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. 2021 Feb 19;12(2):199.
doi: 10.1038/s41419-021-03487-0.

Positive feedback of SuFu negating protein 1 on Hedgehog signaling promotes colorectal tumor growth

Zhengwei Yan #  1 , Minzhang Cheng #  1 , Guohui Hu #  1 , Yao Wang  1   2 , Shaopeng Zeng  1 , Aidi Huang  1 , Linlin Xu  1 , Yuan Liu  3 , Chao Shi  1 , Libin Deng  4 , Quqin Lu  5 , Hai Rao  6 , Hua Lu  7   8 , Ye-Guang Chen  9 , Shiwen Luo  10
Affiliations

Positive feedback of SuFu negating protein 1 on Hedgehog signaling promotes colorectal tumor growth

Zhengwei Yan et al. Cell Death Dis. .

Abstract

Hedgehog (Hh) signaling plays a critical role in embryogenesis and tissue homeostasis, and its deregulation has been associated with tumor growth. The tumor suppressor SuFu inhibits Hh signaling by preventing the nuclear translocation of Gli and suppressing cell proliferation. Regulation of SuFu activity and stability is key to controlling Hh signaling. Here, we unveil SuFu Negating Protein 1 (SNEP1) as a novel Hh target, that enhances the ubiquitination and proteasomal degradation of SuFu and thus promotes Hh signaling. We further show that the E3 ubiquitin ligase LNX1 plays a critical role in the SNEP1-mediated degradation of SuFu. Accordingly, SNEP1 promotes colorectal cancer (CRC) cell proliferation and tumor growth. High levels of SNEP1 are detected in CRC tissues and are well correlated with poor prognosis in CRC patients. Moreover, SNEP1 overexpression reduces sensitivity to anti-Hh inhibitor in CRC cells. Altogether, our findings demonstrate that SNEP1 acts as a novel feedback regulator of Hh signaling by destabilizing SuFu and promoting tumor growth and anti-Hh resistance.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. SNEP1 is a downstream target gene of the Gli transcriptional factor.
A, B Screening for novel downstream target genes of Hh signaling. Venn diagram (A) and heatmap (B) of differentially expressed genes (DEGs) (fold change ≥2 or ≤ 0.05, adjusted p < 0.05) in HT-29 cells treated with GANT61 or expressing Gli2 and cluster analysis of these genes with Gene Ontology (GO) annotation. C Gli2 expression affects SNEP1 mRNA levels. HT-29 cells were transfected with Myc-vector, Myc-Gli2, or Myc-Gli2A for 48 h and harvested for qPCR analysis of SNEP1 mRNA. Bcl2, Ptch1, Sox2 and Gli1, well-known Hh signaling target genes, were used as positive controls. D Gli2 expression affects SNEP1 protein levels. HT-29 cells were transfected with Gli2 constructs for 48 h and harvested for IB with the indicated antibodies. E Gli2 knockdown reduces SNEP1 protein levels. HT-29 cells were transfected with shRNAi-Gli2 plasmids for 72 h and harvested for IB analysis with the indicated antibodies. F Pharmacological repression of Gli2 reduces SNEP1 mRNA levels. HT-29 cells were treated with GANT61 for 48 h and harvested for RNA extraction and qPCR. G Chromatin immunoprecipitation (ChIP) assays for Gli2 at the SNEP1 promoter. Upper: schematic representation of the SNEP1 promoter region showing the putative transcription factor-binding sites. Lower: HT-29 cells were harvested for ChIP assays as described in the Materials and Methods with IgG or anti-Gli2 antibodies for IP followed by qPCR with specific primers for each putative-binding element as indicated. H Luciferase assays for Gli2 transcriptional activity at the SNEP1 promoter. A series of SNEP1-luciferase constructs (left) were transfected into HEK-293T cells, and relative Gli2 transcript levels were measured 48 h after transfection (right). Each experiment was performed in triplicate. *p < 0.05, **p < 0.01.
Fig. 2
Fig. 2. SNEP1 promotes CRC cell proliferation and tumor growth.
A SNEP1 promotes the colony formation of CRC cells. HCT-116 or Caco2 cells stably expressing LV-SNEP1 were seeded in a 6-well plate for 14 days. The cells were then stained with 0.5% crystal violet (w/v). B Quantitative analysis of HCT-116 and Caco2 cells stably overexpressing SNEP1. The bar graph displays the means ± SD, n = 3, **p < 0.01. C SNEP1 knockdown inhibits colony formation of CRC cells. HT-29 or SW620 cells stably expressing LV-shSNEP1 were seeded in a 6-well plate for 14 days. The cells were then stained with 0.5% crystal violet (w/v). D Quantitative analysis of HT-29 and SW620 cell lines with stable SNEP1 knockdown was performed using ImageJ software. The bar graph displays the mean ± SD, n = 3, **p < 0.01. E, F SNEP1 overexpression promotes tumor growth ex vivo. HCT-116 stable cell lines (2 × 107 cells) that