Enhancement of TGF-β signaling responses by the E3 ubiquitin ligase Arkadia provides tumor suppression in colorectal cancer

Abstract

TGF-β signaling provides tumor protection against colorectal cancer (CRC). Mechanisms that support its tumor-suppressive properties remain unclear. The ubiquitin ligase Arkadia/RNF111 enhances TGF-β signaling responses by targeting repressors of the pathway for degradation. The corepressors SnoN/Ski, critical substrates of Arkadia, complex with the activated TGF-β signaling effectors Smad2/3 (pSmad2/3) on the promoters of target genes and block their transcription. Arkadia degrades this complex including pSmad2/3 and unblocks the promoter. Here, we report that Arkadia is expressed highly in the mouse colonic epithelium. Heterozygous Akd(+/-) mice are normal but express less Arkadia. This leads to reduced expression of several TGF-β target genes, suggesting that normal levels of Arkadia are required for efficient signaling responses. Critically, Akd(+/-) mice exhibit increased susceptibility to azoxymethane/dextran sodium sulfate carcinogen-induced CRC, as they develop four-fold more tumors than wild-type mice. Akd(+/-) tumors also exhibit a more aggressive pathology, higher proliferation index, and reduced cytostasis. Therefore, Arkadia functions as a tumor suppressor whose peak expression is required to suppress CRC development and progression. The accumulation of nuclear SnoN and pSmad2, along with the downregulation of TGF-β target genes observed in Akd(+/-) colon and tumors, suggest that tumor-suppressing properties of Arkadia are mediated by its ability to derepress TGF-β signaling. Consistent with this likelihood, we identified mutations in primary colorectal tumors from human patients that reduce Arkadia function and are associated with the accumulation of nuclear SNON. Collectively, our findings reveal that Arkadia enhances TGF-β signaling responses and supports its tumor-suppressing properties in CRC.

Figure 1. Deep sequencing of AKD C-terminus from CRC patients

(A) Scatter plot correlating the relative level of SNON expression (qPCR) and nuclear SNON staining (IHC) from human CRC samples. Patients with high SNON stability highlighted in black were selected for deep sequencing. (B) Protein alignment of the C-terminus of AKD from human (Q6ZNA4), mouse (Q99ML9), chick (Q90ZT8), Xenopus (Q0V9R0), Drosophila (Q9VGI6) and C.elegans (Q9XUM8). The grey lines indicate the NRG (NRGASQG), TIER (TIERCTY) and NLS (PHKYKKV) domains and the black line highlights the RING domain. Numbers indicate four key mutations identified. (C) Luciferase CAGA₁₂ reporter assay values from three biological repeat experiments, each in quadruplicate. The Q899STOP mutation exhibits a dominant-negative effect, similar to the W972R control. (D) Immunoblot shows relative stability of GFP-AKD proteins (top bands) compared to GFP control (bottom bands). GFP-Q899STOP is stable at approximately 140kDa. H3 was used as a loading control. 40μg of protein extract was loaded in each lane.

Figure 2. Expression of Arkadia in the mouse colon

(A) wt and (B) Akd^+/− colon sections stained with X-gal. (C) anti-Arkadia IHC in wt colon. (D) qPCR expression of Akd and (E) six TGF-β target genes in wt and Akd^+/− colon tissue. (F) Immunoblot with pSmad2 antibody and H3 loading control. Expression of SnoN in (G) wt and (H) Akd^+/− colon tissue. Elevated SnoN staining is observed in Akd^+/− colons, particularly in the differentiated epithelial cells at the surface of the crypts. All images are shown at x40 magnification; bar 25μm.

Figure 3. AOM/DSS induction of CRC in wt and Akd^+/− mice

(A) Macroscopic view of representative X-gal stained mouse colons (129SVcc background) at end of protocol (distal end at top; bar 1cm). (B) Percentage of mice that developed tumors. (C) Mean number of tumors observed from wt (n=12) and Akd^+/− (n=7) mice in a 129SVcc background and wt (n=6) and Akd^+/− (n=6) mice in a 129SVcc/CD1 background. (D-I) H&E staining of colons and tumors from wt and Akd^+/− treated mice. wt: (D) normal colon, (E) adenoma with severe dysplasia and (F) intramucosal carcinoma. Akd^+/−: (G) intramucosal carcinoma with mucus production, (H) invasive adenocarcinoma and (I) vessel invasion. Black arrowheads mark sites of invasion. (D-H) shown at x2.5 magnification; bar 250μm and (I) at x20 magnification; bar 50μm.

Figure 4. Immunoblot analysis of wt and Akd^+/− tumors

(A) Immunoblotting with SnoN, pSmad2, active beta-catenin (β-catⁿ) and PCNA. Lanes N, treated adjacent normal colon tissue; lanes T, representative wt (T¹-T³) and Akd^+/− (T^A-T^C) tumor samples. H3 was used as a loading control. Protein levels normalized to H3 are shown in the three charts. (B) Analysis of p21^WAF, SnoN and pSmad2 protein levels in six wt (T¹-T⁶) and six Akd^+/− (T^A-T^F) tumors. 10μg of protein extract was loaded in each lane for all gels. Due to the enhanced stability of pSmad2 in Akd^+/− tumors, we also observed more stable levels of total Smad2. (C) qPCR expression levels of various genes (as indicated) in the above analyzed tumors. The difference is PAI-1 expression was calculated as P=0.0532. Average fold changes are shown in the charts on the right.

Figure 5. IHC for SnoN, pSmad2 and Arkadia in wt and Akd^+/− tumors

Representative sections of adenocarcinomas stained with anti-pSmad2: (A) wt and (B) Akd^+/−; anti-SnoN: (C) wt and (D) Akd^+/−; and anti-Arkadia: (E) wt and (F) Akd^+/−. All images are shown at x40 magnification; bar 25μm.