CSN6-TRIM21 axis instigates cancer stemness during tumorigenesis

Abstract

Background: Cancer stem cells (CSCs) are responsible for tumour initiation, metastasis and recurrence. However, the mechanism of CSC formation, maintenance and expansion in colorectal cancer (CRC) remains poorly characterised.

Methods: The role of COP9 signalosome subunit 6 (CSN6) in regulating cancer stemness was evaluated by organoid formation and limited dilution analysis. The role of CSN6-TRIM21-OCT1-ALDH1A1 axis in CSC formation was evaluated in vitro and in vivo. The association of CSN6, TRIM21 and ALDH1A1 expression was validated by a tissue microarray with 267 CRC patients.

Results: The results showed that CSN6 is critical for sphere formation and maintaining the growth of patient-derived organoids (PDOs). We characterised the role of CSN6 in regulating cancer stemness, which involves the TRIM21 E3 ubiquitin ligase, transcription factor POU class 2 homeobox 1 (OCT1) and cancer stem cell marker aldehyde dehydrogenase 1 A1 (ALDH1A1). Our data showed that CSN6 facilitates ubiquitin-mediated degradation of TRIM21, which in turn decreases TRIM21-mediated OCT1 ubiquitination and subsequently stabilises OCT1. Consequently, OCT1 stabilisation leads to ALDH1A1expression and promotes cancer stemness. We further showed that the protein expression levels of CSN6, TRIM21 and ALDH1A1 can serve as prognostic markers for human CRC.

Conclusions: In conclusion, we validate a pathway for cancer stemness regulation involving ALDH1A1 levels through the CSN6-TRIM21 axis, which may be utilised as CRC molecular markers and be targeted for therapeutic intervention in cancers.

Fig. 1. CSN6 is required for sphere formation and initiates stemness through ALDH1A1.

a Sphere-formation assay of DLD-1, HCT116 and HCT-8 cells carrying scrambled or CSN6-specific shRNA. b DLD-1 cells carrying scrambled or CSN6-specific shRNA were dissociated into a single-cell suspension, seeded in 96-well plates with an ultra-low attachment surface at a density of 2, 10, 50 or 100 cells per well and cultured for 12 days. The frequency of sphere-initiating cells was estimated using the ELDA software. c Quantitative RT-PCR analysis was performed to measure the mRNA levels of stem cell markers (Aldh1a1, Lgr5, Cd133 and Cd44), embryonic stem cell components (Nanog and Oct4), WNT pathway components (Vegf and Ccnd1), Notch pathway components (Notch1, Hey1 and Nrarp) and BMP family genes (Bmp2 and Bmp4) in DLD-1 cells and HCT116 cells carrying scrambled or CSN6-specific shRNA. d Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in DLD-1, HCT116 and HCT-8 cells with CSN6 knockdown or CSN6 overexpression. e Quantitative RT-PCR analysis was performed to measure the mRNA levels of colorectal cancer and adjacent colorectal tissues. The levels of Csn6 were positively correlated with the expression of Aldh1a1 at mRNA levels in 13 pairs of human colorectal carcinomas (T) with matched normal tissues (N). f Kaplan–Meier survival curves of relapse-free survival time based on Csn6 and Aldh1a1 expression in CRC tissues. *P < 0.05, **P < 0.01 and ***P < 0.001. g Knockdown of CSN6 affected patient-derived tumour organoid (tumour PDO) growth. The morphology of the organoids is shown. The number of organoids growing to a size of >25 μm was calculated. Scale bars, 25 μm.

Fig. 2. CSN6 interacts with the TRIM21 E3 ligase and regulates ALDH1A1 through regulating TRIM21.

