Aberrant expression of MYD88 via RNA‐controlling CNOT4 and EXOSC3 in colonic mucosa impacts generation of colonic cancer

Abstract In 2020, the worldwide incidence and mortality of colorectal cancer (CRC) were third and second, respectively. As the 5‐y survival rate is low when CRC is diagnosed at an advanced stage, a reliable method to predict CRC susceptibility is important for preventing the onset and development and improving the prognosis of CRC. Therefore, we focused on the normal colonic mucosa to investigate changes in gene expression that may induce subsequent genetic alterations that induce malignant transformation. Comprehensive gene expression profiling in the normal mucosa adjacent to colon cancer (CC) compared with tissue from non‐colon cancer patients was performed. PCR arrays and qRT‐PCR revealed that the expression of 5 genes involved in the immune response, including MYD88, was increased in the normal mucosa of CC patients. The expression levels of MYD88 were strikingly increased in precancerous normal mucosa specimens, which harbored no somatic mutations, as shown by immunohistochemistry. Microarray analysis identified 2 novel RNA‐controlling molecules, EXOSC3 and CNOT4, that were significantly upregulated in the normal mucosa of CC patients and were clearly visualized in the nuclei. Forced expression of EXOSC3 and CNOT4 in human colonic epithelial cells increased the expression of IFNGR1, MYD88, NFκBIA, and STAT3 and activated ERK1/2 and JNK in 293T cells. Taken together, these results suggested that, in the inflamed mucosa, EXOSC3‐ and CNOT4‐mediated RNA stabilization, including that of MYD88, may trigger the development of cancer and can serve as a potential predictive marker and innovative treatment to control cancer development.


| INTRODUC TI ON
Colorectal cancer (CRC) is the second and third most common cancer diagnosed in females and males, respectively, with an estimated 1.94 million cases and 929 500 deaths in 2020 worldwide. 1 When CRC is diagnosed at an early stage, the 5-y relative survival rate is very high, 2 and early resection of CRC allows for almost 100% recovery of the patients. 3 Therefore, a reliable way to predict CRC candidates is critical for cancer prevention and the early detection, diagnosis, and appropriate treatment of CRC.
Both genetic and epigenetic alterations commonly occur in CRC and are the driving force of tumorigenesis. 4-6 CRC arises from 1 or a combination of 3 different mechanisms: chromosomal instability (CIN), the CIMP, and microsatellite instability (MSI). 7,8 The CIN pathway is also known as the adenoma-carcinoma sequence. 9 CRC is generally considered to develop from benign lesions through the adenoma-carcinoma sequence and another tumorigenic pathway of de novo carcinogenesis, suggesting the development of tumors from normal (or intact) colonic mucosa without the intervening step of an adenoma. 10 It is well established that germline mutations in MSIrelated genes occur in Lynch syndrome and are closely associated with colon cancer predisposition. In addition to traditional MSI testing and IHC analysis of mismatch repair (MMR) proteins, the evaluation of MSI by next-generation sequencing has been used to verify the presence of Lynch syndrome pancancer. [11][12][13] Chronic inflammation has been shown to promote colon carcinogenesis by inducing genetic mutations, inhibiting apoptosis, and stimulating angiogenesis and cell proliferation. 14 Inflammation is an adaptive response that is triggered by a variety of abnormal conditions, including infection of the microbiome and tissue injury, as well as more subtle changes in tissue homeostasis. [15][16][17] Microbial infection or tissue injury triggers inflammation by activating the TLR/adaptor molecule myeloid differentiation factor 88 (MYD88) signaling pathway. MYD88 is a TLR adapter molecule that is important for the activation of downstream NF-κB and MAPK pathways, 18 contributing to carcinogenesis and prognosis in various cancers, including skin, liver, pancreatic, breast, lung, gastric, and colon cancer. [19][20][21][22][23][24][25] High expression of MYD88 was frequently detected in CRC with liver metastasis and significantly related to poor prognosis. 26 Experimentally, mice lacking MYD88 formed fewer tumors compared with wild-type mice with azoxymethane (AOM)-induced intestinal tumorigenesis. In APC MIN mice, MYD88-dependent signaling was required for the progression of tumors derived from epithelial cells. 27 The role of MYD88 in controlling mucosal homeostasis depends on the effect of the transcription factor NF-κB on enterocyte survival, which is implicated in the inflammation associated with cancer. 25 Recently, MYD88 gene mutations have been reported in hematological malignancies, 28,29 and increased protein expression of MYD88 with no somatic mutation was observed in gastric cancer. 30 Multiple molecular events that occur in CRC have been thoroughly investigated by colon cancer cells with paired normal mucosa. Conversely, increasing evidence has indicated that clonal expansion of phenotypically normal or noncancerous tissues is biologically significant in cancer development. 31,32 Therefore, attention should be given to the molecular events occurring in the normal colonic mucosa to prevent CRC development. Therefore, we performed comprehensive molecular analyses using histologically normal colonic mucosa adjacent to colon cancer as a feasible precancerous lesion and compared these tissues to those without colon cancer. We identified 2 kinds of RNA-controlling components, EXOSC3 and CNOT4, that were markedly increased in the normal colonic mucosa of colon cancer patients with enhanced MYD88. Forced expression of EXOSC3 and CNOT4 increased the expression levels of IFNGR1, MYD88, NFκBIA, and STAT3 in human colonic epithelial cells, leading to activation of the MAPK and JNK pathways. Therefore, disturbances in comprehensive RNA control in the normal colonic mucosa might allow for the onset of colon cancer, and EXOSC3 and CNOT4 would be potential predictive markers of colon cancer and contribute to the onset by stabilizing RNAs including MYD88.