overexpressed SNEP1 were subcutaneously injected into eight nude mice on each side of the inguinal region. Xenografts were harvested after 2 weeks. Tumor sizes on either side were monitored every other day and tumor weights are shown in F. Data are presented as mean ± SD (n = 8). G, H SNEP1 knockdown leads to suppression of tumor growth ex vivo. HT-29 stable cell lines (2 × 107 cells) with LV-shSNEP1 were subcutaneously injected into eight nude mice in each side of the inguinal region. Xenografts were harvested after 2 weeks. Tumor sizes on either side were monitored every alternate day and the tumor weights were shown in H. Data are presented as mean ± SD (n = 8). I Schematic showing mouse treatment for the inflammation-dependent CRC model. J–L SNEP1 overexpression promotes tumor growth in an inflammation-dependent CRC model. Tumor sizes and weights are shown in K and L) Data are presented as the mean ± SD.
Fig. 3
Fig. 3. SNEP1-induced activation of Hh signaling and proliferation of CRC cell is mediated by SuFu degradation.
A SNEP1 activates Hh signaling. HCT-116 cells with ectopic SNEP1 expression were harvested for qPCR. Data are presented as mean ± SD (n = 3). **p < 0.01. B SNEP1 facilitates CRC cell proliferation dependent on activation of the Hh Pathway. Caco2 cells were infected with LV-SNEP1 or control lentivirus, and treated with 2.5 μM GANT61. The cells were then stained with 0.5% crystal violet (w/v). The bar graph displays the mean ± SD, n = 3, **p < 0.01, N.S., not significance. C Exogenous expression of SNEP1 reduces SuFu protein levels. Protein levels of Gli2 and SuFu were detected via IB analysis of lysates isolated from lentivirus-transduced cell lines stably overexpressing SNEP1. D SNEP1 knockdown enhances SuFu protein levels. Protein levels of Gli2 and SuFu were measured by IB analysis of lysates isolated from lentivirus-transduced cells with stable SNEP1 knockdown. E MG132 rescues the reduction of SuFu protein levels by SNEP1. HEK-293T cells were transfected with a gradient Flag-SNEP1 construct for 48 h and were pretreated with the proteasome inhibitor MG132 overnight before harvesting. Lysates were examined via IB with the indicated antibodies. F SuFu degradation was attenuated by SNEP1 depletion. Cycloheximide (CHX) (100 μg/ml) was incubated for the indicated period with HEK-293T cells transfected with shRNA-SNEP1. Cell lysates were harvested for IB with anti-SuFu antibody. G Quantitative analysis of SuFu protein levels shown in F using ImageJ software. H Overexpression of SuFu decreased SNEP1-induced activation of Hh signaling. HCT-116 cells were infected with LV-SNEP1 or control lentivirus, and transfected with vector or SuFu plasmids for 48 h. qPCR were performed. Data are presented as mean ± SD (n = 3). **p < 0.01. I Endogenous complexes of SNEP1 with SuFu. Cell lysates of HT-29 cells were subjected to IP analysis with the indicated antibodies and protein-A/G beads overnight. Bound proteins were analyzed via IB. J Ectopically expressed SNEP1 interacts with SuFu. HEK-293T cells transfected with GFP-SNEP1 and Flag-SuFu plasmids were subjected to a Co-IP assay. K GST pull-down assay of SNEP1 and SuFu. The prokaryotically expressed GST-SNEP1 was incubated with cell lysates of GFP-SuFu-transfected HEK-293T cells. The protein complex pulled down by GST was detected via IB.
Fig. 4
Fig. 4. SNEP1 enhances LNX1-SuFu interactions and facilitates LNX1-mediated SuFu proteolysis.
A An IP assay of SNEP1 with LNX1. After transfection with the indicated plasmids, HEK-293T cells were harvested for a Co-IP assay with an antibody recognizing Flag-tag, and binding proteins were detected via IB. B An IP assay of SNEP1 with LNX1. HT-29 cell lysates were subjected to a Co-IP assay with the indicated antibodies and protein-A/G beads overnight. Bound proteins were detected via IB. C GST pull-down assay of SNEP1 with LNX1. The prokaryotically expressed GST-SNEP1 was incubated with HEK-293T cell lysates overexpressing Flag-LNX1. The protein complexes pulled down with GST were detected via IB. D LNX1 knockdown increases SuFu protein levels. HEK-293T cells were transfected with different doses of sh-LNX1 construct for 48 h and harvested for IB analysis. E SNEP1-mediated SuFu degradation depends on LNX1. IB analysis of HEK-293T cell lysates that expressed GFP-SNEP1 with LNX1 knockdown or not. F Ectopic SNEP1 increases SuFu-LNX1 interactions. HEK-293T cells were cotransfected with HA-SuFu and Flag-LNX1 with or without GFP-SNEP1 plasmids and treated with MG132 