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Gjb4 serves as a novel biomarker for lung cancer and promotes metastasis and chemoresistance via Src activation

Oncogene volume 38, pages 822–837 (2019)Cite this article

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Abstract

Most lung cancer patients are diagnosed late with metastasis, which is the major cause of cancer-related death and recurrent tumors that often exhibit chemoresistance. In the present study, we initially identified gap junction beta-4 protein (Gjb4) to be overexpressed in highly metastatic cancer cells selected by their enhanced binding to serum components. Overexpression or knockdown of Gjb4 increased or decreased lung metastasis of syngeneic mice, respectively. We found that Gjb4 expression was higher in lung tumors than normal tissues (p = 0.0026), and Gjb4 levels in blood buffy coat samples showed significant performance in diagnosing stage I–III (p = 0.002814) and stage IV (p < 0.0001) lung cancer. Moreover, high Gjb4 expression levels were correlated with poor prognosis (p = 1.4e−4) and recurrence (p = 1.9e−12). Using syngeneic mouse model, we observed that Gjb4 was able to promote tumor growth. High molecular weight serum fraction containing the major growth factor component IGF1 was able to induce Gjb4 via PKC pathway. Gjb4 activated Src signaling via MET, and overexpression of Gjb4 enhanced sphere-forming ability and anchorage-independent growth, which were reversed by inhibition of Src. In addition, we demonstrated that Gjb4-mediated Src activation enhanced chemoresistance of cancer cells toward gemcitabine and etoposide. The combination of Gjb4 knockdown, gemcitabine, and dasatinib further enhanced the inhibition of cancer cell viability. Together, our study has identified Gjb4 as a potential novel diagnostic and prognostic biomarker for lung cancer. Targeting Gjb4 may be exploited as a modality for improving lung cancer therapy.

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References

  1. Duggan MA, Anderson WF, Altekruse S, Penberthy L, Sherman ME. The Surveillance, Epidemiology, and End Results (SEER) Program and Pathology: toward strengthening the critical relationship. Am J Surg Pathol. 2016;40:e94–102.

    Article  Google Scholar 

  2. Popper HH. Progression and metastasis of lung cancer. Cancer Metastas. 2016;35:75–91.

    Article  CAS  Google Scholar 

  3. Uramoto H, Tanaka F. Recurrence after surgery in patients with NSCLC. Transl Lung Cancer Res. 2014;3:242–9.

    PubMed  PubMed Central  Google Scholar 

  4. Kalemkerian GP. Combination chemotherapy for relapsed small-cell lung cancer. Lancet Oncol. 2016;17:1033–5.

    Article  Google Scholar 

  5. Teleki I, Szasz AM, Maros ME, Gyorffy B, Kulka J, Meggyeshazi N, et al. Correlations of differentially expressed gap junction connexins Cx26, Cx30, Cx32, Cx43 and Cx46 with breast cancer progression and prognosis. PLoS ONE. 2014;9:e112541.

    Article  Google Scholar 

  6. Spray DC. Gap junction proteins: where they live and how they die. Circ Res. 1998;83:679–81.

    Article  CAS  Google Scholar 

  7. Losa D, Chanson M, Crespin S. Connexins as therapeutic targets in lung disease. Expert Opin Ther Targets. 2011;15:989–1002.

    Article  CAS  Google Scholar 

  8. Gielen PR, Aftab Q, Ma N, Chen VC, Hong X, Lozinsky S, et al. Connexin43 confers temozolomide resistance in human glioma cells by modulating the mitochondrial apoptosis pathway. Neuropharmacology. 2013;75:539–48.

    Article  CAS  Google Scholar 

  9. Kyo N, Yamamoto H, Takeda Y, Ezumi K, Ngan CY, Terayama M, et al. Overexpression of connexin 26 in carcinoma of the pancreas. Oncol Rep. 2008;19:627–31.

    PubMed  Google Scholar 

  10. Elzarrad MK, Haroon A, Willecke K, Dobrowolski R, Gillespie MN, Al-Mehdi A-B. Connexin-43 upregulation in micrometastases and tumor vasculature and its role in tumor cell attachment to pulmonary endothelium. BMC Med. 2008;6:20.

    Article  Google Scholar 

  11. Ezumi K, Yamamoto H, Murata K, Higashiyama M, Damdinsuren B, Nakamura Y, et al. Aberrant expression of connexin 26 is associated with lung metastasis of colorectal cancer. Clin Cancer Res. 2008;14:677–84.

