Downregulation of RNF138 in CRC tumors correlates with poor prognosis
In analyzing the data collected from The Cancer Genome Atlas (TCGA) database, we noticed that RNF138 is downregulated in different subsets of cancers, particularly in CRC, liver hepatocellular carcinoma, and thyroid carcinoma, to a less extent in cholangiocarcinoma and head and neck squamous cell carcinoma (Fig. 1a; Supplementary Table 1). This unique expression pattern was also found when using the data derived from additional independent GEO datasets (GDS4382, GDS4718, and GDS2947) (Fig. 1b). In addition, RNF138 was confirmed to be downregulated in 20 randomized CRC samples compared with the matched adjacent normal tissues at transcriptional level (Fig. 1c; Supplementary Table 2) as well as at translational level (Fig. 1d) (Supplementary Fig. S1a–c). Similar results were obtained when analyzing additional 5 CRC samples in pairs by immunofluorescence (IF) (Fig. 1e; Supplementary Fig. S1d), consistent with the results from further examining 134 pairs of CRC tissue specimens by immunohistochemistry (IHC) (Fig. 1f; Supplementary Table 3). Therefore, RNF138 downregulation is closely associated with CRC tumorigenesis.
We probed the expression of RNF138 in tissue microarrays (TMAs) of 420 CRC samples (Supplementary Table 4) with the clinicopathological parameter setup established (Supplementary Fig. S2a). The reduction in RNF138 expression was found specifically associated with more aggressive tumor phenotypes (P = 0.027), reflected by both the increase in the size and extent of primary tumors (P = 0.003) and lymph node invasion (P = 0.003), while no significant correlation was observed with CRC tumor metastasis (P = 0.246) (Supplementary Fig. S2b–d; Supplementary Table 4). Importantly, the Kaplan–Meier survival analysis revealed that the increase in RNF138 expression closely correlates with enhanced disease-free survival (DFS) and improved overall survival (OS) prognoses of patients (Fig. 1g–i), suggesting RNF138 expression level as a prognosis indicator of CRC.
Ablation of RNF138 expression exacerbates the progression from colitis to aggressive malignancy
Chronic inflammation, often in the form of colitis in colorectal tissue, plays important roles in the colonic neoplastic transformation and progression.6,29 We next interrogated the possible role of RNF138 in the colitis-associated colorectal tumorigenesis. The chronic colitis and the colitis-associated colorectal cancer (CAC) animal models were developed as illustrated in Fig. 2a. Upon the colitis occurrence, RNF138−/− mice deteriorated more rapidly than RNF138fl/fl mice under co-housing condition, which was reflected in both the change in clinical scores and weight loss (Fig. 2b; Supplementary Fig. S3a), and ultimately suffered a higher mortality rate (Fig. 2c). The colorectum in RNF138−/− mice were significantly reduced in length and exhibited splenomegaly as opposed to the control (Fig. 2d; Supplementary Fig. S3b–d). In RNF138−/− mice, extensive inflammation was manifested throughout the mucosa along with reinforced lymphocytic infiltration as revealed by IHC, which coincided with altered epithelial structures and loss of crypts (Fig. 2e). In contrast, only inflammatory lesions were found in the control mice. Furthermore, precancerous lesions in the colorectal tissue were more evident in the RNF138−/− (57.14%) (Fig. 2f). Two out of seven RNF138−/− mice developed substantial CRC tumors with high-grade dysplasia (Supplementary Fig. S3e). Meanwhile, excessive cell proliferation indicated by Ki67 occurred in the colonic tissues of RNF138−/− mice (Fig. 2g).
