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Comparative Study
. 2011 Aug 1;71(11):1198-209.
doi: 10.1002/pros.21335. Epub 2011 Jan 12.

Reactivation of embryonic nodal signaling is associated with tumor progression and promotes the growth of prostate cancer cells

Mitchell G Lawrence  1 , Naira V Margaryan, Daniela Loessner, Angus Collins, Kris M Kerr, Megan Turner, Elisabeth A Seftor, Carson R Stephens, John Lai; APC BioResource; Lynne-Marie Postovit, Judith A Clements, Mary J C Hendrix
Affiliations
Comparative Study

Reactivation of embryonic nodal signaling is associated with tumor progression and promotes the growth of prostate cancer cells

Mitchell G Lawrence et al. Prostate. .

Abstract

Background: Nodal is a member of the transforming growth factor β (TGFβ) superfamily that directs embryonic patterning and promotes the plasticity and tumorigenicity of tumor cells, but its role in the prostate is unknown. The goal of this study was to characterize the expression and function of Nodal in prostate cancer and determine whether, like other TGFβ ligands, it modulates androgen receptor (AR) activity.

Methods: Nodal expression was investigated using immunohistochemistry of tissue microarrays and Western blots of prostate cell lines. The functional role of Nodal was examined using Matrigel and soft agar growth assays. Cross-talk between Nodal and AR signaling was assessed with luciferase reporter assays and expression of endogenous androgen regulated genes.

Results: Significantly increased Nodal expression was observed in cancer compared with benign prostate specimens. Nodal was only expressed by DU145 and PC3 cells. All cell lines expressed Nodal's co-receptor, Cripto-1, but lacked Lefty, a critical negative regulator of Nodal signaling. Recombinant human Nodal triggered downstream Smad2 phosphorylation in DU145 and LNCaP cells, and stable transfection of pre-pro-Nodal enhanced the growth of LNCaP cells in Matrigel and soft agar. Finally, Nodal attenuated AR signaling, reducing the activity of a PSA promoter construct in luciferase assays and down-regulating the endogenous expression of androgen regulated genes.

Conclusions: An aberrant Nodal signaling pathway is re-expressed and functionally active in prostate cancer cells.

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Figures

Figure 1
Figure 1. Nodal Expression in Prostate Tissues
(A) Representative images of Nodal immunohistochemistry (brown color) showing stronger staining in regions of Gleason pattern 3 to 5 cancer compared to normal glands. No immunoreactivity was observed with the isotype control antibody (Iso Ctl; Grade 5 inset). Scale bars, 50 μm. (B) Benign glands with p63- and high molecular weight cytokeratin-positive (brown staining) basal cells adjacent to AMACR-positive (red staining) malignant glands. (C) Stronger Nodal staining was apparent in malignant cells compared with benign glands. For B and C, scale bars for low magnification images on the left are 500 μm, and scale bars for high magnification images on the right are 50 μm. These are serial sections. (D) A graph comparing Nodal immunoreactivity, scored as no staining (0), weak (1), moderate (2) or strong (3), in cores of benign prostate and Gleason score 3+3, 3+4, 4+3 and ≥ 4+4 cancer. There was a significant correlation between Nodal staining and cancer versus benign cores (χ2 test, P=0.4×10−21) as well as between high grade (Gleason scores 7 and higher) compared with low grade (Gleason score 6) prostate cancer (χ2 test, P=0.9×10−4).
Figure 2
Figure 2. Nodal Expression in Prostate Cancer Cell Lines
(A) Real-time PCR of Nodal expression in prostate cancer cell lines normalized to H9 hESCs (n=3). (B) Western blots of lysates from prostate cancer cell lines immunoprecipitated using rabbit or goat anti-Nodal antibodies. LNCaP cells stably transfected with Nodal were used as a positive control for the Western blot (Nodal Input) and IP (Nodal IP). All blots were probed with a mouse anti-Nodal antibody. Nodal was detected as a single ~42 kDa band. The unbound fractions of IP samples were probed for GAPDH to compare initial protein concentrations. (C) RT-PCR showing that Cripto-1 was expressed in all prostate cell lines. H9 hESC was used as a positive control and GAPDH as the house-keeping gene. (D) Western blots of prostate cancer and MEL1 hESC lysates where Cripto-1 was detected as two bands of approximately 17 and 25 kDa. GAPDH was used as a loading control. (E) RT-PCR showing negligible Lefty and Cerberus expression in prostate cancer cells compared to H9 hESCs. Low levels of TMEFF1 were expressed in 22Rv1 and DU145 cells. LN = LNCaP, 22 = 22Rv1, DU = DU145, MD = MDAPCa2b, SC = H9 or MEL1 hESCs, RTase = reverse transcriptase, and NTC = no template control.
Figure 3
Figure 3. Nodal Signaling in Prostate Cancer Cell Lines
(A) DU145 cells were treated for 5–60 mins with 1 μg/mL recombinant Nodal or 30 min with 1 ng/mL TGFβ1. Western blots of pSmad2 and total Smad2 levels were quantified using densitometry. Nodal significantly increased Smad2 phosphorylation at 60 mins (One Way Anova with Tukey’s posthoc analysis, n=2, *P<0.05). (B) DU145 cells were treated for 60 min with 10–1000 ng/mL Nodal and 10 μM SB431542 (+) or DMSO control (−). LNCaP cells were treated with 500 ng/mL Nodal for 6 hrs with fresh Nodal added every 2 hrs. There was a significant increase in pSmad2 with 1 μg/mL Nodal in DU145 cells and significant decrease when SB431542 was added (One Way Anova with Tukey’s posthoc analysis, n=3, ***P<0.001). There was also a significant increase in Smad2 phosphorylation in LNCaP cells treated with Nodal (t test, n=3, **P<0.01).
Figure 4
Figure 4. Nodal Increases the Growth of Prostate Cancer Cells
(A) A Western blot with a FLAG antibody confirming exogenous Nodal expression in LNCaP-Nodal, but not in LNCaP-Vector cells. The membrane was reprobed for GAPDH to verify equal loading. (B) The growth of LNCaP-Vector and LNCaP-Nodal cells in Matrigel constructs measured by DNA content after 1–12 days culture. Data are normalized to day 1. By day 12, there was a significant increase in the number of LNCaP-Nodal cells compared to the vector control (Two Way Anova with Tukey’s posthoc analysis, n=3, *P<0.05). (C) Representative phase contrast images showing that LNCaP-Nodal cells formed larger clusters than LNCaP-Vector cells embedded within Matrigel constructs. Scale bars, 250 μm. (D) In soft agar assays, a significantly higher proportion of LNCaP-Nodal cells formed colonies compared with LNCaP-Vector cells (t test, n=3, *P<0.05). (E) Representative phase contrast images confirming that LNCaP-Nodal cells formed more prominent colonies. Scale bars, equal 500 μm.
Figure 5
Figure 5. Nodal Antagonizes Androgen Receptor Signaling
(A) LNCaP cells transfected with 10–250 ng of Nodal and treated with 1 nM R1881. PSA luciferase activity relative to Renilla is significantly reduced by 250 ng of Nodal (Two way Anova with Tukey’s posthoc analysis, n=3, *P<0.05). (B) Real-time PCR of PSA, KLK2, TMPRSS2, and NKX3.1 which were all significantly down-regulated in LNCaP cells stably transfected with Nodal compared with the vector control (t test, n=3, **P<0.01, ***P<0.001).
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