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. 2024 Aug 20;10(1):369.
doi: 10.1038/s41420-024-02147-4.

Linear polyubiquitylation of Gli protein regulates its protein stability and facilitates tumor growth in colorectal cancer

Junyao Cheng #  1   2 , Linlin Xu #  1   3 , Yanlu Xuan #  1   2 , Feifei Zhou #  1   4 , Aidi Huang  1   3 , Shaopeng Zeng  1 , Hailong Wang  1   5 , Yiting Wang  6 , Yuan Zhan  3 , Xiaohua Yan  7 , Shiwen Luo  1   3 , Yuan Liu  8 , Minzhang Cheng  9   10
Affiliations

Linear polyubiquitylation of Gli protein regulates its protein stability and facilitates tumor growth in colorectal cancer

Junyao Cheng et al. Cell Death Discov. .

Abstract

The linear ubiquitin chain assembly complex (LUBAC) mediates the linear ubiquitination of various proteins and is involved in NF-κB signaling and immune regulation. However, the function and mechanism of linear ubiquitination in regulating oncogenic signaling and tumor growth have remained poorly understood. Herein, we identified Gli proteins, key transcription factors in the Hedgehog (Hh) signaling pathway, as novel substrates of LUBAC. Linear ubiquitination stabilizes Gli proteins, leading to the noncanonical activation of Hh signaling in CRC cells. Furthermore, LUBAC facilitates tumor growth in CRC cells. Additionally, elevated expression of LUBAC components in CRC tissues was observed, and higher expression levels of these components correlated with poor prognosis in CRC patients. Interestingly, inhibition of LUBAC using either a small molecule agonist or RNA silencing specifically suppressed cell growth in CRC cells but had no effect on normal intestinal cells. Taken together, aberrant expression of LUBAC components activates Hh signaling noncanonically by mediating linear ubiquitination, promoting tumor growth in CRC, demonstrating the novel function of linear ubiquitination in regulating the protein stability of its substrates and highlighting the potential of targeting LUBAC as a therapeutic strategy in CRC.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. HOIP is highly expressed in CRC tissues.
A Representative images of IHC staining of both human CRC tissues and adjacent tissues in the same section stained for HOIP. B HOIP expression was plotted per the IHC scores in each carcinoma and adjacent tissue. C Immunoblotting of 10 pairs of randomly chosen CRC samples. D Quantification of the protein level of HOIP in (C). Kaplan‒Meier estimates of the overall survival (E) and disease-free survival (F) of CRC patients between the low and high expression groups for HOIP.
Fig. 2
Fig. 2. HOIP facilitates CRC cell growth.
A HOIP promotes the colony formation of CRC cells. HCT-116 or SW480 cells stably expressing LV-HOIP were seeded in a 6-well plate for 14 days. B Quantitative analysis of HCT-116 and SW480 cells stably overexpressing HOIP. Data are shown as the means ± SDs, n = 3. C HOIP promotes the sphere formation of CRC cells. HCT-116 or SW480 cells stably expressing LV-HOIP were mixed with soft agar (0.3%) and seeded in a 12-well plate for 14 days. D Quantitative analysis of HCT-116 and SW480 cells stably overexpressing HOIP. Data are shown as the means ± SDs. Growth curve of CRC cells. HCT-116 (E) or SW480 (F) cells stably expressing LV-HOIP were seeded in a 96-well plate, and the cells were counted by Cell Counting Kit (CCK)-8. Data are shown as the means ± SDs, n = 5. G–J HOIP overexpression promotes tumor growth ex vivo. HCT-116 stable cell lines (1 × 107 cells) that overexpressed HOIP 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 (H). Tumor size (I) and tumor weight (J) were determined. Data are shown as the means ± SDs, n = 8.
Fig. 3
Fig. 3. HOIP inhibition suppresses CRC cell growth.
A HOIPIN-1 inhibited the colony formation of CRC cells. HCT-116 or HT-29 cells were seeded in a 6-well plate and then treated with HOIPIN-1 at the indicated concentration for 14 days. B Quantitative analysis of HCT-116 and HT-29 cells treated with HOIPIN-1. Data are shown as the means ± SDs, n = 3. Growth curve of CRC cells. HCT-116 (C) or HT-29 (D) cells were seeded in a 96-well plate and then treated with or without HOIPIN-1 (5 μM). The cells were counted by CCK-8. Data are shown as the means ± SDs, n = 3. E HOIPIN-1 inhibited the colony formation of PDOs derived from CRC. PDOs treated with HOIPIN-1 at the indicated concentration were cultured for 11 days, and images were taken at the end point. F Viability of PDOs treated with HOIPIN-1 were measured by CCK-8. Data are shown as the means ± SDs, n = 3. G Expression of indicated genes in PDOs were determined by qPCR. Data are shown as the means ± SDs, n = 3. H HOIPIN-1 cooperates with cisplatin in suppressing cell growth. HCT-116 cells were seeded in a 96-well plate and then treated with HOIPIN-1 (10 μM) and cisplatin (1 μM) or not. The cells were counted by CCK-8. Data are shown as the means ± SDs, n = 5.