    Article  CAS  Google Scholar 

  12. Murphy SF, Varghese RT, Lamouille S, Guo S, Pridham KJ, Kanabur P, et al. Connexin 43 inhibition sensitizes chemoresistant glioblastoma cells to temozolomide. Cancer Res. 2016;76:139–49.

    Article  CAS  Google Scholar 

  13. Munoz JL, Rodriguez-Cruz V, Greco SJ, Ramkissoon SH, Ligon KL, Rameshwar P. Temozolomide resistance in glioblastoma cells occurs partly through epidermal growth factor receptor-mediated induction of connexin 43. Cell Death Dis. 2014;5:e1145.

    Article  CAS  Google Scholar 

  14. López-Bigas N, Melchionda S, Gasparini P, Borragán A, Arbonés ML, Estivill X. A common frameshift mutation and other variants in GJB4 (connexin 30.3): analysis of hearing impairment families. Hum Mutat. 2002;19:458.

    Article  Google Scholar 

  15. Common JEA, O’Toole EA, Leigh IM, Thomas A, Griffiths WAD, Venning V, et al. Clinical and genetic heterogeneity of erythrokeratoderma variabilis. J Invest Dermatol. 2005;125:920–7.

    Article  CAS  Google Scholar 

  16. Ganapathy V, Moghe PV, Roth CM. Targeting tumor metastases: drug delivery mechanisms and technologies. J Control Release. 2015;219:215–23.

    Article  CAS  Google Scholar 

  17. Hedberg KK, Stauff C, Høyer-Hansen G, Rønne E, Griffith OH. High-molecular-weight serum protein complexes differentially promote cell migration and the focal adhesion localization of the urokinase receptor in human glioma cells. Exp Cell Res. 2000;257:67–81.

    Article  CAS  Google Scholar 

  18. Bausch-Fluck D, Hofmann A, Bock T, Frei AP, Cerciello F, Jacobs A, et al. A mass spectrometric-derived cell surface protein atlas. PLoS ONE. 2015;10:e0121314.

    Article  Google Scholar 

  19. Győrffy B, Surowiak P, Budczies J, Lánczky A. Online survival analysis software to assess the prognostic value of biomarkers using transcriptomic data in non-small-cell lung cancer. PLoS ONE. 2013;8:e82241.

    Article  Google Scholar 

  20. Li X, Asmitananda T, Gao L, Gai D, Song Z, Zhang Y, et al. Biomarkers in the lung cancer diagnosis: a clinical perspective. Neoplasma. 2012;59:500–7.

    Article  CAS  Google Scholar 

  21. Zaghloul MZ. Cytokeratin 19 (CK19) as a tumor marker in pleural effusion. Trop Med Surg 2015; https://doi.org/10.4172/2329-9088.1000e122.

  22. Manegold C. Gemcitabine (Gemzar) in non-small cell lung cancer. Expert Rev Anticancer Ther. 2004;4:345–60.

    Article  CAS  Google Scholar 

  23. Reck M, Groth G, Buchholz E, Goetz E, Gatzemeier U, Manegold C. Topotecan and etoposide as first-line therapy for extensive disease small cell lung cancer: a phase II trial of a platinum-free regimen. Lung Cancer. 2005;48:409–13.

    Article  Google Scholar 

  24. Geimonen E, Jiang W, Ali M, Fishman GI, Garfield RE, Andersen J. Activation of protein kinase C in human uterine smooth muscle induces connexin-43 gene transcription through an AP-1 site in the promoter sequence. J Biol Chem. 1996;271:23667–74.

    Article  CAS  Google Scholar 

  25. Beattie J, Allan GJ, Lochrie JD, Flint DJ. Insulin-like growth factor-binding protein-5 (IGFBP-5): a critical member of the IGF axis. Biochem J. 2006;395:1–19.

    Article  CAS  Google Scholar 

  26. Chu C-H, Tzang B-S, Chen L-M, Kuo C-H, Cheng Y-C, Chen L-Y, et al. IGF-II/mannose-6-phosphate receptor signaling induced cell hypertrophy and atrial natriuretic peptide/BNP expression via Galphaq interaction and protein kinase C-alpha/CaMKII activation in H9c2 cardiomyoblast cells. J Endocrinol. 2008;197:381–90.

    Article  CAS  Google Scholar 

  27. Touyz RM, Schiffrin EL. Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells. Pharmacol Rev. 2000;52:639–72.

    CAS  PubMed  Google Scholar 

  28. Kløverpris S, Skov LL, Glerup S, Pihl K, Christiansen M, Oxvig C. Formation of high-molecular-weight angiotensinogen during pregnancy is a result of competing redox reactions with the proform of eosinophil major basic protein. Biochem J. 2013;449:209–17.

