Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Phenotype-assisted transcriptome analysis identifies FOXM1 downstream from Ras–MKK3–p38 to regulate in vitro cellular invasion

Abstract

The Ras oncogene is known to activate three major MAPK pathways, ERK, JNK, p38 and exert distinct cellular phenotypes, that is, apoptosis and invasion through the Ras–MKK3–p38-signaling cascade. We attempted to identify the molecular targets of this pathway that selectively govern the invasive phenotype. Stable transfection of NIH3T3 fibroblasts with MKK3act cDNA construct revealed similar p38-dependent in vitro characteristics observed in Ha-RasEJ-transformed NIH3T3 cells, including enhanced invasiveness and anchorage-independent growth correlating with p38 phosphorylation status. To identify the consensus downstream targets of the Ras–MKK3–p38 cascade involved in invasion, in vitro invasion assays were used to isolate highly invasive cells from both, MKK3 and Ha-RasEJ transgenic cell lines. Subsequently a genome-wide transcriptome analysis was employed to investigate differentially regulated genes in invasive Ha-RasEJ- and MKK3act-transfected NIH3T3 fibroblasts. Using this phenotype-assisted approach combined with system level protein-interaction network analysis, we identified FOXM1, PLK1 and CDK1 to be differentially regulated in invasive Ha-RasEJ-NIH3T3 and MKK3act–NIH3T3 cells. Finally, a FOXM1 RNA-knockdown approach revealed its requirement for both invasion and anchorage-independent growth of Ha-RasEJ- and MKK3act–NIH3T3 cells. Together, we identified FOXM1 as a key downstream target of Ras and MKK3-induced cellular in vitro invasion and anchorage-independent growth signaling.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  • Adam AP, George A, Schewe D, Bragado P, Iglesias BV, Ranganathan AC et al (2009). Computational identification of a p38SAPK-regulated transcription factor network required for tumor cell quiescence. Cancer Res 69: 5664–5672.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Behren A, Binder K, Vucelic G, Herberhold S, Hirt B, Loewenheim H et al (2005). The p38 SAPK pathway is required for Ha-ras induced in vitro invasion of NIH3T3 cells. Exp Cell Res 303: 321–330.

    Article  CAS  PubMed  Google Scholar 

  • Bradley MO, Kraynak AR, Storer RD, Gibbs JB . (1986). Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes. Proc Natl Acad Sci USA 83: 5277–5281.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Demuth T, Reavie LB, Rennert JL, Nakada M, Nakada S, Hoelzinger DB et al (2007). MAP-ing glioma invasion: mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival. Mol Cancer Ther 6: 1212–1222.

    Article  CAS  PubMed  Google Scholar 

  • Deng Q, Liao R, Wu BL, Sun P . (2004). High intensity ras signaling induces premature senescence by activating p38 pathway in primary human fibroblasts. J Biol Chem 279: 1050–1059.

    Article  CAS  PubMed  Google Scholar 

  • Denhardt DT . (1996). Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. Biochem J 318 (Part 3): 729–747.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Downward J . (2003). Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 3: 11–22.

    Article  CAS  PubMed  Google Scholar 

  • Gemenetzidis E, Bose A, Riaz AM, Chaplin T, Young BD, Ali M et al (2009). FOXM1 upregulation is an early event in human squamous cell carcinoma and it is enhanced by nicotine during malignant transformation. PLoS One 4: e4849.

    Article  PubMed  PubMed Central  Google Scholar 

  • Gusarova GA, Wang IC, Major ML, Kalinichenko VV, Ackerson T, Petrovic V et al (2007). A cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma treatment. J Clin Invest 117: 99–111.

    Article  CAS  PubMed  Google Scholar 

  • Han J, Lee JD, Jiang Y, Li Z, Feng L, Ulevitch RJ . (1996). Characterization of the structure and function of a novel MAP kinase kinase (MKK6). J Biol Chem 271: 2886–2891.

    Article  CAS  PubMed  Google Scholar 

  • Haq R, Brenton JD, Takahashi M, Finan D, Finkielsztein A, Damaraju S et al (2002). Constitutive p38HOG mitogen-activated protein kinase activation induces permanent cell cycle arrest and senescence. Cancer Res 62: 5076–5082.

    CAS  PubMed  Google Scholar 

  • Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T et al (1997). Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science 275: 90–94.

    Article  CAS  PubMed  Google Scholar 

  • Janulis M, Silberman S, Ambegaokar A, Gutkind JS, Schultz RM . (1999). Role of mitogen-activated protein kinases and c-Jun/AP-1 transactivating activity in the regulation of protease mRNAs and the malignant phenotype in NIH 3T3 fibroblasts. J Biol Chem 274: 801–813.

    Article  CAS  PubMed  Google Scholar 

  • Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S et al (1996). Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta). J Biol Chem 271: 17920–17926.

    Article  CAS  PubMed  Google Scholar 

  • Jin K, Lim S, Mercer SE, Friedman E . (2005). The survival kinase Mirk/dyrk1B is activated through Rac1–MKK3 signaling. J Biol Chem 280: 42097–42105.

    Article  CAS  PubMed  Google Scholar 

  • Junttila MR, Ala-Aho R, Jokilehto T, Peltonen J, Kallajoki M, Grenman R et al (2007). p38alpha and p38delta mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells. Oncogene 26: 5267–5279.

    Article  CAS  PubMed  Google Scholar 

  • Kalin TV, Wang IC, Ackerson TJ, Major ML, Detrisac CJ, Kalinichenko VV et al (2006). Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Res 66: 1712–1720.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kalinina OA, Kalinin SA, Polack EW, Mikaelian I, Panda S, Costa RH et al (2003). Sustained hepatic expression of FoxM1B in transgenic mice has minimal effects on hepatocellular carcinoma development but increases cell proliferation rates in preneoplastic and early neoplastic lesions. Oncogene 22: 6266–6276.