Similarly, RNF138−/− mice exhibited higher clinical scores and suffered more significant weight loss and higher mortality rate during the induction of CAC with azoxymethane (AOM) and dextran sodium sulfate (DSS) (Fig. 2h, i; Supplementary Fig. S3f). In these mice, both colon and spleen tissues were also significantly shortened in length and displayed more severe damage (Fig. 2j; Supplementary Fig. S3g, h). Particularly, all RNF138−/− mice (23/23) developed CRC tumors as opposed to 86.7% in the control mice (15/17) (Supplementary Fig. S3i). Furthermore, we found that both the tumor number and tumor load were nearly doubled in the RNF138−/− mice (Fig. 2k–l) along with significant increase in size (Fig. 2m). The tumors in these mice appeared to be more advanced (Fig. 2n), developing higher levels of intramucosal carcinoma and adenocarcinoma that displayed lower degree of differentiation (Fig. 2o, p; Supplementary Fig. S3j). Meanwhile, augmented cell proliferation, highlighted by the Ki67-staining, was also identified in the RNF138−/− mice tissues (Fig. 2q). Thus, we propose that RNF138 suppresses the chronic colitis switch to colonic neoplastic transformation.
RNF138 deletion reinforces tumorigenesis
We constructed the tumor organoids system to understand RNF138’s role in the CRC tumorigenesis and progression (Fig. 3a), in which the growth of organoids was monitored for 9 days from day 0.5 post transplantation. From day 7, larger in diameter became apparent in the organoids derived from the RNF138−/− tumors (Fig. 3b; Supplementary Fig. S4a) along with alternated morphology and more rapid increase in number (Fig. 3c; Supplementary Fig. S4b). The xenografts from the RNF138−/− organoids developed larger in volume, on average two times of the ones from the control (Fig. 3d; Supplementary Fig. S4c, d), and exhibited a higher level of excessive growth indicated by Ki67-staining (Fig. 3e). Altogether, these results confirmed that RNF138 suppresses the tumor growth independent of the orthotopic microenvironment.
RNF138 dysregulation gives rise to aberrant NF-кB signaling underlying chronic inflammation, colonic neoplastic transformation, and progression
We systematically examined the global changes imposed by RNF138 deficiency in either the colon tissues dissected from the colitis and CAC models or the organoids derived from the CAC model. In all three sets of samples, substantial differences were identified at transcriptomic level between the RNF138fl/fl and RNF138−/− genetic backgrounds (Fig. 3f; Supplementary Fig. S5a). As revealed in the gene ontology (GO) term analysis, these differences primarily attributed to changes in the intestinal epithelial cell differentiation, proliferation, apoptosis, and cellular senescence (Supplementary Fig. S5b). Further analysis of signaling networks underlying these functional changes illuminated the central role of the NF-κB signaling interconnecting with the diverse functional elements (Fig. 3g, h; Supplementary Fig. S5c, d). Nuclear staining of p65 as an indicator downstream of NF-κB signaling were significantly enhanced upon RNF138 deletion (Fig. 3i, j). Thus, RNF138 is functionally linked to the NF-κB signaling that is critical for a cascade of events from the onset of colitis to the colitis-to-tumor transition and further CAC tumor progression.
We also recapitulated this phenomenon in human CRC cell lines where the RNF138 expression was suppressed via specific small interfering RNA (siRNA). In both HCT116 and RKO cells, upon RNF138 RNAi, p65 levels were moderately increased though with significant increase in the phosphorylation of p65 (pp65) (Supplementary Fig. S5e), consistent with results observed when disrupting RNF138 by CRISPR-Cas9 (Fig. 3k). Furthermore, RNF138 deficiency enhanced the nuclear translocation of p65 (Fig. 3l) that led to the increase in the downstream gene expression (NFκB1, IκBα, CXCL1, and IL8), overall highlighting enhanced activation of the NF-κB signaling pathway (Fig. 3m; Supplementary Fig. S5f). In brief, RNF138 negatively regulates the NF-κB signaling during colorectal tumorigenesis.
RNF138 restrains the activation of NF-κB signaling by retaining NIBP in the nucleus
RNF138 seems not engaged in the canonical NF-κB signaling pathway directly (Supplementary Table 5; Supplementary Fig. S6a) but through the association with NIK- and IKK-β-binding protein (NIBP), a NF-κB signaling transduction regulator identified previously.30,31 The interaction between RNF138 and NIBP was confirmed by immunoprecipitation, and most likely occurs in the nucleus (Fig. 4a) as NIBP distributes in both the nucleus and cytoplasm whereas RNF138 resides primarily in the nucleus (Fig. 4b). As revealed in the proximity ligation assay, NIBP interacts with RNF138 directly in the nucleus (Fig. 4c) whereas it is associated with IKKβ (Fig. 4d), in the cytoplasm.30 Upon the RNAi against NIBP, the activation of NF-κB was significantly compromised, which was reflected in the reduction both in the phosphorylation of p65, (Fig. 4e) and in the transcription of downstream genes featured by NFкB1, IκBα, CXCL1, and IL8 (Fig. 4f), suggesting that NIBP likely mediates the regulation of NF-κB by RNF138. Furthermore, the loss of NIBP restored the NF-кB signaling that was distorted due to the defective RNF138 (Fig. 4g). In summary, RNF138 regulates the NF-κB signaling at least partially via NIBP.