    Article  Google Scholar 

  29. Matta C, Mobasheri A. Regulation of chondrogenesis by protein kinase C: emerging new roles in calcium signalling. Cell Signal. 2014;26:979–1000.

    Article  CAS  Google Scholar 

  30. Jia G, Mitra AK, Cheng G, Gangahar DM, Agrawal DK. Angiotensin II and IGF-1 regulate connexin43 expression via ERK and p38 signaling pathways in vascular smooth muscle cells of coronary artery bypass conduits. J Surg Res. 2007;142:137–42.

    Article  CAS  Google Scholar 

  31. Leto SM, Trusolino L. Primary and acquired resistance to EGFR-targeted therapies in colorectal cancer: impact on future treatment strategies. J Mol Med. 2014;92:709–22.

    Article  CAS  Google Scholar 

  32. Gavalas NG, Liontos M, Trachana S-P, Bagratuni T, Arapinis C, Liacos C, et al. Angiogenesis-related pathways in the pathogenesis of ovarian cancer. Int J Mol Sci. 2013;14:15885–909.

    Article  Google Scholar 

  33. Dorey K, Amaya E. FGF signalling: diverse roles during early vertebrate embryogenesis. Development. 2010;137:3731–42.

    Article  CAS  Google Scholar 

  34. Lin JH-C, Takano T, Cotrina ML, Arcuino G, Kang J, Liu S, et al. Connexin 43 enhances the adhesivity and mediates the invasion of malignant glioma cells. J Neurosci. 2002;22:4302–11.

    Article  CAS  Google Scholar 

  35. Zhang W, Nwagwu C, Le DM, Yong VW, Song H, Couldwell WT. Increased invasive capacity of connexin43-overexpressing malignant glioma cells. J Neurosurg. 2003;99:1039–46.

    Article  CAS  Google Scholar 

  36. Jaraíz-Rodríguez M, Tabernero MD, González-Tablas M, Otero A, Orfao A, Medina JM, et al. A short region of connexin43 reduces human glioma stem cell migration, invasion, and survival through Src, PTEN, and FAK. Stem Cell Rep. 2017;9:451–63.

    Article  Google Scholar 

  37. van Oosterwijk JG, MAJH vanRuler, Briaire-de Bruijn IH, Herpers B, Gelderblom H, van de Water B, et al. Src kinases in chondrosarcoma chemoresistance and migration: dasatinib sensitises to doxorubicin in TP53 mutant cells. Br J Cancer. 2013;109:1214–22.

    Article  Google Scholar 

  38. George TJ Jr, Trevino JG, Liu C. Src inhibition is still a relevant target in pancreatic cancer. Oncologist. 2014;19:211.

    Article  CAS  Google Scholar 

  39. Zhang S, Yu D. Targeting Src family kinases in anti-cancer therapies: turning promise into triumph. Trends Pharmacol Sci. 2012;33:122–8.

    Article  Google Scholar 

  40. Thakur R, Trivedi R, Rastogi N, Singh M, Mishra DP. Inhibition of STAT3, FAK and Src mediated signaling reduces cancer stem cell load, tumorigenic potential and metastasis in breast cancer. Sci Rep. 2015;5:10194.

    Article  CAS  Google Scholar 

  41. Silva CM. Role of STATs as downstream signal transducers in Src family kinase-mediated tumorigenesis. Oncogene. 2004;23:8017–23.

    Article  CAS  Google Scholar 

  42. Steinman RA, Wentzel A, Lu Y, Stehle C, Grandis JR. Activation of Stat3 by cell confluence reveals negative regulation of Stat3 by cdk2. Oncogene. 2003;22:3608–15.

    Article  CAS  Google Scholar 

  43. González-Sánchez A, Jaraíz-Rodríguez M, Domínguez-Prieto M, Herrero-González S, Medina JM, Tabernero A. Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes. Oncotarget. 2016;7:49819–33.

    Article  Google Scholar 

  44. Roskoski R Jr. Src protein-tyrosine kinase structure, mechanism, and small molecule inhibitors. Pharmacol Res. 2015;94:9–25.

    Article  CAS  Google Scholar 

  45. Longati P, Bardelli A, Ponzetto C, Naldini L, Comoglio PM. Tyrosines1234-1235 are critical for activation of the tyrosine kinase encoded by the MET proto-oncogene (HGF receptor). Oncogene. 1994;9:49–57.

