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Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3

Abstract

Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1−/− mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK’s effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment.

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References

  1. Vaahtomeri K, Makela TP . Molecular mechanisms of tumor suppression by LKB1. FEBS Lett 2011; 585: 944–951.

    CAS  Article  Google Scholar 

  2. Hardie DG . New roles for the LKB1–AMPK pathway. Curr Opin Cell Biol 2005; 17: 167–173.

    CAS  Article  Google Scholar 

  3. Jenne DE, Reimann H, Nezu J, Friedel W, Loff S, Jeschke R et al. Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase. Nat Genet 1998; 18: 38–43.

    CAS  Article  Google Scholar 

  4. Morton JP, Jamieson NB, Karim SA, Athineos D, Ridgway RA, Nixon C et al. LKB1 haploinsufficiency cooperates with Kras to promote pancreatic cancer through suppression of p21-dependent growth arrest. Gastroenterology 2010; 139: 586–597 597 e581–586.

    CAS  Article  Google Scholar 

  5. Andrade-Vieira R, Xu Z, Colp P, Marignani PA . Loss of LKB1 expression reduces the latency of ErbB2-mediated mammary gland tumorigenesis, promoting changes in metabolic pathways. PLoS One 2013; 8: e56567.

    CAS  Article  Google Scholar 

  6. Wong DJ, Liu H, Ridky TW, Cassarino D, Segal E, Chang HY . Module map of stem cell genes guides creation of epithelial cancer stem cells. Cell stem cell 2008; 2: 333–344.

    CAS  Article  Google Scholar 

  7. Andrade-Vieira R, Goguen D, Bentley HA, Bowen CV, Marignani PA . Pre-clinical study of drug combinations that reduce breast cancer burden due to aberrant mTOR and metabolism promoted by LKB1 loss. Oncotarget 2014; 5: 12738–12752.

    Article  Google Scholar 

  8. Avtanski DB, Nagalingam A, Bonner MY, Arbiser JL, Saxena NK, Sharma D . Honokiol inhibits epithelial-mesenchymal transition in breast cancer cells by targeting signal transducer and activator of transcription 3/Zeb1/E-cadherin axis. Mol Oncol 2014; 8: 565–580.

    CAS  Article  Google Scholar 

  9. Yan D, Avtanski D, Saxena NK, Sharma D . Leptin-induced epithelial-mesenchymal transition in breast cancer cells requires beta-catenin activation via Akt/GSK3- and MTA1/Wnt1 protein-dependent pathways. J Biol Chem 2012; 287: 8598–8612.

    CAS  Article  Google Scholar 

  10. Suzuki A, Raya A, Kawakami Y, Morita M, Matsui T, Nakashima K et al. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells. Proc Natl Acad Sci USA 2006; 103: 10294–10299.

    CAS  Article  Google Scholar 

  11. Zhou JJ, Chen RF, Deng XG, Zhou Y, Ye X, Yu M et al. Hepatitis C virus core protein regulates NANOG expression via the stat3 pathway. FEBS Lett 2014; 588: 566–573.

    CAS  Article  Google Scholar 

  12. Foshay KM, Gallicano GI . Regulation of Sox2 by STAT3 initiates commitment to the neural precursor cell fate. Stem Cells Dev 2008; 17: 269–278.

    CAS  Article  Google Scholar 

  13. Do DV, Ueda J, Messerschmidt DM, Lorthongpanich C, Zhou Y, Feng B et al. A genetic and developmental pathway from STAT3 to the OCT4-NANOG circuit is essential for maintenance of ICM lineages in vivo. Genes Dev 2013; 27: 1378–1390.

    CAS  Article  Google Scholar 

  14. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet 2008; 40: 499–507.

    CAS  Article  Google Scholar 

  15. Fenton H, Carlile B, Montgomery EA, Carraway H, Herman J, Sahin F et al. LKB1 protein expression in human breast cancer. Appl Immunohistochem Mol Morphol 2006; 14: 146–153.

    CAS  Article  Google Scholar 

  16. Li J, Liu J, Li P, Mao X, Li W, Yang J et al. Loss of LKB1 disrupts breast epithelial cell polarity and promotes breast cancer metastasis and invasion. J Exp Clin Cancer Res 2014; 33: 70.

    Article  Google Scholar 

  17. Ylikorkala A, Rossi DJ, Korsisaari N, Luukko K, Alitalo K, Henkemeyer M et al. Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice. Science 2001; 293: 1323–1326.

    CAS  Article  Google Scholar 

  18. Rossi DJ, Ylikorkala A, Korsisaari N, Salovaara R, Luukko K, Launonen V et al. Induction of cyclooxygenase-2 in a mouse model of Peutz-Jeghers polyposis. Proc Natl Acad Sci USA 2002; 99: 12327–12332.