RNF138 serves as a Ub-E3 ligase in DNA damage response.22,23,32,33 In contrast, the level of NIBP remained unchanged upon the disruption of RNF138 by either RNAi or knockout (Supplementary Fig. S6b), suggesting the functional association between two is likely independent of ubiquitination and protein-degradation. Interestingly, the paradigm of NIBP subcellular distribution was shifted upon RNF138 depletion, with significant increase in the nuclear partitioning coincided with the decreased cytoplasmic presence (Fig. 4h), which consequently enhanced the co-localization of NIBP and IKKβ as observed in the RNF138-deficient HCT116 and RKO cells (Fig. 4i).
Thus, we propose that the nuclear-cytoplasmic partitioning of NIBP contributes significantly to the key mechanism by which RNF138 regulates the NF-κB signaling transduction.
The Ub-E3 ligase activity is dispensable for the regulation of NIBP by RNF138
The ubiquitin interacting motif (UIM) located in RNF138 extreme C-terminus (AA225-243) is essential for its interaction with NIBP as opposed to the RING domain (AA18-58) and triple Zinc finger (ZNF) (ZNF1 AA86-105, ZNF2 AA159-180, and ZNF3 AA189-215) (Fig. 5a, c). However, little impact on the interaction between RNF138 and NIBP was observed when RNF138, as a Ub-E3 ligase, became inactive with specific mutations introduced to the catalytic cysteines (C18A/C54A) (Fig. 5c). We also identified the second (AA360-669) and the third Trs120 domain (AA882-1100) in NIBP were primarily responsible for its interaction with RNF138 (Fig. 5b, d).
This molecular connection was also reflected at functional level. The distorted nuclear partitioning of NIBP due to RNF138 deficiency could be restored by ectopically expressing either the full-length protein (FL), RING domain protein-truncated protein (ΔRING) or the catalytic cysteines C/A mutant (Mutant), but much less effective with the UIM-truncated protein (Fig. 5e). Furthermore, in contrast to the UIM-truncated protein, either the full length, the RING domain-truncated, or the catalytic cystine C/A mutant could restore the NF-κB signaling distorted by the loss of RNF138, which was manifested first by changes in the phosphorylation of p65 (Fig. 5f), then the expression of downstream genes including NF-кB1, IκBα, CXCL1, and IL8 (Fig. 5g), and further in cell proliferation (Fig. 5h). Importantly, the UIM-truncated RNF138 failed to exert impact on the proliferation as opposed to the full-length protein, the RING domain-truncated, and the catalytic cystine C/A mutant RNF138. Therefore, RNF138 regulates NF-κB signaling through its physical and functional interaction with NIBP independent of its Ub-E3 ligase activity.
