As the genomic region containing the Bcl-2-related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non-small-cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node-positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFβ-induced migration and epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma-derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low-BOK-expressing tumors might favor the overall survival of NSCLC patients.

Department of Cardiothoracic Surgery School of Medicine University of Pittsburgh PA

Department of Medicine School of Medicine University of Pittsburgh PA

Department of Pathology Hospital Bulovka Prague Czech Republic

Institute of Biochemistry and Experimental Oncology 1st Faculty of Medicine Charles University Prague Czech Republic

Institute of Pharmacology Faculty of Medicine University of Bern Bern Switzerland

See more in PubMed

Llambi F, Wang YM, Victor B, Yang M, Schneider DM, Gingras S, Parsons MJ, Zheng JH, Brown SA, Pelletier S, Moldoveanu T, Chen T, et al. BOK Is a Non-canonical BCL-2 Family Effector of Apoptosis Regulated by ER-Associated Degradation. Cell. 2016;165(2):421–33. PubMed PMC

Echeverry N, Bachmann D, Ke F, Strasser A, Simon HU, Kaufmann T. Intracellular localization of the BCL-2 family member BOK and functional implications. Cell Death Differ. 2013;20(6):785–99. PubMed PMC

Hsu SY, Kaipia A, McGee E, Lomeli M, Hsueh AJ. Bok is a pro-apoptotic Bcl-2 protein with restricted expression in reproductive tissues and heterodimerizes with selective anti-apoptotic Bcl-2 family members. Proc Natl Acad Sci U S A. 1997;94(23):12401–6. PubMed PMC

Inohara N, Ekhterae D, Garcia I, Carrio R, Merino J, Merry A, Chen S, Nunez G. Mtd, a novel Bcl-2 family member activates apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL. J Biol Chem. 1998;273(15):8705–10. PubMed

Einsele-Scholz S, Malmsheimer S, Bertram K, Stehle D, Johanning J, Manz M, Daniel PT, Gillissen BF, Schulze-Osthoff K, Essmann F. Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Bak. J Cell Sci. 2016;129(11):2213–23. PubMed

Carpio MA, Michaud M, Zhou W, Fisher JK, Walensky LD, Katz SG. BCL-2 family member BOK promotes apoptosis in response to endoplasmic reticulum stress. Proc Natl Acad Sci U S A. 2015;112(23):7201–6. PubMed PMC

Ke F, Grabow S, Kelly GL, Lin A, O'Reilly LA, Strasser A. Impact of the combined loss of BOK, BAX and BAK on the hematopoietic system is slightly more severe than compound loss of BAX and BAK. Cell Death Dis. 2015;6:e1938. PubMed PMC

Ke F, Bouillet P, Kaufmann T, Strasser A, Kerr J, Voss AK. Consequences of the combined loss of BOK and BAK or BOK and BAX. Cell Death Dis. 2013;4:e650. PubMed PMC

Ke F, Voss A, Kerr JB, O'Reilly LA, Tai L, Echeverry N, Bouillet P, Strasser A, Kaufmann T. BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice. Cell Death Differ. 2012;19(6):915–25. PubMed PMC

Fernandez-Marrero Y, Bleicken S, Das KK, Bachmann D, Kaufmann T, Garcia-Saez AJ. The membrane activity of BOK involves formation of large, stable toroidal pores and is promoted by cBID. FEBS J. 2017;284(5):711–24. PubMed

Bartholomeusz G, Wu Y, Ali SM, Xia W, Kwong KY, Hortobagyi G, Hung MC. Nuclear translocation of the pro-apoptotic Bcl-2 family member Bok induces apoptosis. Mol Carcinog. 2006;45(2):73–83. PubMed

Ray JE, Garcia J, Jurisicova A, Caniggia I. Mtd/Bok takes a swing: proapoptotic Mtd/Bok regulates trophoblast cell proliferation during human placental development and in preeclampsia. Cell Death Differ. 2010;17(5):846–59. PubMed