    CAS  PubMed  Google Scholar 

  46. Tapper H, Sundler R. Bafilomycin A1 inhibits lysosomal, phagosomal, and plasma membrane H(+)-ATPase and induces lysosomal enzyme secretion in macrophages. J Cell Physiol. 1995;163:137–44.

    Article  CAS  Google Scholar 

  47. Han YH, Moon HJ, You BR, Park WH. The effect of MG132, a proteasome inhibitor on HeLa cells in relation to cell growth, reactive oxygen species and GSH. Oncol Rep. 2009;22:215–21.

    CAS  PubMed  Google Scholar 

  48. Steinberg M. Dasatinib: a tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia and philadelphia chromosome-positive acute lymphoblastic leukemia. Clin Ther. 2007;29:2289–308.

    Article  CAS  Google Scholar 

  49. Wan Y, Yuan Y, Pan Y, Zhang Y. Antitumor activity of high-dose pulsatile gefitinib in non-small-cell lung cancer with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Exp Ther Med. 2017;13:3067–74.

    Article  CAS  Google Scholar 

  50. Miyata T, Yoshimatsu T, So T, Oyama T, Uramoto H, Osaki T, et al. Cancer stem cell markers in lung cancer. Pers Med Universe. 2015;4:40–45.

    Article  Google Scholar 

  51. Li Y, Rogoff HA, Keates S, Gao Y, Murikipudi S, Mikule K, et al. Suppression of cancer relapse and metastasis by inhibiting cancer stemness. Proc Natl Acad Sci. 2015;112:1839–44.

    Article  CAS  Google Scholar 

  52. Zhao J. Cancer stem cells and chemoresistance: the smartest survives the raid. Pharmacol Ther. 2016;160:145–58.

    Article  CAS  Google Scholar 

  53. Wang S. Anchorage-independent growth of prostate cancer stem cells. Methods Mol Biol. 2009;568:151–60.

    Article  Google Scholar 

  54. Huang G, Ye S, Zhou X, Liu D, Ying Q-L. Molecular basis of embryonic stem cell self-renewal: from signaling pathways to pluripotency network. Cell Mol Life Sci. 2015;72:1741–57.

    Article  CAS  Google Scholar 

  55. Labelle M, Begum S, Hynes RO. Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell. 2011;20:576–90.

    Article  CAS  Google Scholar 

  56. Guan P-P, Yu X, Guo J-J, Wang Y, Wang T, Li J-Y, et al. By activating matrix metalloproteinase-7, shear stress promotes chondrosarcoma cell motility, invasion and lung colonization. Oncotarget. 2015;6:9140–59.

    PubMed  PubMed Central  Google Scholar 

  57. Celià-Terrassa T, Kang Y. Distinctive properties of metastasis-initiating cells. Genes Dev. 2016;30:892–908.

    Article  Google Scholar 

  58. Mor-Vaknin N, Punturieri A, Sitwala K, Markovitz DM. Vimentin is secreted by activated macrophages. Nat Cell Biol. 2003;5:59–63.

    Article  CAS  Google Scholar 

  59. Shigyo M, Kuboyama T, Sawai Y, Tada-Umezaki M, Tohda C. Extracellular vimentin interacts with insulin-like growth factor 1 receptor to promote axonal growth. Sci Rep. 2015;5:12055.

    Article  CAS  Google Scholar 

  60. Giuffrida ML, Caraci F, De Bona P, Pappalardo G, Nicoletti F, Rizzarelli E, et al. The monomer state of beta-amyloid: where the Alzheimer’s disease protein meets physiology. Rev Neurosci. 2010;21:83–93.

    Article  CAS  Google Scholar 

  61. Banerjee D. Connexin’s connection in breast cancer growth and progression. Int J Cell Biol. 2016;2016:9025905.

    Article  Google Scholar 

  62. Kotini M, Mayor R. Connexins in migration during development and cancer. Dev Biol. 2015;401:143–51.

    Article  CAS  Google Scholar 

  63. Naoi Y, Miyoshi Y, Taguchi T, Kim SJ, Arai T, Tamaki Y, et al. Connexin26 expression is associated with lymphatic vessel invasion and poor prognosis in human breast cancer. Breast Cancer Res Treat. 2007;106:11–17.