a DLD-1 cells were transfected with Flag-CSN6 plasmids. The protein level of TRIM21 was immunoblotted with anti-TRIM21 antibodies. b DLD-1 cells were infected with scrambled or CSN6-specific shRNA lentivirus. The protein level of TRIM21 was immunoblotted with anti-TRIM21 antibodies. c Quantitative RT-PCR analysis was performed to measure the mRNA levels of Trim21 in cells transfected with empty vector or Flag-CSN6. d Flag-TRIM21 was expressed in HCT116 cells. Flag-TRIM21 was immunoprecipitated with anti-Flag, and the associated CSN6 was detected by western blotting. e HEK293T cell lysates were immunoprecipitated with an anti-TRIM21 antibody and immunoblotted with anti-TRIM21 and anti-CSN6 antibodies. f HEK293T cells were co-transfected with Flag-TRIM21 and the HA-CSN6-WT, CSN6-N terminal or CSN6-C terminal construct. Cell lysates were immunoprecipitated with anti-Flag and subsequently immunoblotted with anti-Flag and anti-HA antibodies. Heavy heavy immunoglobulin chain, Light light immunoglobulin chain. g HEK293T cells were co-transfected with HA-CSN6 and the Flag-TRIM21-WT, TRIM21-N terminal or TRIM21-C terminal construct. Cell lysates were immunoprecipitated with anti-Flag and subsequently immunoblotted with anti-Flag and anti-HA antibodies. h Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells carrying scrambled or TRIM21-specific siRNA. Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells carrying empty vector or Flag-TRIM21 plasmids. i Sphere-formation assay of DLD-1 cells transduced with control or Flag-TRIM21 lentivirus. j HCT116 cells were transfected with Flag-CSN6 plasmids. The protein level of TRIM21, OCT1 and ALDH1A1 was immunoblotted with anti-TRIM21, anti-OCT1 and anti-ALDH1A1 antibodies. k Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells with TRIM21 knockout. l Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells stably expressing empty vector or Flag-CSN6 and rescued with Flag-TRIM21 plasmids. m Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in HCT116 cells stably expressing scrambled or CSN6-specific shRNA and rescued with TRIM21-specific siRNA. n The knockout efficiency of TRIM21 in HCT116 cells by TRIM21 sgRNAs was evaluated by western blotting. Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1 in control or HCT116 cells with TRIM21 knockout and then transfected with empty vector or Flag-CSN6. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 3. CSN6 and CUL1 affect the steady-state expression, ubiquitination level and turnover rate of TRIM21.

a HEK293T cells were co-transfected with Flag-TRIM21, HA-CUL1 and Myc-CSN6. Flag-TRIM21 was immunoprecipitated with anti-Flag, and the associated Cullin1 and CSN6 were detected by western blotting with anti-Flag, anti-HA and anti-Myc antibodies. b HEK293T cells were co-transfected with the indicated plasmids. Flag-TRIM21 was immunoprecipitated with anti-Flag, and the associated Cullin1 and CSN6 were detected by western blotting with anti-Flag and anti-HA antibodies. c DLD-1 and HCT116 cells were transfected with HA-CUL1 plasmids. The protein levels of TRIM21 were immunoblotted with anti-TRIM21 antibodies. d HEK293T cells were co-transfected with His-Ubi, Flag-TRIM21 and Myc-CUL1. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with Ni-NTA beads and immunoblotted with anti-Flag and anti-Myc antibodies. e HEK293T cells were transfected with Flag-TRIM21 and HA-CUL1. Overexpression of CUL1 increased the turnover rate of the TRIM21 protein. CHX cycloheximide, IOD integrated optical density. f HCT116 cells were treated with MLN4924 (3 μM) at the indicated time points, and cell lysates were immunoblotted with the indicated antibodies. g HCT116 cells were transfected with Flag-CSN6 plasmids, and MLN4924 (3 μM) was added to the cells 24 h before they were harvested. The protein levels of TRIM21 were immunoblotted with anti-TRIM21 antibodies. h HEK293T cells were co-transfected with His-Ubi and Flag-TRIM21, and MLN4924 (3 μM) was then added to the cells at the indicated time points. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down (PD) with Ni-NTA beads and the ubiquitination of TRIM21 was immunoblotted with anti-Flag antibodies. i HEK293T cells were co-transfected with His-Ubi, Flag-TRIM21 and Myc-CSN6, and MLN4924 (3 μM) was then added to the cells 24 h before they were harvested. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with Ni-NTA beads and immunoblotted with anti-Flag and anti-Myc antibodies.

Fig. 4. CSN6 facilitates ubiquitination-mediated degradation of TRIM21.