    CAS  Article  Google Scholar 

  19. Xu Q, Yi LT, Pan Y, Wang X, Li YC, Li JM et al. Antidepressant-like effects of the mixture of honokiol and magnolol from the barks of Magnolia officinalis in stressed rodents. Prog Neuropsychopharmacol Biol Psychiatry 2008; 32: 715–725.

    CAS  Article  Google Scholar 

  20. Oh JH, Kang LL, Ban JO, Kim YH, Kim KH, Han SB et al. Anti-inflammatory effect of 4-O-methylhonokiol, compound isolated from Magnolia officinalis through inhibition of NF-kappaB [corrected]. Chem Biol Interact 2009; 180: 506–514.

    CAS  Article  Google Scholar 

  21. Choi DY, Lee YJ, Hong JT, Lee HJ . Antioxidant properties of natural polyphenols and their therapeutic potentials for Alzheimer's disease. Brain Res Bull 2012; 87: 144–153.

    CAS  Article  Google Scholar 

  22. Leeman-Neill RJ, Cai Q, Joyce SC, Thomas SM, Bhola NE, Neill DB et al. Honokiol inhibits epidermal growth factor receptor signaling and enhances the antitumor effects of epidermal growth factor receptor inhibitors. Clin Cancer Res 2010; 16: 2571–2579.

    CAS  Article  Google Scholar 

  23. Prasad R, Kappes JC, Katiyar SK . Inhibition of NADPH oxidase 1 activity and blocking the binding of cytosolic and membrane-bound proteins by honokiol inhibit migratory potential of melanoma cells. Oncotarget 2016; 7: 7899–7912.

    PubMed  PubMed Central  Google Scholar 

  24. Averett C, Arora S, Zubair H, Singh S, Bhardwaj A, Singh AP . Molecular targets of Honokiol: a promising phytochemical for effective cancer management. Enzymes 2014; 36: 175–193.

    CAS  Article  Google Scholar 

  25. Nagalingam A, Arbiser JL, Bonner MY, Saxena NK, Sharma D . Honokiol activates AMP-activated protein kinase in breast cancer cells via an LKB1-dependent pathway and inhibits breast carcinogenesis. Breast Cancer Res 2012; 14: R35.

    CAS  Article  Google Scholar 

  26. Sinibaldi D, Wharton W, Turkson J, Bowman T, Pledger WJ, Jove R . Induction of p21WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling. Oncogene 2000; 19: 5419–5427.

    CAS  Article  Google Scholar 

  27. Yu H, Jove R . The STATs of cancer—new molecular targets come of age. Nat Rev Cancer 2004; 4: 97–105.

    CAS  Article  Google Scholar 

  28. Buettner R, Mora LB, Jove R . Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clin cancer res 2002; 8: 945–954.

    CAS  PubMed  Google Scholar 

  29. Yue P, Turkson J . Targeting STAT3 in cancer: how successful are we? Expert Opin Investig Drugs 2009; 18: 45–56.

    CAS  Article  Google Scholar 

  30. Bowman T, Garcia R, Turkson J, Jove R . STATs in oncogenesis. Oncogene 2000; 19: 2474–2488.

    CAS  Article  Google Scholar 

  31. Burdelya L, Catlett-Falcone R, Levitzki A, Cheng F, Mora LB, Sotomayor E et al. Combination therapy with AG-490 and interleukin 12 achieves greater antitumor effects than either agent alone. Mol Cancer Ther 2002; 1: 893–899.

    CAS  PubMed  Google Scholar 

  32. Grandis JR, Drenning SD, Chakraborty A, Zhou MY, Zeng Q, Pitt AS et al. Requirement of Stat3 but not Stat1 activation for epidermal growth factor receptor- mediated cell growth in vitro. J Clin Invest 1998; 102: 1385–1392.

    CAS  Article  Google Scholar 

  33. Song H, Wang R, Wang S, Lin J . A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells. Proc Natl Acad Sci USA 2005; 102: 4700–4705.

    CAS  Article  Google Scholar 

  34. Bai X, Cerimele F, Ushio-Fukai M, Waqas M, Campbell PM, Govindarajan B et al. Honokiol, a small molecular weight natural product, inhibits angiogenesis in vitro and tumor growth in vivo. J Biol Chem 2003; 278: 35501–35507.

    CAS  Article  Google Scholar 

  35. Avtanski DB, Nagalingam A, Kuppusamy P, Bonner MY, Arbiser JL, Saxena NK et al. Honokiol abrogates leptin-induced tumor progression by inhibiting Wnt1-MTA1-beta-catenin signaling axis in a microRNA-34a dependent manner. Oncotarget 2015; 6: 16396–16410.

    PubMed  PubMed Central  Google Scholar 

  36. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci USA 2004; 101: 3329–3335.

    CAS  Article  Google Scholar 

  37. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C et al. Stat3 as an oncogene. Cell 1999; 98: 295–303.