overnight before harvesting. Cell lysates were subjected to a co-IP-IB assay. G SNEP1 is required for the interaction between SuFu and LNX1. HT-29 cells stably expressing LV-shSNEP1 were treated with MG132 overnight before harvest. Cell lysates were subjected to a co-IP-IB assay. H SNEP1 knockdown reverses the repression of the action of LNX1 on SuFu. IB analysis of HEK-293T cell lysates expressing Flag-LNX1 with or without shRNA-SNEP1. I SuFu knockdown promotes translocation of Gli2 into the nucleus. HCT-116 cells were transfected with Flag-SNEP1, Flag-LNX1 or shRNA-SuFu for 48 h and subjected to IB for detection of cytoplasmic and nuclear fractions of Gli2. GAPDH and PARP-1 were used as cytoplasmic and nuclear loading controls, respectively. J Quantification of I using ImageJ software. Data are shown as the mean ± SD, n = 3, **p < 0.01, ***p < 0.001.
Fig. 5
Fig. 5. SNEP1 facilitates LNX1-mediated ubiquitination of SuFu.
A SNEP1 knockdown impairs LNX1-mediated ubiquitination of SuFu. HEK-293T cells were transfected with Flag-LNX1, shRNA control, or shSNEP1 for 48 h and treated with MG132 overnight before harvest. Cell lysates were subjected to IP with anti-SuFu antibody and protein A/G beads overnight. Polyubiquitin chains bound to SuFu were assessed via IB. B SNEP1 increases LNX1-mediated ubiquitination of SuFu. HEK-293T cells were transfected with Flag-LNX1, GFP-vector, or GFP-SNEP1 for 48 h and treated with MG132 overnight before harvest. Cell lysates were subjected to IP with anti-SuFu antibody and protein A/G beads overnight. Polyubiquitin chains bound to SuFu were assessed via IB. C SNEP1 enhances LNX1-mediated SuFu ubiquitination in vitro. GST-SuFu, GST-LNX1 (1-600), and SNEP1 were expressed in and purified from E. coli. Purified proteins were mixed with E1, E2, and ubiquitin purchased from Enzo and incubated at 37 °C for 4 h. Polyubiquitin chains were assessed via IB. D The SNEP1-LNX1 complex targets K59 and K470 for SuFu ubiquitination. HEK-293T cells were transfected with GFP-SuFu, GFP-SuFu (K59R), GFP-SuFu (K470R), or GFP-SuFu (K59/470R) and Flag-LNX1 and treated with MG132 overnight before harvest. Cell lysates were subjected to IP with an anti-SuFu antibody and protein A/G beads overnight. Polyubiquitin chains bound to SuFu were assessed via IB. E SuFu (K59/470R) is resistant to degradation mediated by SNEP1. HEK-293T cells were transfected with GFP-SuFu or GFP-SuFu (K59/470R) and Flag-vector or SNEP1 for 48 h before harvest. Cell lysates were assessed via IB. F SuFu (K59/470R) displays a prolonged half-life in cells. CHX (100 μg/ml) was incubated for different time points with GFP-SuFu or GFP-SuFu (K59/470R) and Flag-SNEP1-transfected HEK-293T cells. Cell lysates were harvested for IB with the indicated antibodies. G Quantitative analysis of SuFu protein levels shown in F using ImageJ software. H SuFu (K59/470R) is inactivated in response to LNX1-promoted cell proliferation. HT-29 cells transfected with GFP-SuFu or GFP-SuFu (K59/470R) were treated with EdU for 4 h before fixation with paraformaldehyde. Cells were then stained with rhodamine-tagged anti-EdU antibody and DAPI. I Quantitative analysis of the ratio of EdU+ cells shown in H, **p < 0.01.
Fig. 6
Fig. 6. SNEP1 expression is elevated, but SuFu is decreased in primary CRC tissues.
A Representative images of IHC staining of both human CRC tissues and adjacent tissues in the same section stained for SNEP1 and SuFu. B, C SNEP1 (B) and SuFu (C) expression was plotted per the IHC scores in each carcinoma and adjacent tissue. D–E Kaplan–Meier estimates of the overall survival of CRC patients between the negative/low and medium/high expression groups for SNEP1 (D) and SuFu (E).
Fig. 7
Fig. 7. SNEP1 induces vismodegib-resistance in colorectal cancer cells.
A–D HCT-116 or Caco2 cells stably expressing LV-SNEP1 were seeded in a 6-well plate for 14 days, and treated with 2.5 μM vismodegib. The cells were then stained with 0.5% crystal violet (w/v) (A and C). Quantitative analysis was performed using Image J software (B and D). The bar graph displays the mean ± SD, n = 3, **p < 0.01, N.S., not significance. E, F Relative cell viability and IC50 of vismodegib in control or SNEP1-overexpressing HCT-116 cells (E), Caco2 cells (F). Data represent mean ± SD of three separate experiments. G Schematic showing how SNEP1 and LNX1 work together to degrade SuFu via a ubiquitin-dependent mechanism to promote CRC development by activating the Hh pathway.
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