| Patients and human tissues
Here, 17 CC patients and 15 non-colon cancer (NCC) patients who were diagnosed between 2008 and 2014 at the Department of Cancer Pathology, Faculty of Medicine, Hokkaido University, and Sapporo Tokushukai Hospital (Table 1) were included in the study.
Among them, 2 representative cases were selected for PCR array and microarray analyses, which had the best quality of extracted RNAs and less infiltration of inflammatory cells in the normal mucosa. The 2 CC patients (CC11 and CC14) had sigmoid colon cancer with tumor sizes of 5.0 cm or less, and normal colonic mucosa 2.0 cm on the anal side from the tumor was used for the analyses. The 2 NCC patients (NCC1 and NCC2) had sigma elongatum or diverticulitis of the sigmoid colon. In each case, normal colonic mucosa without malignancy or inflammation, as observed microscopically, was used.

| RNA extraction from FFPE samples and quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections of normal colonic mucosa from CC patients (CC11 and CC14) or NCC patients (NCC1 and NCC2) using the RNeasy FFPE Kit (Qiagen). All samples were cut at a thickness of 10 µm, attached to a glass slide, and trimmed only the normal mucosa area.
cDNA was synthesized using a SuperScript VILO cDNA Synthesis Kit (Invitrogen), and qRT-PCR was performed using StepOnePlus™ instrument (Applied Biosystems, Thermo Fisher Scientific Inc.) as described previously. 33 Gene expression was determined using

| PCR array
Total RNA was extracted from the normal colonic mucosa of CC (CC11 and CC14) or NCC (NCC1 and NCC2) patients as described above, and PCR array analysis was performed using RT 2 Profiler™ PCR Array Human Innate & Adaptive Immune Responses (Qiagen;

| Microarray analysis
Total RNA was extracted from the normal colonic mucosa of CC (CC11 and CC14) or NCC (NCC1 and NCC2) patients as described above, and microarray analysis was performed using SurePrint G3

| Transfection of 293T cells with EXOSC3 and CNOT4
Total RNA was extracted from human embryonic kidney 293F cells

