Research ArticleCancer

The transcription factor FOXF1 promotes prostate cancer by stimulating the mitogen-activated protein kinase ERK5

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Science Signaling  10 May 2016:
Vol. 9, Issue 427, pp. ra48
DOI: 10.1126/scisignal.aad5582
  • Fig. 1 Overexpression of FOXF1 in PCa tumor cells increases prostate carcinogenesis in a mouse model.

    (A) FOXF1 staining in normal prostate epithelium and prostate tumors. Images are representative of normal murine prostates from four 9-week-old mice, murine prostate tumors from five TRAMP transgenic mice and four Hi-Myc/ARR2PB-c-Myc transgenic mice, and human prostate cancer sections from 10 patients. Images are magnified at ×200 (×400, inset); scale bars, 50 μm. (B) Myc-CaP murine PCa cells were transduced with lentiviruses to overexpress Foxf1 (FoxF1-OE) and the luciferase reporter. The mRNA and protein levels of FOXF1 were measured by qRT-PCR and Western blot (n = 4 experiments). (C) Mass of orthotopic control or FoxF1-OE Myc-CaP xenografts at 4 weeks after implantation. Tumor growth was monitored using bioluminescent images from luciferase-labeled Myc-CaP tumor cells (panel). Data are means ± SEM from 12 mice per group. (D) Percent of mice that developed metastasis from control or FoxF1-OE Myc-CaP tumors. Data are means ± SEM from 10 mice per group. (E) Weights of control and FoxF1-expressing TRAMP tumors upon harvest at 4 weeks after implantation. (F) Number of peritoneal metastasis in orthotopic control and FoxF1-expressing TRAMP tumors. Data are means ± SEM from 12 mice per group. *P < 0.05, unpaired Student’s t test. N.D., not detected.

  • Fig. 2 FoxF1 induces cellular proliferation in Myc-CaP and TRAMP orthotopic tumors.

    (A and B) qRT-PCR analysis of the expression of mRNAs encoding Foxf1 and cell cycle–associated genes in control and FoxF1-OE Myc-CaP tumors (A) and TRAMP (B), normalized against the expression of mRNA encoding β-actin. Total mRNA was isolated from prostate tumors 4 weeks after orthotopic inoculation of Myc-CaP or TRAMP cells, respectively. Data are means ± SEM from 10 tumors per group. (C) Immunohistochemical staining for FoxF1, Ki-67, phosphorylated histone 3 (PH3), and cleaved caspase-3 in control and FoxF1-OE Myc-CaP and TRAMP tumors harvested at 4 weeks after orthotopic inoculation of cells. Data are means ± SEM from 10 random microscope fields in tumors from 8 to 10 mice per group. Images are magnified at ×200; scale bar, 50 μm. *P < 0.05, unpaired Student’s t test.

  • Fig. 3 FoxF1 induces prostate cancer invasion and metastasis.

    (A) H&E staining of prostates bearing control or Foxf1-overexpressing (FoxF1-OE) Myc-CaP tumor cells. Images are magnified at ×100; scale bar, 100 μm. (B) Peritoneal metastatic lesions per mouse bearing control or Foxf1-overexpressing Myc-CaP tumors. Data are means ± SEM from 10 mice per group. (C) qRT-PCR assessing the expression of mRNAs of invasion-associated genes (Mmp2, Mmp9, Mmp10, Mmp13, Etv1, S100A8, and Stmn1) in control and FoxF1-overexpressing Myc-CaP tumors. mRNA abundance was normalized to that encoding β-actin. Data are means ± SEM from 10 mice per group. (D and E) As in (B) and (C), respectively, for control or FoxF1-overexpressing TRAMP tumors. *P < 0.05, unpaired Student’s t test.

  • Fig. 4 FoxF1 changes gene expression in prostate tumors.

    (A) RNA sequencing analysis for differentially expressed genes in control and FoxF1-OE TRAMP tumors. Heat map represents fold changes between control TRAMP and FoxF1-OE TRAMP tumors. mRNAs were pooled from five tumors per genotype. (B) Biological processes regulated by FoxF1 in tumor cells identified using ToppGene Suite (https://toppgene.cchmc.org/). Statistical significance of each functional category was determined using negative log2 transformation of P value. (C) mRNA fold changes and P values from RNA sequencing mapped in (A). (D) qRT-PCR validation of several genes found to be differentially expressed by RNA sequencing analysis. Expression was normalized to that of mRNA encoding β-actin. Data are means ± SEM of 10 tumors per group. *P < 0.05, unpaired Student’s t test.

  • Fig. 5 FoxF1 induces the ERK5-MAPK pathway through transcriptional activation of the Wnk1 and Map3k2 promoters.