    CAS  Article  Google Scholar 

  38. Taliaferro-Smith L, Nagalingam A, Zhong D, Zhou W, Saxena NK, Sharma D . LKB1 is required for adiponectin-mediated modulation of AMPK-S6K axis and inhibition of migration and invasion of breast cancer cells. Oncogene 2009; 28: 2621–2633.

    CAS  Article  Google Scholar 

  39. Saxena NK, Taliaferro-Smith L, Knight BB, Merlin D, Anania FA, O'Regan RM et al. Bidirectional crosstalk between leptin and insulin-like growth factor-I signaling promotes invasion and migration of breast cancer cells via transactivation of epidermal growth factor receptor. Cancer Res 2008; 68: 9712–9722.

    CAS  Article  Google Scholar 

  40. Sharma D, Saxena NK, Davidson NE, Vertino PM . Restoration of tamoxifen sensitivity in estrogen receptor-negative breast cancer cells: tamoxifen-bound reactivated ER recruits distinctive corepressor complexes. Cancer res 2006; 66: 6370–6378.

    CAS  Article  Google Scholar 

  41. Saxena NK, Vertino PM, Anania FA, Sharma D . leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3. J biol chem 2007; 282: 13316–13325.

    CAS  Article  Google Scholar 

  42. Hung WC, Chen SH, Paul CD, Stroka KM, Lo YC, Yang JT et al. Distinct signaling mechanisms regulate migration in unconfined versus confined spaces. J Cell Biol 2013; 202: 807–824.

    CAS  Article  Google Scholar 

  43. Wang P, Chen SH, Hung WC, Paul C, Zhu F, Guan PP et al. Fluid shear promotes chondrosarcoma cell invasion by activating matrix metalloproteinase 12 via IGF-2 and VEGF signaling pathways. Oncogene 2015; 34: 4558–4569.

    CAS  Article  Google Scholar 

  44. Hung WC, Yang JR, Yankaskas CL, Wong BS, Wu PH, Pardo-Pastor C et al. Confinement sensing and signal optimization via Piezo1/PKA and Myosin II pathways. Cell Rep 2016; 15: 1430–1441.

    CAS  Article  Google Scholar 

  45. Saxena NK, Sharma D, Ding X, Lin S, Marra F, Merlin D et al. Concomitant activation of the JAK/STAT, PI3K/AKT, and ERK signaling is involved in leptin-mediated promotion of invasion and migration of hepatocellular carcinoma cells. Cancer Res 2007; 67: 2497–2507.

    CAS  Article  Google Scholar 

  46. Avtanski DB, Nagalingam A, Kuppusamy P, Bonner MY, Arbiser JL, Saxena NK et al. Honokiol abrogates leptin-induced tumor progression by inhibiting Wnt1-MTA1-beta-catenin signaling axis in a microRNA-34a dependent manner. Oncotarget 2015; 6: 16396–16410.

    PubMed  PubMed Central  Google Scholar 

  47. Boudeau J, Scott JW, Resta N, Deak M, Kieloch A, Komander D et al. Analysis of the LKB1-STRAD-MO25 complex. J Cell Sci 2004; 117: 6365–6375.

    CAS  Article  Google Scholar 

  48. Nath-Sain S, Marignani PA . LKB1 catalytic activity contributes to estrogen receptor alpha signaling. Mol Biol Cell 2009; 20: 2785–2795.

    CAS  Article  Google Scholar 

  49. Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 2012; 490: 61–70.

    Article  Google Scholar 

  50. Azare J, Doane A, Leslie K, Chang Q, Berishaj M, Nnoli J et al. Stat3 mediates expression of autotaxin in breast cancer. PLoS One 2011; 6: e27851.

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by NCI NIH, R21CA185943 (to NKS); OTKA K108655 (to BG); NCI NIH R01AR47901 (to JLA); The Nova Scotia Health Research Foundation (DL); The Canadian Breast Cancer Foundation and The Nova and the Dalhousie Medical Research Foundation (PAM); NCI NIH R01CA131294, NCI NIH R21CA155686, Avon Foundation, Breast Cancer Research Foundation (BCRF) 90047965 and The Fetting Fund (to DS).

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Correspondence to N K Saxena or D Sharma.

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SS, AN, NM, MYB, PM, AA, PK, DL, PK, SC, MS, AB, VFM, C-YH, WM, BG, KK, SS, PAM, NKS and DS declare no conflict of interest. JLA is listed as an inventor on patents filed by Emory University. Emory has licensed its HNK technologies to Naturopathic Pharmacy. JLA has received stock in Naturopathic Pharmacy, which to the best of knowledge is not publically traded.

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Sengupta, S., Nagalingam, A., Muniraj, N. et al. Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3. Oncogene 36, 5709–5721 (2017). https://doi.org/10.1038/onc.2017.164

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