| Inflammation-related genes, including MYD88, are increased in paracancerous normal colonic mucosa
To determine the molecular changes that occur in precancerous colonic mucosa, we focused on identifying gene expression profiles in the paracancerous normal mucosa of CC patients and compared them to those in the normal mucosa of NCC patients. The CC patients were confirmed not to have Lynch syndrome by family history and IHC analysis for mismatch repair (MMR) proteins such as MLH1, MSH2, PMS2, and MSH6 (data not shown; Table 1). In addition, only CC patients with tumor sizes of 5.0 cm or less were selected for analysis. As representative samples with best quality of extracted RNAs and less infiltration of inflammatory cells in the normal mucosa, 2 CC patients with sigmoid colon cancer (CC11 and CC14) were selected, and normal colonic mucosa 2.0 cm on the anal side away from the tumor (NC11 and NC14) was used for the molecular analysis to exclude unpredictable effects of passage obstacles ( Figure 1A). The normal mucosa was evaluated microscopically by pathologists and determined to be normal lesions without any inflammation or tumor components. In comparison, normal colonic mucosa from non-colon cancer patients (NCC1 and NCC2) were used. NCC1 and NCC2 had megacolon and diverticulitis of the sigmoid colon, respectively, and normal colonic mucosa close to the surgically resected margin in each patient was used (N1 and N2, Figure 1B).
For gene expression analyses, we first examined the relationship between inflammation and carcinogenesis by evaluating gene expression linked to inflammation using RT 2 Profiler™ PCR Array Human Innate & Adaptive Immune Responses in 4 sample sets ( Figure 1C; Table S2). In the normal mucosa of 2 CC patients (NC11 and NC14), 21 and 12 genes were upregulated more than 2-fold relative to those in the non-colon cancer patients NCC1 (N1; Figure 2A) and NCC2 (N2; Figure 2B), respectively. The latter 12 genes were commonly and highly expressed than those in N1 and N2 ( Figure 2C).

| MYD88 is increased in the normal colonic mucosa of colon cancer patients
Because the adaptor protein MYD88 has been shown to contribute to carcinogenesis and is related to prognosis in many cancers, including skin, liver, pancreatic, breast, lung, gastric, and colon cancer, 19 14 cases (93%), average score: 0.93; Figure 3B,C). Therefore, consistent with the gene expression data, MYD88 protein expression was higher in normal colonic mucosa in CC patients than in NCC patients ( Figure 3C).

| Mutation analysis of MYD88 in colon cancer tissues
Recent studies have revealed the MYD88 L265P somatic mutation in hematological malignancies that induces constitutive activation of NF-κB signaling and tumorigenesis. 28,29 To examine the mutation status of MYD88 in colon cancer patients, we screened the L265P hot-spot mutation in 17 colon cancer samples by direct sequencing, but no somatic mutations were found in any samples ( Figure 3D).

| Comprehensive gene expression profiling identified increases in RNA-controlling EXOSC3 and CNOT4 in paracancerous normal colonic mucosa
To comprehensively investigate the gene expression profile in the mucosa of colon cancer patients, microarray analysis was performed using normal colonic mucosa with or without colon cancer.

| EXOSC3 and CNOT4 are increased in paracancerous normal colonic mucosa
To verify the microarray analysis data, immunohistochemical analysis of EXOSC3 and CNOT4 was performed. Both EXOSC3 and CNOT4 were strongly localized to the nucleus ( Figures 5A and 6A Figure 6C,D). We also investigated the expression of EXOSC3, CNOT4 and MYD88 in 17 cases with SSL ( Figure S5A), because a majority of colon cancer develops also through the serrated-neoplasia pathway in addition to the adenoma-carcinoma sequence. The expression of MYD88 and EXOSC3 was almost equal between SSL and the corresponding adjacent normal mucosa. In addition, the expression of MYD88 and EXOSC3 is likely to correlate with each other.
Conversely, the expression levels of CNOT4 were low in normal mucosa in all cases (1+ or 0), whereas they were clearly enhanced in SSL, which is specific for the serrated pathway but not in the adenomacarcinoma sequence. Therefore, CNOT4 might be dominant in controlling the expression levels of genes in the SSL ( Figure S5B).