Schulman JJ, Wright FA, Han X, Zluhan EJ, Szczesniak LM, Wojcikiewicz RJ. The Stability and Expression Level of Bok Are Governed by Binding to Inositol 1,4,5-Trisphosphate Receptors. J Biol Chem. 2016;291(22):11820–8. PubMed PMC

Schulman JJ, Wright FA, Kaufmann T, Wojcikiewicz RJ. The Bcl-2 protein family member Bok binds to the coupling domain of inositol 1,4,5-trisphosphate receptors and protects them from proteolytic cleavage. J Biol Chem. 2013;288(35):25340–9. PubMed PMC

D'Orsi B, Engel T, Pfeiffer S, Nandi S, Kaufmann T, Henshall DC, Prehn JH. Bok Is Not Pro-Apoptotic But Suppresses Poly ADP-Ribose Polymerase-Dependent Cell Death Pathways and Protects against Excitotoxic and Seizure-Induced Neuronal Injury. J Neurosci. 2016;36(16):4564–78. PubMed PMC

Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J, Barretina J, Boehm JS, Dobson J, Urashima M, Mc Henry KT, Pinchback RM, et al. The landscape of somatic copy-number alteration across human cancers. Nature. 2010;463(7283):899–905. PubMed PMC

Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin. 2016;66(1):7–30. PubMed

Travis WD, Brambilla E, Nicholson AG, Yatabe Y, Austin JH, Beasley MB, Chirieac LR, Dacic S, Duhig E, Flieder DB, Geisinger K, Hirsch FR, et al. The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. J Thorac Oncol. 2015;10(9):1243–60. PubMed

Detterbeck FC, Boffa DJ, Tanoue LT. The new lung cancer staging system. Chest. 2009;136(1):260–71. PubMed

Gurzeler U, Rabachini T, Dahinden CA, Salmanidis M, Brumatti G, Ekert PG, Echeverry N, Bachmann D, Simon HU, Kaufmann T. In vitro differentiation of near-unlimited numbers of functional mouse basophils using conditional Hoxb8. Allergy. 2013;68(5):604–13. PubMed

Krepela E, Dankova P, Moravcikova E, Krepelova A, Prochazka J, Cermak J, Schutzner J, Zatloukal P, Benkova K. Increased expression of inhibitor of apoptosis proteins, survivin and XIAP, in non-small cell lung carcinoma. Int J Oncol. 2009;35(6):1449–62. PubMed

Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;11(8):783–4. PubMed PMC

Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002;18(11):1427–31. PubMed

Feng YX, Sokol ES, Del Vecchio CA, Sanduja S, Claessen JH, Proia TA, Jin DX, Reinhardt F, Ploegh HL, Wang Q, Gupta PB. Epithelial-to-mesenchymal transition activates PERK-eIF2alpha and sensitizes cells to endoplasmic reticulum stress. Cancer Discov. 2014;4(6):702–15. PubMed

Donnenberg AD, Meyer EM, Rubin JP, Donnenberg VS. The cell-surface proteome of cultured adipose stromal cells. Cytometry A. 2015;87(7):665–74. PubMed

Reference Database for Gene Expression Analysis. [Accessed: September 30, 2016]; available at: http://sbmdb.genome.rcast.u-tokyo.ac.jp/refexa/main_search.jsp.

Gao S, Fu W, Durrenberger M, De Geyter C, Zhang H. Membrane translocation and oligomerization of hBok are triggered in response to apoptotic stimuli and Bnip3. Cell Mol Life Sci. 2005;62(9):1015–24. PubMed PMC

Petryszak R, Keays M, Tang YA, Fonseca NA, Barrera E, Burdett T, Fullgrabe A, Fuentes AM, Jupp S, Koskinen S, Mannion O, Huerta L, et al. Expression Atlas update--an integrated database of gene and protein expression in humans, animals and plants. Nucleic Acids Res. 2016;44(D1):D746–D752. PubMed PMC

