Overexpression of PIK3CA in murine head and neck epithelium drives tumor invasion and metastasis through PDK1 and enhanced TGFβ signaling

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Abstract

Head and neck squamous cell carcinoma (HNSCC) patients have a poor prognosis, with invasion and metastasis as major causes of mortality. The phosphatidylinositol 3-kinase (PI3K) pathway regulates a wide range of cellular processes crucial for tumorigenesis, and PIK3CA amplification and mutation are among the most common genetic alterations in human HNSCC. Compared with the well-documented roles of the PI3K pathway in cell growth and survival, the roles of the PI3K pathway in tumor invasion and metastasis have not been well delineated. We generated a PIK3CA genetically engineered mouse model (PIK3CA-GEMM) in which wild-type PIK3CA is overexpressed in head and neck epithelium. Although PIK3CA overexpression alone was not sufficient to initiate HNSCC formation, it significantly increased tumor susceptibility in an oral carcinogenesis mouse model. PIK3CA overexpression in mouse oral epithelium increased tumor invasiveness and metastasis by increasing epithelial–mesenchymal transition and by enriching a cancer stem cell phenotype in tumor epithelial cells. In addition to these epithelial alterations, we also observed marked inflammation in tumor stroma. AKT is a central signaling mediator of the PI3K pathway. However, molecular analysis suggested that progression of PIK3CA-driven HNSCC is facilitated by 3-phosphoinositide-dependent protein kinase (PDK1) and enhanced transforming growth factor β (TGFβ) signaling rather than by AKT. Examination of human HNSCC clinical samples revealed that both PIK3CA and PDK1 protein levels correlated with tumor progression, highlighting the significance of this pathway. In summary, our results offer significant insight into how PIK3CA overexpression drives HNSCC invasion and metastasis, providing a rationale for targeting PI3K/PDK1 and TGFβ signaling in advanced HNSCC patients with PIK3CA amplification.

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References

  1. 1

    Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D . Global cancer statistics. CA Cancer J Clin 2011; 61: 69–90.

  2. 2

    Siegel R, Naishadham D, Jemal A . Cancer statistics, 2013. CA Cancer J Clin 2013; 63: 11–30.

  3. 3

    Leemans CR, Braakhuis BJ, Brakenhoff RH . The molecular biology of head and neck cancer. Nat Rev Cancer 2011; 11: 9–22.

  4. 4

    Engelman JA, Luo J, Cantley LC . The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 2006; 7: 606–619.

  5. 5

    Liu P, Cheng H, Roberts TM, Zhao JJ . Targeting the phosphoinositide 3-kinase pathway in cancer. Nat Rev Drug Discov 2009; 8: 627–644.

  6. 6

    Du L, Shen J, Weems A, Lu SL . Role of phosphatidylinositol-3-kinase pathway in head and neck squamous cell carcinoma. J Oncol 2012; 2012: 450179.

  7. 7

    Vogt PK, Gymnopoulos M, Hart JR . PI 3-kinase and cancer: changing accents. Curr Opin Genet Dev 2009; 19: 12–17.

  8. 8

    Vasudevan KM, Barbie DA, Davies MA, Rabinovsky R, McNear CJ, Kim JJ et al. AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer. Cancer Cell 2009; 16: 21–32.

  9. 9

    Marsh Durban V, Deuker MM, Bosenberg MW, Phillips W, McMahon M . Differential AKT dependency displayed by mouse models of BRAFV600E-initiated melanoma. J Clin Invest 2013; 123: 5104–5118.

  10. 10

    Lui VW, Hedberg ML, Li H, Vangara BS, Pendleton K, Zeng Y et al. Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers. Cancer Discov 2013; 3: 761–769.

  11. 11

    Pickering CR, Zhang J, Yoo SY, Bengtsson L, Moorthy S, Neskey DM et al. Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers. Cancer Discov 2013; 3: 770–781.