Fig. 4
Fig. 4. HOIP interacts with Gli proteins.
A Ectopically expressed HOIP interacts with Gli proteins. HEK-293T cells transfected with HA-HOIP and Flag-Gli2 or Flag-Gli3 plasmids were subjected to a co-IP assay. B Endogenous complexes of HOIP with Gli proteins. Cell lysates of HCT-116 or HT-29 cells were subjected to IP analysis with the indicated antibodies and protein A/G beads overnight. Bound proteins were analysed via IB. C Gli2 interacts with LUBAC components. HEK-293T cells transfected with HA-HOIP, Sharping or HOIL and Flag-Gli2 plasmids were subjected to a co-IP assay. D Schema of domains of HOIP. E HOIP binds to Gli2 through its PUB domain. HEK-293T cells transfected with Flag-Gli2 and HA-HOIP or its truncated mutation plasmids were subjected to a co-IP assay. F Hh signaling affects the interaction between HOIP and Gli2. HEK-293T cells transfected with Flag-Gli2 and HA-HOIP plasmids, followed by treatment with N-Shh for 30 min, were subjected to a co-IP assay.
Fig. 5
Fig. 5. HOIP mediates the linear polyubiquitylation of Gli proteins.
A HOIP activates Hh signaling. HCT-116 cells were harvest after transfected with 8×GBS-Luc, pRL-TK, and HA-HOIP for 48 hours, and the cell lysate were subjected to a dual-luciferase reporter assay according to the manual. The firefly luciferase activity was normalized to Renila luciferase activity. Data are shown as the means ± SDs, n = 3, and ANOVA with multiple comparisons was investigated. B HOIP elevates the protein level of exogenous Gli2. HEK-293T cells transfected with Flag-Gli2 and HA-HOIP (0, 0.15 μg, 0.3 μg and 0.6 μg per well in 12 well-plate) plasmids were subjected to IB with the indicated antibodies. C HOIP elevates the protein level of endogenous Gli2 and Gli3. HEK-293T cells transfected with HA-HOIP (0, 0.15 μg, 0.3 μg and 0.6 μg per well in 12 well-plate) plasmids were subjected to IB with the indicated antibodies. D HOIP impairs the degradation of Gli2. Cycloheximide (CHX) (100 μg/ml) was incubated for the indicated period with HEK-293T cells transfected with or without Flag-Gli2 and HA-HOIP. Cell lysates were harvested for IB with the indicated antibody. E LUBAC activates Hh signaling. HCT-116 cells transfected with 8×GBS-Luc, pRL-TK, HA-HOIP, Sharpin and HOIL were subjected to a dual-luciferase reporter assay. Data are shown as the means ± SDs, n = 3. F, G LUBAC stabilizes Gli2. HEK-293T cells transfected with Flag-Gli2 and HA-Sharpin or HOIL (0, 0.15 μg, 0.3 μg and 0.6 μg per well in 12 well-plate) plasmids were subjected to IB with the indicated antibodies.
Fig. 6
Fig. 6. HOIP mediates the linear polyubiquitylation of Gli proteins.
A HOIP mediates the polyubiquitylation of Gli proteins. HEK-293T cells transfected with Flag-Gli2 or Gli3 and HA-HOIP or its point mutation plasmids were subjected to a denatured co-IP assay. B HOIP mediates the linear ubiquitylation of Gli proteins. HEK-293T cells transfected with Flag-Gli2 or Gli3 and HA-HOIP or its point mutation plasmids were subjected to a denatured co-IP assay. Antibodies specifically recognizing M1-polyubiquitylation were used to identify linear ubiquitin chains. C E3 ligase activity of HOIP is essential for the stabilization of Gli proteins. HEK-293T cells transfected with Flag-Gli2 or Gli3 and HA-HOIP or its point mutation plasmids were subjected to IB with the indicated antibodies. D HOIP mediates linear ubiquitylation but impairs the K63 ubiquitination of Gli proteins. HEK-293T cells transfected with Flag-Gli2 and HA-HOIP or its point mutant, together with GFP-UBANNEMO or GFP-NZFTAB2 plasmids, were subjected to a denatured co-IP assay. UBANNEMO was used to recognize M1 ubiquitination, and NZFTAB2 was used to specifically recognize K63 ubiquitination.
Fig. 7
Fig. 7. Linear ubiquitylation of Gli proteins facilitates CRC growth.
LUBAC mediates the linear ubiquitylation of Gli proteins, which regulates their protein stability and activates Hh signalling, therefore facilitates cell growth in CRC.
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