    Article  CAS  Google Scholar 

  64. Lin R, Warn-Cramer BJ, Kurata WE, Lau AF. v-Src phosphorylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctional communication. J Cell Biol. 2001;154:815–27.

    Article  CAS  Google Scholar 

  65. Aasen T, Mesnil M, Naus CC, Lampe PD, Laird DW. Gap junctions and cancer: communicating for 50 years. Nat Rev Cancer. 2016;16:775–88.

    Article  CAS  Google Scholar 

  66. Meşe G, Richard G, White TW. Gap junctions: basic structure and function. J Invest Dermatol. 2007;127:2516–24.

    Article  Google Scholar 

  67. Novello S, Barlesi F, Califano R, Cufer T, Ekman S, Levra MG, et al. Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2016;27:v1–v27.

    Article  CAS  Google Scholar 

  68. Feliciano P. CXCL1 and CXCL2 link metastasis and chemoresistance. Nat Genet. 2012;44:840.

    Google Scholar 

  69. Acharyya S, Oskarsson T, Vanharanta S, Malladi S, Kim J, Morris PG, et al. A CXCL1 paracrine network links cancer chemoresistance and metastasis. Cell. 2012;150:165–78.

    Article  CAS  Google Scholar 

  70. Montuenga LM, Pio R. Current challenges in lung cancer early detection biomarkers. Eur J Cancer. 2009;45(Suppl 1):377–8.

    Article  Google Scholar 

  71. Tung S-L, Huang W-C, Hsu F-C, Yang Z-P, Jang T-H, Chang J-W, et al. miRNA-34c-5p inhibits amphiregulin-induced ovarian cancer stemness and drug resistance via downregulation of the AREG-EGFR-ERK pathway. Oncogenesis. 2017;6:e326.

    Article  CAS  Google Scholar 

  72. Lin K-T, Gong J, Li C-F, Jang T-H, Chen W-L, Chen H-J, et al. Vav3-rac1 signaling regulates prostate cancer metastasis with elevated Vav3 expression correlating with prostate cancer progression and posttreatment recurrence. Cancer Res. 2012;72:3000–9.

    Article  CAS  Google Scholar 

  73. Yeh Y-M, Chuang C-M, Chao K-C, Wang L-H. MicroRNA-138 suppresses ovarian cancer cell invasion and metastasis by targeting SOX4 and HIF-1α. Int J Cancer. 2013;133:867–78.

    Article  CAS  Google Scholar 

  74. Huang L, Hu C, Di Benedetto M, Varin R, Liu J, Wang L, et al. Induction of multiple drug resistance in HMEC-1 endothelial cells after long-term exposure to sunitinib. Onco Targets Ther. 2014;7:2249–55.

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by grants from the National Health Research Institutes, Taiwan (06A1-MGPP09-014), the Ministry of Education, Taiwan (CMRC-CHM-7), and the Ministry of Science and Technology, Taiwan (MOST 104-2320-B-039-055-MY3, MOST 104-2320-B-039-054-MY3 and MOST 106-2320-B-039-059-). We thank Core Laboratory of Microarray, Cell Sorting, Pathology, Confocal Microscopy as well as Proteomics and chemistry of National Health Research Institutes (NHRI) for experiments involving exon array analysis, cell sorting, H&E staining, Immunohistochemistry, confocal microscopy and mass spectrometry, respectively. We thank Dr. Yi Rong Chen of IMGM, NHRI, Taiwan for the gift of H1650 human lung cancer cell line. We also thank Dr. Chin Fu Hsiao of Division of Biostatistics and Bioinformatics of NHRI, Taiwan for the clinical samples from lung cancer tissue bank. We thank Dr. Sheng-Chieh Lin for the assistance of manuscript preparation.

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  1. These authors contributed equally: Yi-Pei Lin, Jun-I Wu

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  1. Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan

    Yi-Pei Lin, Jun-I Wu, Chien-Wei Tseng, Huei-Jane Chen & Lu-Hai Wang

  2. Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan

    Yi-Pei Lin & Lu-Hai Wang

  3. Department of Life Sciences, National Central University, Taoyuan, Taiwan

    Jun-I Wu & Lu-Hai Wang

  4. Chinese Medicine Research Center, China Medical University, Taichung, Taiwan

    Chien-Wei Tseng & Lu-Hai Wang

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Lin, YP., Wu, JI., Tseng, CW. et al. Gjb4 serves as a novel biomarker for lung cancer and promotes metastasis and chemoresistance via Src activation. Oncogene 38, 822–837 (2019). https://doi.org/10.1038/s41388-018-0471-1

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