    Article  CAS  PubMed  Google Scholar 

  • Korver W, Roose J, Heinen K, Weghuis DO, de Bruijn D, van Kessel AG et al (1997). The human TRIDENT/HFH-11/FKHL16 gene: structure, localization, and promoter characterization. Genomics 46: 435–442.

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, McDonnell PC, Gum RJ, Hand AT, Lee JC, Young PR . (1997). Novel homologues of CSBP/p38 MAP kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles. Biochem Biophys Res Commun 235: 533–538.

    Article  CAS  PubMed  Google Scholar 

  • Laoukili J, Kooistra MR, Bras A, Kauw J, Kerkhoven RM, Morrison A et al (2005). FoxM1 is required for execution of the mitotic programme and chromosome stability. Nat Cell Biol 7: 126–136.

    Article  CAS  PubMed  Google Scholar 

  • Marshall CJ . (1996). Ras effectors. Curr Opin Cell Biol 8: 197–204.

    Article  CAS  PubMed  Google Scholar 

  • Moon A . (2006). Differential functions of Ras for malignant phenotypic conversion. Arch Pharm Res 29: 113–122.

    Article  CAS  PubMed  Google Scholar 

  • Ozanne BW, Spence HJ, McGarry LC, Hennigan RF . (2007). Transcription factors control invasion: AP-1 the first among equals. Oncogene 26: 1–10.

    Article  CAS  PubMed  Google Scholar 

  • Park HJ, Carr JR, Wang Z, Nogueira V, Hay N, Tyner AL et al (2009). FoxM1, a critical regulator of oxidative stress during oncogenesis. EMBO J 28: 2908–2918.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pilarsky C, Wenzig M, Specht T, Saeger HD, Grutzmann R . (2004). Identification and validation of commonly overexpressed genes in solid tumors by comparison of microarray data. Neoplasia 6: 744–750.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qi X, Tang J, Loesch M, Pohl N, Alkan S, Chen G . (2006). p38gamma mitogen-activated protein kinase integrates signaling crosstalk between Ras and estrogen receptor to increase breast cancer invasion. Cancer Res 66: 7540–7547.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B, Davis RJ . (1996). MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol 16: 1247–1255.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Raman M, Chen W, Cobb MH . (2007). Differential regulation and properties of MAPKs. Oncogene 26: 3100–3112.

    Article  CAS  PubMed  Google Scholar 

  • Reunanen N, Li SP, Ahonen M, Foschi M, Han J, Kahari VM . (2002). Activation of p38 alpha MAPK enhances collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J Biol Chem 277: 32360–32368.

    Article  CAS  PubMed  Google Scholar 

  • Schaeffer HJ, Weber MJ . (1999). Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 19: 2435–2444.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schwacke JH, Voit EO . (2007). The potential for signal integration and processing in interacting MAP kinase cascades. J Theor Biol 246: 604–620.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shin I, Kim S, Song H, Kim HR, Moon A . (2005). H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells. J Biol Chem 280: 14675–14683.

    Article  CAS  PubMed  Google Scholar 

  • Song H, Ki SH, Kim SG, Moon A . (2006). Activating transcription factor 2 mediates matrix metalloproteinase-2 transcriptional activation induced by p38 in breast epithelial cells. Cancer Res 66: 10487–10496.

    Article  CAS  PubMed  Google Scholar 

  • Stacey DW, Kung HF . (1984). Transformation of NIH 3T3 cells by microinjection of Ha-ras p21 protein. Nature 310: 508–511.

    Article  CAS  PubMed  Google Scholar 

  • Sun P, Yoshizuka N, New L, Moser BA, Li Y, Liao R et al (2007). PRAK is essential for ras-induced senescence and tumor suppression. Cell 128: 295–308.

    Article  CAS  PubMed  Google Scholar 

  • Wierstra I, Alves J . (2007). FOXM1, a typical proliferation-associated transcription factor. Biol Chem 388: 1257–1274.

    CAS  PubMed  Google Scholar 

  • Yoshida Y, Wang IC, Yoder HM, Davidson NO, Costa RH . (2007). The forkhead box M1 transcription factor contributes to the development and growth of mouse colorectal cancer. Gastroenterology 132: 1420–1431.

    Article  CAS  PubMed  Google Scholar 

  • Zhang JY, Schultz RM . (1992). Fibroblasts transformed by different ras oncogenes show dissimilar patterns of protease gene expression and regulation. Cancer Res 52: 6682–6689.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Thomas Regiert (DKFZ) for excellent technical assistance with microarray analysis. We also thank J Han (Scripps Research Institute) for providing us with the MKK3(b) expression construct and RH Medema (Laboratory of Experimental Oncology, University Medical Center Utrecht) for the FoxM1 promoter construct. This work was supported by the German Krebshilfe (Deutsche Krebshilfe, 107691, to CS, PH, PP and AA), NSCOR NNJ04HJ12G and DFG-SPP1190 (to AA and PH) and DFG-Si634-5/1 (to CS and PP).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to C Simon.

Additional information

Data deposition footnote: Microarray data under accession number E-TABM-561.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Behren, A., Mühlen, S., Acuna Sanhueza, G. et al. Phenotype-assisted transcriptome analysis identifies FOXM1 downstream from Ras–MKK3–p38 to regulate in vitro cellular invasion. Oncogene 29, 1519–1530 (2010). https://doi.org/10.1038/onc.2009.436

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2009.436

Keywords

This article is cited by

Search

Quick links