  12. 12

    Iglesias-Bartolome R, Martin D, Gutkind JS . Exploiting the head and neck cancer oncogenome: widespread PI3K-mTOR pathway alterations and novel molecular targets. Cancer Discov 2013; 3: 722–725.

  13. 13

    Morris LG, Taylor BS, Bivona TG, Gong Y, Eng S, Brennan CW et al. Genomic dissection of the epidermal growth factor receptor (EGFR)/PI3K pathway reveals frequent deletion of the EGFR phosphatase PTPRS in head and neck cancers. Proc Natl Acad Sci U S A 2011; 108: 19024–19029.

  14. 14

    Woenckhaus J, Steger K, Werner E, Fenic I, Gamerdinger U, Dreyer T et al. Genomic gain of PIK3CA and increased expression of p110alpha are associated with progression of dysplasia into invasive squamous cell carcinoma. J Pathol 2002; 198: 335–342.

  15. 15

    Kozaki K, Imoto I, Pimkhaokham A, Hasegawa S, Tsuda H, Omura K et al. PIK3CA mutation is an oncogenic aberration at advanced stages of oral squamous cell carcinoma. Cancer Sci 2006; 97: 1351–1358.

  16. 16

    Estilo CL, O-Charoenrat P, Ngai I, Patel SG, Reddy PG, Dao S et al. The role of novel oncogenes squamous cell carcinoma-related oncogene and phosphatidylinositol 3-kinase p110alpha in squamous cell carcinoma of the oral tongue. Clin Cancer Res 2003; 9: 2300–2306.

  17. 17

    Fenic I, Steger K, Gruber C, Arens C, Woenckhaus J . Analysis of PIK3CA and Akt/protein kinase B in head and neck squamous cell carcinoma. Oncol Rep 2007; 18: 253–259.

  18. 18

    Lu SL, Reh D, Li AG, Woods J, Corless CL, Kulesz-Martin M et al. Overexpression of transforming growth factor beta1 in head and neck epithelia results in inflammation, angiogenesis, and epithelial hyperproliferation. Cancer Res 2004; 64: 4405–4410.

  19. 19

    Vitale-Cross L, Czerninski R, Amornphimoltham P, Patel V, Molinolo AA, Gutkind JS . Chemical carcinogenesis models for evaluating molecular-targeted prevention and treatment of oral cancer. Cancer Prev Res (Phila) 2009; 2: 419–422.

  20. 20

    Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY et al. The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 2008; 133: 704–715.

  21. 21

    Iliopoulos D, Polytarchou C, Hatziapostolou M, Kottakis F, Maroulakou IG, Struhl K et al. MicroRNAs differentially regulated by Akt isoforms control EMT and stem cell renewal in cancer cells. Sci Signal 2009; 2: ra62.

  22. 22

    Marvel D, Gabrilovich DI . Myeloid-derived suppressor cells in the tumor microenvironment: expect the unexpected. J Clin Invest 2015; 125: 3356–3364.

  23. 23

    Lu SL, Herrington H, Reh D, Weber S, Bornstein S, Wang D et al. Loss of transforming growth factor-beta type II receptor promotes metastatic head-and-neck squamous cell carcinoma. Genes Dev 2006; 20: 1331–1342.

  24. 24

    Bornstein S, White R, Malkoski S, Oka M, Han G, Cleaver T et al. Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation. J Clin Invest 2009; 119: 3408–3419.

  25. 25

    Liu P, Cheng H, Santiago S, Raeder M, Zhang F, Isabella A et al. Oncogenic PIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and PI3K pathway-independent mechanisms. Nat Med 2011; 17: 1116–1120.

  26. 26

    Klarenbeek S, van Miltenburg MH, Jonkers J . Genetically engineered mouse models of PI3K signaling in breast cancer. Mol Oncol 2013; 7: 146–164.

  27. 27

    Engelman JA, Chen L, Tan X, Crosby K, Guimaraes AR, Upadhyay R et al. Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers. Nat Med 2008; 14: 1351–1356.

