Targeting posttranslational modifications of RIOK1 inhibits the progression of colorectal and gastric cancers
- Key Laboratory of Basic Pharmacology, Ministry of Education, Zunyi Medical College, China
- Zhongshan Hospital of Xiamen University, China
- Medical College of Xiamen University, China
- Xiamen Municipal Key Laboratory of Gastrointestinal Oncology, China
- Xiangya School of Medicine, Central South University, China
- Second Affiliated Hospital of Xinjiang Medical University, China
- Rutgers University, United States
- The First Affiliated Hospital of Harbin Medical University, China
- Hongqi Hospital, Mudanjiang Medical University, China
- Zhongshan Hospital of Xiamen University, Fujian, China
- The First Affiliated Hospital of Anhui Medical University, China
- The Third Affiliated Hospital of Harbin Medical University, China
- Rutgers University, The State University of New Jersey, United States
Figures
Figure 1 with 1 supplement
RIOK1 is significantly upregulated in CRC and associated with an aggressive and poor survival.
(A) RIOK1 expression in five paired human CRC biopsies and matched normal mucosa analyzed by Western-blot. (B) Comparison of RIOK1 mRNA expression level in human CRC tissues (with and without …
Figure 1—figure supplement 1
RIOK1 expression in GC patients.
Immunohistochemical analysis and statistic calculation of RIOK1 on a group of patients with GC (n = 20) and healthy adjacent tissue (n = 20).
Figure 2 with 2 supplements
RIOK1 promotes growth and metastasis of CRC cells in vitro and in vivo.
(A) The expression levels of RIOK1 in 7 CRC cell lines and normal intestinal epithelial cells (IECs) were analyzed by Western blot. (B) Western blot for RIOK1 in HCT116 and SW480 cells infected with …
Figure 2—figure supplement 1
RIOK1 promotes the proliferation, invasion and metastasis of GC in vitro and in vivo.
(A) Western blot for RIOK1 in MKN45 cells infected with shRIOK1, or control shRNA lentiviral vector, as well as in SGC7091 infected with the RIOK1-expressing or empty control vector (n = 4).And the …
Figure 2—figure supplement 2
PI3K/AKT pathway is required for RIOK1-mediated CRC and GC cell proliferation and migration.
(A) CRC and GC cells were transfected with RIOK1 plasmid, and treated with LY29004 (30 μM) or A443654 (1 μM). Then cell proliferation (A1), migration(A2) and invasion (A3) were determined. (B) The …
Figure 3 with 2 supplements
SETD7 monomethylates RIOK1 on lysine 411 in vitro and in vivo.
(A) Anti-RIOK1 immunoprecipitates from high-density Setd7+/+ and Setd7−/− MEFs were immunoblotted for RIOK1, SETD7, methyl-lysine (mono-, dimethylated) (ab23366), and dimethyl-lysine (ab7315) …
Figure 3—figure supplement 1
SET7/9 directly interacts with RIOK1 in vitro and in vivo.
(A) Whole-cell lysates of RKO cells transfected with GFP-SET7/9 and His-RIOK1 were precipitated with an anti-GFP or anti-His antibody, and the interactive components were analyzed by Western blot. (B…
Figure 3—figure supplement 2
Validation of the antibodies’ specificity.
To easily find whether methylation of RIOK1 occurred in cells, we prepared a modification-specific antibody, anti-RIOK1-K411me1, which specifically recognized monomethylated RIOK1-K411 but not the …
Figure 4
SET7/9 significantly reduces the stability of RIOK1.
(A) His-RIOK1-WT or His-RIOK1-K411R was cotransfected into HEK293T cells for 48 hr with or without Flag-SET7/9. Cell lysates were examined by western blot with indicated antibodies. (B) His-RIOK1-WT …
Figure 5 with 1 supplement
SET7/9-mediated methylation of RIOK1 is reversed by LSD1, which increases RIOK1 stability.
