2003) Evidence that bone morphogenetic protein 4 has multiple biological functions during kidney and urinary tract development. Kidney Int 63:835–844PubMedCrossRefGoogle Scholar
  34. Miyazono K, Miyazawa K (2002) Id: a target of BMP signaling. Science’s STKE 2002(151):pe40.  https://doi.org/10.1126/stke.2002.151.pe40 CrossRefPubMedGoogle Scholar
  35. Miyazono K, Maeda S, Imamura T (2005) BMP receptor signaling: transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine Growth Factor Rev 16:251–263PubMedCrossRefGoogle Scholar
  36. Miyazono K, Kamiya Y, Morikawa M (2010) Bone morphogenetic protein receptors and signal transduction. J Biochem 147:35–51PubMedCrossRefGoogle Scholar
  37. Müller P, Doliva R, Busch M, Philippeit C, Stephan H, Dünker N, Makishima M (2015) Additive effects of retinoic acid (RA) and bone morphogenetic protein 4 (BMP-4) apoptosis signaling in retinoblastoma cell lines. PLoS One 10:1–22Google Scholar
  38. Müller-Klieser W (1987) Multicellular spheroids. A review on cellular aggregates in cancer research. J Cancer Res Clin Oncol 113:101–122CrossRefGoogle Scholar
  39. Park KS, Dubon MJ, Gumbiner BM (2015) N-cadherin mediates the migration of MCF-10A cells undergoing bone morphogenetic protein 4-mediated epithelial mesenchymal transition. Tumor Biol 36:3549–3556CrossRefGoogle Scholar
  40. Pickup MW, Hover LD, Guo Y, Gorska AE, Novitskiy SV, Moses HL, Owens P (2015) Deletion of the BMP receptor BMPR1a impairs mammary tumor formation and metastasis. Oncotarget 6:22890–22904PubMedPubMedCentralCrossRefGoogle Scholar
  41. Qi X, Li TG, Hao J, Hu J, Wang J, Simmons H, Miura S, Mishina Y, Zhao GQ (2004) BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc Natl Acad Sci USA 101:6027–6032PubMedCrossRefGoogle Scholar
  42. Reungwetwattana T, Weroha SJ, Molina JR (2012) Oncogenic pathways, molecularly targeted therapies, and highlighted clinical trials in non-small-cell lung cancer (NSCLC). Clin Lung Cancer 13:252–266PubMedCrossRefGoogle Scholar
  43. Richter A, Valdimarsdottir L, Hrafnkelsdottir HE, Runarsson JF, Omarsdottir AR, Oostwaard DW, Mummery C, Valdimarsdottir G (2014) BMP4 promotes EMT and mesodermal commitment in human embryonic stem cells via SLUG and MSX2. Stem Cells 32:636–648PubMedCrossRefGoogle Scholar
  44. Robert B (2007) Bone morphogenetic protein signaling in limb outgrowth and patterning. Dev Growth Differ 49:455–468PubMedCrossRefGoogle Scholar
  45. Rothhammer T, Poser I, Soncin F, Bataille F, Moser M, Bosserhoff AK (2005) Bone morphogenic proteins are overexpressed in malignant melanoma and promote cell invasion and migration. Cancer Res 65:448–456PubMedGoogle Scholar
  46. Sadlon TJ, Lewis ID, D’Andrea RJ (2004) BMP4: its role in development of the hematopoietic system and potential as a hematopoietic growth factor. Stem Cells 22:457–474PubMedCrossRefGoogle Scholar
  47. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to imagej: 25 years of image analysis. Nat Methods 9:671–675PubMedPubMedCentralCrossRefGoogle Scholar
  48. Shepherd TG, Nachtigal MW (2003) Identification of a putative autocrine bone morphogenetic protein-signaling pathway in human ovarian surface epithelium and ovarian cancer cells. Endocrinology 144:3306–3314PubMedCrossRefGoogle Scholar
  49. Shirai YT, Ehata S, Yashiro M, Yanagihara K, Hirakawa K, Miyazono K (2011) Bone morphogenetic protein-2 and -4 play tumor suppressive roles in human diffuse-type gastric carcinoma. Am J Pathol 179:2920–2930PubMedPubMedCentralCrossRefGoogle Scholar
  50. Su D, Zhu S, Han X, Feng Y, Huang H, Ren G, Pan L, Zhang Y, Lu J, Huang B (2009) BMP4-Smad signaling pathway mediates adriamycin-induced premature senescence in lung cancer cells. J Biol Chem 284:12153–12164PubMedPubMedCentralCrossRefGoogle Scholar
  51. Thawani JP, Wang AC, Than KD, Lin CY, La Marca F, Park P (2010) Bone morphogenetic proteins and cancer: review of the literature. Neurosurgery 66:233–246PubMedCrossRefGoogle Scholar
  52. Virtanen S, Alarmo EL, Sandström S, Ampuja M, Kallioniemi A (2011) Bone morphogenetic protein-4 and -5 in pancreatic cancer–novel bidirectional players. Exp Cell Res 317:2136–2146PubMedCrossRefGoogle Scholar
  53. Winnier G, Blessing M, Labosky PA, Hogan BL (1995) Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev 9:2105–2116PubMedCrossRefGoogle Scholar
  54. Xu T, Yu C-Y, Sun J-J, Liu Y, Wang X-W, Pi L-M, Tian Y-Q, Zhang X (2011) Bone morphogenetic protein-4-induced epithelial-mesenchymal transition and invasiveness through Smad1-mediated signal pathway in squamous cell carcinoma of the head and neck. Arch Med Res 42:128–137PubMedCrossRefGoogle Scholar
  55. Ye XY, Niu XM, Tang NW, Xu YH, Li ZM, Yu YF, Lu S, Chen ZW (2012) Adenovirus mediated knockdown of bone morphogenetic protein 2 inhibits human lung cancer growth and invasion in vitro and in vivo. Int J Immunopathol Pharmacol 25:967–976PubMedCrossRefGoogle Scholar
  56. Yokoyama Y, Watanabe T, Tamura Y, Hashizume Y, Miyazono K, Ehata S (2017) Autocrine BMP-4 signaling is a therapeutic target in colorectal cancer. Cancer Res 77:4026–4038PubMedCrossRefGoogle Scholar
  57. Zeng S, Zhang Y, Ma J et al (2017) BMP4 promotes metastasis of hepatocellular carcinoma by an induction of epithelial–mesenchymal transition via upregulating ID2. Cancer Lett 390:67–76PubMedCrossRefGoogle Scholar
  58. Zhang J, Li L (2005) BMP signaling and stem cell regulation. Dev Biol 284:1–11PubMedCrossRefGoogle Scholar
  59. Zhang L, Ye Y, Long X, Xiao P, Ren X, Yu J (2016) BMP signaling and its paradoxical effects in tumorigenesis and dissemination. Oncotarget 7:78206–78218PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Klinik Innere Medizin II, Abteilung Hämatologie und Internistische OnkologieUniversitätsklinikum JenaJenaGermany

Personalised recommendations