  28. 28

    Kinross KM, Montgomery KG, Kleinschmidt M, Waring P, Ivetac I, Tikoo A et al. An activating Pik3ca mutation coupled with Pten loss is sufficient to initiate ovarian tumorigenesis in mice. J Clin Invest 2012; 122: 553–557.

  29. 29

    Hare LM, Phesse TJ, Waring PM, Montgomery KG, Kinross KM, Mills K et al. Physiological expression of the PI3K-activating mutation Pik3ca(H1047R) combines with Apc loss to promote development of invasive intestinal adenocarcinomas in mice. Biochem J 2014; 458: 251–258.

  30. 30

    Hennessy BT, Gonzalez-Angulo AM, Stemke-Hale K, Gilcrease MZ, Krishnamurthy S, Lee JS et al. Characterization of a naturally occurring breast cancer subset enriched in epithelial-to-mesenchymal transition and stem cell characteristics. Cancer Res 2009; 69: 4116–4124.

  31. 31

    Wallin JJ, Guan J, Edgar KA, Zhou W, Francis R, Torres AC et al. Active PI3K pathway causes an invasive phenotype which can be reversed or promoted by blocking the pathway at divergent nodes. PLoS One 2012; 7: e36402.

  32. 32

    Fan QW, Cheng C, Knight ZA, Haas-Kogan D, Stokoe D, James CD et al. EGFR signals to mTOR through PKC and independently of Akt in glioma. Sci Signal 2009; 2: ra4.

  33. 33

    Lauring J, Cosgrove DP, Fontana S, Gustin JP, Konishi H, Abukhdeir AM et al. Knock in of the AKT1 E17K mutation in human breast epithelial cells does not recapitulate oncogenic PIK3CA mutations. Oncogene 2010; 29: 2337–2345.

  34. 34

    Xue G, Restuccia DF, Lan Q, Hynx D, Dirnhofer S, Hess D et al. Akt/PKB-mediated phosphorylation of Twist1 promotes tumor metastasis via mediating cross-talk between PI3K/Akt and TGF-beta signaling axes. Cancer Discov 2012; 2: 248–259.

  35. 35

    Curry NL, Mino-Kenudson M, Oliver TG, Yilmaz OH, Yilmaz VO, Moon JY et al. Pten-null tumors cohabiting the same lung display differential AKT activation and sensitivity to dietary restriction. Cancer Discov 2013; 3: 908–921.

  36. 36

    Wong KK . Oral-specific chemical carcinogenesis in mice: an exciting model for cancer prevention and therapy. Cancer Prev Res (Phila) 2009; 2: 10–13.

  37. 37

    Weber SM, Bornstein S, Li Y, Malkoski SP, Wang D, Rustgi AK et al. Tobacco-specific carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces AKT activation in head and neck epithelia. Int J Oncol 2011; 39: 1193–1198.

  38. 38

    Moral M, Segrelles C, Lara MF, Martínez-Cruz AB, Lorz C, Santos M et al. Akt activation synergizes with Trp53 loss in oral epithelium to produce a novel mouse model for head and neck squamous cell carcinoma. Cancer Res 2009; 69: 1099–1108.

  39. 39

    Pearce LR, Komander D, Alessi DR . The nuts and bolts of AGC protein kinases. Nat Rev Mol Cell Biol 2010; 11: 9–22.

  40. 40

    Finlay DK, Sinclair LV, Feijoo C, Waugh CM, Hagenbeek TJ, Spits H et al. Phosphoinositide-dependent kinase 1 controls migration and malignant transformation but not cell growth and proliferation in PTEN-null lymphocytes. J Exp Med 2009; 206: 2441–2454.

  41. 41

    Feng Q, Di R, Tao F, Chang Z, Lu S, Fan W et al. PDK1 regulates vascular remodeling and promotes epithelial-mesenchymal transition in cardiac development. Mol Cell Biol 2010; 30: 3711–3721.