(A) Endogenous RIOK1 co-immunoprecipitated reciprocally with endogenous LSD1 from HEK293T cells and subjected to immunoprecipitation followed by immunoblotting with indicated antibodies. (B) …
Figure 5—figure supplement 1
LSD1 interacts with RIOK1 endogenously.
HCT116 cells were extracted and immunoprecipitated with an anti–LSD1 (left) or anti-RIOK1 (right) antibody. IP with rabbit IgG was used as the negative control. Western blot analysis was performed …
Figure 6 with 1 supplement
K411 methylation of RIOK1 by SETD7 promotes the interaction between FBXO6 and RIOK1.
(A) The effect of FBXO6 knockdown on the levels of RIOK1 proteins in HEK293T cells. (B) HEK293T cells were transfected with the indicated siRNAs, and after 48 hr, treated with 160 μM CHX, and …
Figure 6—figure supplement 1
FBXO6 interacts with RIOK1 endogenously.
HCT116 cells were extracted and immunoprecipitated with an anti–FBXO6 (left) or anti-RIOK1 (right) antibody. IP with rabbit IgG was used as the negative control. Western blot analysis was performed …
Figure 7 with 1 supplement
CK2 phosphorylates RIOK1 at T410 both in vitro and in vivo.
(A) RIOK1 consensus phosphorylation sites (shown in red) corresponding to the CK2 consensus motifs S/TXXE/D are presented. These consensus phosphorylation sites are exactly located within the …
Figure 7—figure supplement 1
CK2 interacts with RIOK1 endogenously.
(A) HCT116 cells were extracted and immunoprecipitated with an anti–CK2 (left) or anti-RIOK1 (right) antibody. IP with rabbit IgG was used as the negative control. Western blot analysis was …
Figure 8 with 1 supplement
T410 phosphorylation of RIOK1 antagonists SETD7-mediated K411 methylation, which stabilizes RIOK1 in CRC and GC cells.
(A) The effect of knockdown of CK2α in HCT116 cells on the levels of RIOK1 proteins, T410p, and K411me. (B) The effect of the CK2α-K68A mutant on the levels of RIOK1 proteins, T410p, and K411me. (C) …
Figure 8—figure supplement 1
LSD1, CK2 and RIOK1 were in the same complex.
Two-step co-IP assay. The HCT116 cells were transfected with the HA-LSD1, Flag-RIOK1 and Myc-CK2 plasmids as indicated. Cells were lysed. The first immunoprecipitation was performed with an …
Figure 9 with 2 supplements
Methylation of RIOK1 at K411 and phosphorylation of RIOK1 at T410 oppositely regulated the CRC growth and metastasis.
(A) MTT assays in RKO cells expressing indicated proteins (upper panel). Western blotting was used to detect expression of RIOK1 in RKO cells using anti-RIOK1 antibody (lower panel), and the …
Figure 9—figure supplement 1
Methylation of RIOK1 at K411 and phosphorylation of RIOK1 at T410 oppositely regulated the CRC growth and metastasis.
(A) Photography and statistic results of colony formation assays in HCT116-shRIOK1#1 cells expressing indicated proteins. (B) Photography and statistic results of tumor weight in HCT116-shRIOK1#1 …
Figure 9—figure supplement 2
Clinical relevance of RIOK1, SET7/9, LSD1, FBXO6 and CK2 Expression in patients with CRC.
(A) Immunohistochemical staining of 104 human CRC for LSD1, CK2, SET7/9, and FBXO6 was performed. Representative photos of stains are shown in the groups with high (staining score, 5–8.0) and low …
Additional files
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Supplementary file 1
Univariate analysis and multivariate analysis between RioK1 expresssion and Clinicopathologic Features of CRC Patients (n = 104)
- https://doi.org/10.7554/eLife.29511.022
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Supplementary file 2
Identification of RioK1-interacting proteins via MS
- https://doi.org/10.7554/eLife.29511.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.29511.024