    (A) qRT-PCR for Wnk1 and Map3k2 expression in Myc-CaP tumors, normalized against the expression of mRNA encoding β-actin. (B) Western blot for total and phosphorylated ERK5 (pERK5) in control (Con) and FoxF1-OE Myc-CaP tumors. Blots are representative of six mice per group. (C) Immunohistochemistry for MAP3K2 and phosphorylated ERK5 in control and FoxF1-OE Myc-CaP tumors. Images are representative of 10 mice per group. Images are magnified at ×200 (×400, insets); scale bars, 50 μm. (D and E) A schematic of potential FoxF1-binding sites (white boxes) in the mouse Map3k2 promoter (D) and Wnk1 promoter (E). (F and G) ChIP assays in Myc-CaP cells assessing direct binding of FOXF1 to the indicated regions in the Map3k2 (F) or Wnk1 (G) promoters relative to control immunoglobulin (Ig). (H and I) Relative luciferase signal in HEK293T cells transfected with plasmids encoding either an empty vector (CMV-Empty) or FOXF1 (CMV-Foxf1) and the luciferase reporter driven by either the −5.7-kb Map3k2 promoter region (H) or the −4.5-kb Wnk1 promoter region (I). Data in (F) to (I) are means ± SEM from triplicate experiments. *P < 0.05, Student’s t test.

  • Fig. 6 Inactivation of ERK5 reduces tumor growth and metastasis in FOXF1-positive prostate tumors.

    (A) Effect of shRNA-mediated stable knockdown of ERK5 on the growth and metastasis of orthotopically implanted FoxF1-positive Myc-CaP cells (FoxF1-OE + shErk5). Scramble, control shRNA. Tumors are representative of five mice per group. (B) Western blot for total and phosphorylated ERK5 abundance in orthotopic Myc-CaP prostate tumors 4 weeks after cells were inoculated. (C) Mass of Myc-CaP tumors, represented in (A), measured 4 weeks after cells were inoculated. (D) Number of peritoneal metastases in mice bearing tumors represented in (A). (E) Tumor cell proliferation assessed by Ki-67 staining in the indicated tumor tissues. Images are representative of five mice per group. Images are magnified at ×200; scale bars, 50 μm. (F to H) qRT-PCR for mRNAs associated with proliferation (F), EMT (G), and invasion (H) in the indicated tumor tissues. Expression was normalized to that of mRNA encoding β-actin. Data are means ± SEM of four tumors per group. *P < 0.05, Student’s t test.

  • Fig. 7 MAP3K2 and WNK1 promote growth of FoxF1-OE mouse prostate tumors, and their expression positively correlates with that of FOXF1 in human prostate tumors.

    (A and B) Growth (A) and number of peritoneal metastases (B) of orthotopic FoxF1-overexpressing Myc-CaP tumors stably transduced with shMAP3K2, shWNK1, or both. (C and D) Representative Western blots (C) and densitometry analysis (D) in FoxF1-OE Myc-CaP orthotopic tumors transduced as indicated. Data are means ± SEM from four mice per group. *P < 0.05, t test. (E) Gene expression data from 58 human patient samples were obtained from GEO data set GSE2109. Gene expression was normalized to that of internal housekeeping genes. (F and G) Pearson’s correlation analysis of FOXF1 expression against that of WNK1 (F) and MAP3K2 (G). *P < 0.05, Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/427/ra48/DC1

    Fig. S1. Overexpression of Foxf1 increases cellular proliferation, invasion, and migration of PCa tumor cells in culture.

    Fig. S2. Overexpression of Foxf1 in orthotopic xenografts induces EMT.

    Fig. S3. Overexpression of Foxf1 induces activation of the MAPK pathway in TRAMP orthotopic tumors.

    Fig. S4. Depletion of FOXF1 reduces the growth of human PCa xenografts.

    Fig. S5. Pharmacological inhibition of ERK5 in FoxF1-OE PCa cells decreases tumor cell proliferation, invasion, and migration in culture.

    Fig. S6. FOXF1 expression is increased in a subset of human prostate cancers.

    Table S1. TaqMan probes.

  • Supplementary Materials for:

    The transcription factor FOXF1 promotes prostate cancer by stimulating the mitogen-activated protein kinase ERK5

    Logan Fulford, David Milewski, Vladimir Ustiyan, Navin Ravishankar, Yuqi Cai, Tien Le, Sreeharsha Masineni, Susan Kasper, Bruce Aronow, Vladimir V. Kalinichenko, Tanya V. Kalin*

    *Corresponding author. Email: tatiana.kalin{at}cchmc.org

    This PDF file includes:

    • Fig. S1. Overexpression of Foxf1 increases cellular proliferation, invasion, and migration of PCa tumor cells in culture.
    • Fig. S2. Overexpression of Foxf1 in orthotopic xenografts induces EMT.
    • Fig. S3. Overexpression of Foxf1 induces activation of the MAPK pathway in TRAMP orthotopic tumors.
    • Fig. S4. Depletion of FOXF1 reduces the growth of human PCa xenografts.
    • Fig. S5. Pharmacological inhibition of ERK5 in FoxF1-OE PCa cells decreases tumor cell proliferation, invasion, and migration in culture.
    • Fig. S6. FOXF1 expression is increased in a subset of human prostate cancers.
    • Table S1. TaqMan probes.

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    Citation: L. Fulford, D. Milewski, V. Ustiyan, N. Ravishankar, Y. Cai, T. Le, S. Masineni, S. Kasper, B. Aronow, V. V. Kalinichenko, T. V. Kalin, The transcription factor FOXF1 promotes prostate cancer by stimulating the mitogen-activated protein kinase ERK5. Sci. Signal. 9, ra48 (2016).

    © 2016 American Association for the Advancement of Science

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