a HEK293T cells were transfected with His-Ubi together with Flag-TRIM21 and HA-CSN6. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with Ni-NTA beads and immunoblotted with anti-Flag and anti-HA antibodies. b HEK293T cells were transfected with HA-Ubi and Flag-TRIM21 and subsequently treated with shCSN6. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with the anti-Flag and anti-HA antibodies. c In DLD-1 cells expressing scrambled or CSN6-specific shRNA, CSN6 knockdown reduced the turnover rate of the TRIM21 protein and increased the turnover rate of OCT1. CHX cycloheximide. d DLD-1 cells were seeded into six-well plates. Forty-eight hours later, the cells were treated with MG132 at the indicated time points. e HEK293T cells were co-transfected with HA-Ubi and Flag-TRIM21. Forty-eight hours later, MG132 or NH₄Cl was added to the cells at the indicated time points. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. f HCT116 cells were co-transfected with HA-CSN6 and Flag-TRIM21-WT or Flag-TRIM21-LD (C16A, C31A and H33W) mutant. Cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. g The knockout efficiency of TRIM21 in HEK293T cells by TRIM21 sgRNAs was evaluated by western blotting. h HEK293T cells were co-transfected with the HA-Ubi-WT, Ubi-K48 only or Ubi-K63 only construct and the Flag-TRIM21-WT or Flag-TRIM21-LD construct. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. i HEK293T KO cells were co-transfected with the indicated HA-Ubi constructs and Flag-TRIM21 or the Flag-TRIM21 ΔRING mutant. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-HA antibodies. j Prediction of TRIM21 ubiquitination sites by UbiSite (http://140.138.144.145/~ubinet/index.php) and UbPred (http://www.ubpred.org/index.html). k HEK293T cells were co-transfected with HA-Ubi and Flag-TRIM21-WT or its mutants. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. l HCT116 cells were co-transfected with HA-CSN6 and Flag-TRIM21-WT or the Flag-TRIM21 K214R/K217R mutant. Cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. m HCT116 cells were co-transfected with HA-CSN6 and Flag-TRIM21 or the Flag-TRIM21 K214R/K217R mutant and were then treated with cycloheximide. Cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. n HEK293T KO cells were co-transfected with the indicated HA-Ubi constructs and Flag-TRIM21-WT or the Flag-TRIM21 K214R/K217R mutant. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-HA antibodies.

Fig. 5. The CSN6–TRIM21–OCT1 axis is involved in ALDH1A1 expression.

a HCT116 cells were transfected with the indicated plasmids. Cell lysates were immunoblotted with anti-Flag and anti-OCT1 antibodies. b TRIM21 knockout cells were transfected with the indicated plasmid, and cell lysates were immunoblotted with anti-Flag and anti-OCT1 antibodies. c HEK293T cells were transfected with HA-Ubi and the indicated plasmids. Cell lysates were subjected to IP with anti-Flag and immunoblotted with anti-HA antibodies. d HEK293T cells were transfected with His-Ubi and the indicated plasmids. Cell lysates were subjected to PD with Ni-NTA beads and immunoblotted with anti-Flag and anti-CSN6 antibodies. e HEK293T cells were transfected with the indicated plasmids. Cell lysates were subjected to PD with Ni-NTA beads and immunoblotted with anti-Flag antibodies. f HEK293T cells were transfected with HA-Ubi and Flag-OCT1 and subsequently treated with shCSN6 and siTRIM21. MG132 was added to the cells 6 h before they were harvested. Cell lysates were pulled down with anti-Flag and immunoblotted with anti-Flag and anti-HA antibodies. g DLD-1 cells were engineered to stably express scrambled or CSN6-specific shRNA and were then rescued with Flag-OCT1 plasmids. Sphere formation was measured. Quantitative RT-PCR analysis was performed to measure the mRNA levels of Aldh1a1. *P < 0.05 and **P < 0.01.

Fig. 6. CSN6-mediated regulation of CRC stemness via the TRIM21–OCT1–AlDH1A1 axis is involved in tumorigenesis.

a Frequency of CSCs in DLD-1 cells transduced with control non-silencing shRNA (shCtrl) or shCSN6 lentivirus, as measured by an LDA in vivo. Tumour morphology in each group. b Quantification of tumour initiation. c Tumour growth curve for mice subcutaneously injected with control or shCSN6 DLD-1 cells (1 × 10⁶ cells per mouse, six mice per group). d RNA was extracted from tumour tissues. Quantitative RT-PCR analysis was performed to measure the mRNA level of Aldh1a1. The data are presented as the means ± SDs. e Protein was extracted from the tumour tissues described in c and immunoblotted with the indicated antibodies. f Representative IHC staining for CSN6, TRIM21, OCT1 and ALDH1A1 in tumour tissues from mice. The scale bars represent 100 μm. g Representative IHC staining for CSN6, TRIM21 and ALDH1A1 in human CRC TMAs. Case 1 is representative of a patient with CSN6-high colon cancer. Case 2 is representative of a patient with non-CSN6-high colon cancer. The scale bars represent 100 μm. h Quantification of staining intensities from sections in g. CSN6 and TRIM21 show a negative correlation, while CSN6 and ALDH1A1 show a positive correlation. i Kaplan–Meier survival curves of overall survival time based on CSN6 and TRIM21 expression from TMA analysis. *P < 0.05 and ***P < 0.001.