  42. 42

    Maurer M, Su T, Saal LH, Koujak S, Hopkins BD, Barkley CR et al. 3-Phosphoinositide-dependent kinase 1 potentiates upstream lesions on the phosphatidylinositol 3-kinase pathway in breast carcinoma. Cancer Res 2009; 69: 6299–6306.

  43. 43

    Garcia-Carracedo D, Turk AT, Fine SA, Akhavan N, Tweel BC, Parsons R et al. Loss of PTEN expression is associated with poor prognosis in patients with intraductal papillary mucinous neoplasms of the pancreas. Clin Cancer Res 2013; 19: 6830–6841.

  44. 44

    Scortegagna M, Ruller C, Feng Y, Lazova R, Kluger H, Li JL et al. Genetic inactivation or pharmacological inhibition of Pdk1 delays development and inhibits metastasis of Braf::Pten melanoma. Oncogene 2013; 33: 4330–4339.

  45. 45

    Eser S, Reiff N, Messer M, Seidler B, Gottschalk K, Dobler M et al. Selective requirement of PI3K/PDK1 signaling for Kras oncogene-driven pancreatic cell plasticity and cancer. Cancer Cell 2013; 23: 406–420.

  46. 46

    Bhola NE, Freilino ML, Joyce SC, Sen M, Thomas SM, Sahu A et al. Antitumor mechanisms of targeting the PDK1 pathway in head and neck cancer. Mol Cancer Ther 2012; 11: 1236–1246.

  47. 47

    Raimondi C, Chikh A, Wheeler AP, Maffucci T, Falasca M . A novel regulatory mechanism links PLCgamma1 to PDK1. J Cell Sci 2012; 125: 3153–3163.

  48. 48

    Tan J, Li Z, Lee PL, Guan P, Aau MY, Lee ST et al. PDK1 signaling toward PLK1-MYC activation confers oncogenic transformation, tumor-initiating cell activation, and resistance to mTOR-targeted therapy. Cancer Discov 2013; 3: 1156–1171.

  49. 49

    Zhang L, Zhou F, ten Dijke P . Signaling interplay between transforming growth factor-beta receptor and PI3K/AKT pathways in cancer. Trends Biochem Sci 2013; 38: 612–620.

  50. 50

    Yi JY, Shin I, Arteaga CL . Type I transforming growth factor beta receptor binds to and activates phosphatidylinositol 3-kinase. J Biol Chem 2005; 280: 10870–10876.

  51. 51

    Du Y, Peyser ND, Grandis JR . Integration of molecular targeted therapy with radiation in head and neck cancer. Pharmacol Ther 2014; 142: 88–98.

  52. 52

    Signore M, Pelacchi F, di Martino S, Runci D, Biffoni M, Giannetti S et al. Combined PDK1 and CHK1 inhibition is required to kill glioblastoma stem-like cells in vitro and in vivo. Cell Death Dis 2014; 5: e1223.

  53. 53

    Akhurst RJ, Hata A . Targeting the TGFbeta signalling pathway in disease. Nat Rev Drug Discov 2012; 11: 790–811.

  54. 54

    Han G, Lu SL, Li AG, He W, Corless CL, Kulesz-Martin M et al. Distinct mechanisms of TGF-beta1-mediated epithelial-to-mesenchymal transition and metastasis during skin carcinogenesis. J Clin Invest 2005; 115: 1714–1723.

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Acknowledgements

This work is supported by National Institutes of Health grant R01DE021788 (to S-L Lu), R01DE015953 (to X-J Wang). University of Colorado Academic Enrichment Fund (to S-L Lu), American Cancer Society (to S-L Lu), University of Colorado Cancer Center (to S-L Lu), and Cancer League of Colorado (to S-L Lu). S-L Lu is an investigator of THANC foundation. We thank the Transgenic Core of Oregon Health and Science University for generating founders of the mouse model, and the University of Colorado Skin Disease Research Center Morphology Phenotyping Core (P30 AR057212) for assisting with histological work.

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Correspondence to S-L Lu.

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