Garrison SP, Jeffers JR, Yang C, Nilsson JA, Hall MA, Rehg JE, Yue W, Yu J, Zhang L, Onciu M, Sample JT, Cleveland JL, et al. Selection against PUMA gene expression in Myc-driven B-cell lymphomagenesis. Mol Cell Biol. 2008;28(17):5391–402. PubMed PMC

Greider C, Chattopadhyay A, Parkhurst C, Yang E. BCL-x(L) and BCL2 delay Myc-induced cell cycle entry through elevation of p27 and inhibition of G1 cyclin-dependent kinases. Oncogene. 2002;21(51):7765–75. PubMed

Pierce RH, Vail ME, Ralph L, Campbell JS, Fausto N. Bcl-2 expression inhibits liver carcinogenesis and delays the development of proliferating foci. Am J Pathol. 2002;160(5):1555–60. PubMed PMC

Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA. Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc. 2008;83(5):584–94. PubMed PMC

Sarkar S, Horn G, Moulton K, Oza A, Byler S, Kokolus S, Longacre M. Cancer development, progression, and therapy: an epigenetic overview. Int J Mol Sci. 2013;14(10):21087–113. PubMed PMC

Lu H, Clauser KR, Tam WL, Frose J, Ye X, Eaton EN, Reinhardt F, Donnenberg VS, Bhargava R, Carr SA, Weinberg RA. A breast cancer stem cell niche supported by juxtacrine signalling from monocytes and macrophages. Nat Cell Biol. 2014;16(11):1105–17. PubMed PMC

Vizoso M, Puig M, Carmona FJ, Maqueda M, Velasquez A, Gomez A, Labernadie A, Lugo R, Gabasa M, Rigat-Brugarolas LG, Trepat X, Ramirez J, et al. Aberrant DNA methylation in non-small cell lung cancer-associated fibroblasts. Carcinogenesis. 2015;36(12):1453–63. PubMed PMC

Shintani Y, Fujiwara A, Kimura T, Kawamura T, Funaki S, Minami M, Okumura M. IL-6 Secreted from Cancer-Associated Fibroblasts Mediates Chemoresistance in NSCLC by Increasing Epithelial-Mesenchymal Transition Signaling. J Thorac Oncol. 2016;11(9):1482–92. PubMed

Kim YE, Kim JO, Park KS, Won M, Kim KE, Kim KK. Transforming Growth Factor-beta-Induced RBFOX3 Inhibition Promotes Epithelial-Mesenchymal Transition of Lung Cancer Cells. Mol Cells. 2016;39(8):625–30. PubMed PMC

Li L, Qi L, Liang Z, Song W, Liu Y, Wang Y, Sun B, Zhang B, Cao W. Transforming growth factor-beta1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells. Int J Mol Med. 2015;36(1):113–22. PubMed PMC

Moravcikova E, Krepela E, Prochazka J, Benkova K, Pauk N. Differential sensitivity to apoptosome apparatus activation in non-small cell lung carcinoma and the lung. Int J Oncol. 2014;44(5):1443–54. PubMed PMC

Sun C, Wang L, Huang S, Heynen GJ, Prahallad A, Robert C, Haanen J, Blank C, Wesseling J, Willems SM, Zecchin D, Hobor S, et al. Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma. Nature. 2014;508(7494):118–22. PubMed

Ishiguro F, Murakami H, Mizuno T, Fujii M, Kondo Y, Usami N, Taniguchi T, Yokoi K, Osada H, Sekido Y. Membranous expression of activated leukocyte cell adhesion molecule contributes to poor prognosis and malignant phenotypes of non-small-cell lung cancer. J Surg Res. 2013;179(1):24–32. PubMed

Hansen AG, Arnold SA, Jiang M, Palmer TD, Ketova T, Merkel A, Pickup M, Samaras S, Shyr Y, Moses HL, Hayward SW, Sterling JA, et al. ALCAM/CD166 is a TGF-beta-responsive marker and functional regulator of prostate cancer metastasis to bone. Cancer Res. 2014;74(5):1404–15. PubMed PMC