| Overexpression of EXOSC3 and CNOT4 activates the JNK and MAPK pathways in 293T cells
To clarify the causal relationship between the increased expression of EXOSC3 or CNOT4 and the development of colorectal cancer, we cloned cDNAs of EXOSC3 and CNOT4 and overexpressed them in human colonic epithelial cells (HCoEpiCs, ScienCell Research Laboratories; Figure 7A). Of the 5 genes identified by PCR array and qRT-PCR (Figure 2), IFNGR1, MYD88, NFKB1A, and STAT were increased by the forced expression of EXOSC3 and CNOT4, especially by EXOSC3 ( Figure 7B). Similar results were observed in 293T cells, with slightly lower expression levels than those of HCoEpiC ( Figure S6), suggesting that the HCoEpiC has a more well established machinery for expressing IFNGR1, MYD88, NFKB1A, and STAT3, which might contribute to the onset of colon cancer. To investigate cellular signaling, we used 293T cells because the transfection efficiency F I G U R E 2 Upregulated genes in the paracancerous normal colonic mucosa of colon cancer patients, as determined by PCR array and qRT-PCR. A, B, In the PCR array, 21 and 12 genes were upregulated more than two-fold in the normal colonic mucosa of 2 CC patients (NC11 and NC14) compared with those of NCC1 (N1, A) and NCC2 (N2, B), respectively. C, Venn diagram displaying the upregulated genes in the normal colonic mucosa of 2 CC patients (NC11 and NC14) compared with NCC1 and NCC2. D, mRNA expression levels were determined by qRT-PCR. Among the 12 genes that were commonly upregulated in the PCR array, 5 genes (IFNGR1, IL18, MyD88, NFKBIA, and STAT3) were confirmed to be upregulated more vs NCC1-normal mucosa (N1) in HCoEpiCs was low and insufficient to detect the target factors.
Immunoblotting revealed that the forced expression of EXOSC3 and CNOT4 increased the phosphorylation levels of MAPKs, especially phospho-JNK and phospho-ERK1/2 ( Figure 7A,B). MYD88 expression was increased by the overexpression of EXOSC3 and CNOT4, especially by EXOSC3 ( Figure 7C,D).

F I G U R E 4
Genes that were upregulated in the paracancerous normal colonic mucosa of colon cancer patients, as determined by microarray analysis. A, Venn diagrams displaying the upregulated genes in the normal colonic mucosa of CC11 and CC14 compared with NCC1 and NCC2. In the paracancerous normal mucosa, the expression levels of 257, 98, and 30 genes were increased more than 2-fold, 2.5-fold, and 3-fold in the log2 ratio, respectively. The size of the circles on the Venn diagrams match the number of genes. B, Using the data obtained from the microarray analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the upregulated genes were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). The upregulated genes with log2 ratios >2, >2,5, and >3 are displayed. C, qRT-PCR was performed to analyze EXOSC3 var2 and CNOT4 mRNAs status in the normal colonic mucosa of patients with or without colonic cancer have been reported, 34 this method seems to be inadequate in the clinic.
Individual CRC susceptibility seems to differ in the background normal colonic mucosa under the influence of inflammation. The prediction of CRC susceptibility is valuable for the early detection

RNA degradation R rp40 CNOT4
Relative mRNA expression (-fold change) Recently, somatic mutations in MYD88, such as L265P, have been reported in hematological malignancies, 28,29 and increased protein expression of MYD88 with no mutation was observed in gastric cancer. 30 In this study, we found no somatic mutations in MYD88 in the tumor lesions of all colon cancer patients ( Figure 3D), indicating mutation-independent upregulation of MYD88.
Here, we identified 2 novel RNA-controlling molecules, EXOSC3 and CNOT4, that were upregulated in paracancerous   Figure S5B). Of note, the expression of CNOT4 clearly increased in the SSL compared with the paired adjacent normal mucosa ( Figure S5B), suggesting that CNOT4 might be dominant in controlling RNAs in SSL. We would like to clarify the role of CNOT4 in SSL in more detail in the future.
In this study, we discovered that the overexpression of EXOSC3 and CNOT4 promoted the gene expression of IFNGR1, MYD88, NFKB1A, and STAT3 in human colonic epithelial cells, probably by upregulating the lifespan and metabolism of various mRNAs, and induced the activation of ERK1/2 and JNK in 293F cells (Figure 7), which was linked to the development of CRC. During the chronic inflammation caused by ulcerative colitis, the colonic epithelium is almost entirely remodeled by changes in the expression of numerous genes, which may lead to the onset of clones harboring driver mutations. 31 Colorectal cancer has been suspected to be promoted by commensal bacteria or dysbiotic microbiomes via Colon cancer patients In summary, we revealed that EXOSC3 and CNOT4 would be potential predictive markers of colon cancer and may contribute to the onset by stabilizing RNAs including MYD88. Individuals with high expression should be followed up to enable early detection of CRC. Furthermore, novel molecular therapeutics targeting these molecules would be a valuable strategy to prevent the onset of CRC, which may introduce a new era in preventive medicine for cancer.

STAT3
Relative mRNA expression T/F

D I SCLOS U R E
The authors declare no competing financial interests.