RNF138 downregulation coincides with the aberrant NF-кB signaling in CRC associated with unfavorable clinical outcomes
A significant positive correlation was established between RNF138 expression and p65 total level using human primary CRC TMAs (n = 420) (Supplementary Fig. S7a). Similar association was also established between RNF138 expression with the cytoplasmic accumulation of p65 as opposed to the nuclear translocation of p65 as an indicator of NF-кB activation in CRC tissue specimens (Supplementary Fig. S7b, c), suggesting that RNF138 negatively regulates the NF-кB signaling transduction. We further observed significant downregulation of RNF138 in the CRC tissue specimens that coincided with the hyperactivation of NF-кB signaling pathway indicated by the changes in the p65 nucleic/cytoplasmic (N/C) ratio (Fig. 6a, b). Specifically, the CRC patients were stratified into four groups based on the level of RNF138 and the pp65 N/C ratio, i.e., RNF138high-pp65 N/C ratiolow, RNF138low-pp65 N/C ratiohigh, RNF138high-pp65 N/C ratiohigh, and RNF138low-pp65 N/C ratiolow. The Kaplan–Meier survival analysis shown that CRC patients with RNF138low-pp65 N/C ratiohigh had shorter disease-free survival and overall survival compared with the other three CRC subtypes (Fig. 6c, d). Importantly, RNF138low-pp65 N/C ratiohigh tumors exhibited much more aggressive clinical features (Fig. 6e, f; Supplementary Fig. S7d, e). Moreover, RNF138, the pp65 ratio (N/C), and the progression and prognosis of CRC patients were inversely correlated (Fig. 6g, h; Supplementary Fig. S7f). In particular, the sustained activation of NF-кB signaling occurred in two RNF138-ablated CRC subtypes associated with much more aggressive tumors and adverse outcomes, indicating the inverse correlation between RNF138 expression and the pp65 N/C ratio in tumors (n = 284) (Fig. 6i–l; Supplementary Fig. S7g, h), consistent with the changes in downstream gene expression highlighted in the cases of ICAM1 and PTGS2 (Fig. 6m, n).
In brief, poor prognosis in CRC patients significantly correlates with the elevated NF-кB signaling coincided with RNF138 downregulation in the affiliated tumors. Therefore, we highlighted the possibility of using RNF138 downregulation as indicator for both prognosis and the therapeutic interventions targeting the NF-κB signaling.
Targeting the NF-кB signaling suppresses CRC growth associated with RNF138-ablation
We then further explored the therapeutic potential in targeting the distorted NF-κB signaling associated with RNF138 downregulation. SC75741 appeared effective in suppressing CRC tumor cell growth by the NF-κB pathway (Supplementary Fig. S8a–d), and could restore the aberrant NF-κB signaling with deficient RNF138-NIBP axis (Supplementary Fig. S8e). We therefore selected SC75741 as a specific chemical probe to address the effect of targeting the NF-κB signaling on the CRC progression. The RNF138WT and RNF138KO xenografts (CDX) were generated in parallel by injecting the corresponding HCT116 cells into the nude mice prior to the SC75741 treatment (Fig. 7a). Upon the treatment, the tumor growth was remarkably repressed in the RNF138KO xenografts, reflected in the reduction in both tumor volume and weights, whereas no significant difference was observed in the RNF138WT counterparts (Fig. 7b, c; Supplementary Fig. S9a, b). This inhibition was also marked at molecular level by the significant reduction in the Ki67 and PCNA staining of the RNF138KO xenografts compared with the WT, indicating an impaired proliferation as the result of the inhibition of NF-κB signaling (Fig. 7d; Supplementary Fig. S9c).
We further evaluate the results in a more clinical-relevant context with the CRC patient-derived xenograft (PDX) models (Fig. 7e). Specifically, the corresponding fragments derived from either RNF138high-pp65 N/C ratiolow (patient-1 and patient-2) or RNF138low-pp65 N/C ratiohigh (patient-3 and patient-4) CRC tumors were transplanted into NOD/SCID mice for PDX models (Supplementary Fig. S9f; Supplementary Table 6). SC75741 effectively blocked the activation of NF-κB signaling in all these models and specifically inhibited the growth of the RNF138low-pp65 N/C ratiohigh PDX model (Fig. 7f, g; Supplementary Fig. S9d, e). In contrast, no significant change was observed in the RNF138high-pp65 N/C ratiolow PDX models (Fig. 7f, g; Supplementary Fig. S9e). The Ki67- and PCNA-staining also indicated specific reduction in cell proliferation, accompanied by the diminished NF-κB signaling, in the RNF138low-pp65 N/C ratiohigh model (Fig. 7h; Supplementary Fig. S9g).
Collectively, these results strongly support the role of aberrant activation of NF-κB pathway in the CRC progression that is specially associated with RNF138 dysregulation. We propose NF-κB signaling-targeted therapy as the potential effective clinical intervention for the CRC with poor prognosis specifically associated with RNF138 downregulation (Fig. 7i).