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Prognostic role of tumor mutation burden (TMB) stratified by microsatellite instability (MSI) status.

Supplementary Figure legends; Supplementary Materials and Methods: Detailed and expanded methods on cell lines, in-vitro assays, in-vivo tumor model, histology, and tumor specimens for all data presented.

PDF file - 456K

Generation and characterization of villin-KITENIN/APCmin/+ mice.

PDF file - 206K, Figure 6. Modulation of multiple RTKs in KITENIN-overexpressed Caco2 or KITENIN-knockdown HCT116 CRC cells.

PDF file - 126K, Figure 1. EGF induces synergic AP-1 activation under elevated KITENIN in an EGFR- and EGFR-kinase-independent manner.

Purpose:Despite the well-known prognostic value of the tumor–immune microenvironment (TIME) in colorectal cancers, objective and readily applicable methods for quantifying tumor-infiltrating lymphocytes (TIL) and the tumor–stroma ratio (TSR) are not yet available.Experimental Design:We established an open-source software-based analytic pipeline for quantifying TILs and the TSR from whole-slide images obtained after CD3 and CD8 IHC staining. Using a random forest classifier, the method separately quantified intraepithelial TILs (iTIL) and stromal TILs (sTIL). We applied this method to discovery and validation cohorts of 578 and 283 stage III or high-risk stage II colorectal cancers patients, respectively, who were subjected to curative surgical resection and oxlaliplatin-based adjuvant chemotherapy.Results:Automatic quantification of iTILs and sTILs showed a moderate concordance with that obtained after visual inspection by a pathologist. The K-means–based consensus clustering of 197 TIME parameters that showed robustness against interobserver variations caused colorectal cancers to be grouped into five distinctive subgroups, reminiscent of those for consensus molecular subtypes (CMS1-4 and mixed/intermediate group). In accordance with the original CMS report, the CMS4-like subgroup (cluster 4) was significantly associated with a worse 5-year relapse-free survival and proved to be an independent prognostic factor. The clinicopathologic and prognostic features of the TIME subgroups have been validated in an independent validation cohort.Conclusions:Machine-learning–based image analysis can be useful for extracting quantitative information about the TIME, using whole-slide histopathologic images. This information can classify colorectal cancers into clinicopathologically relevant subgroups without performing a molecular analysis of the tumors.

Purpose:Recent sequencing studies revealed that a subset of colorectal cancer harbors a significantly higher number of somatic mutations. These hypermutated tumors show distinct clinicopathologic features. However, the prognostic impact of the hypermutated tumors is not clearly established.Experimental Design:We analyzed tumor mutation burden (TMB) from targeted next-generation sequencing data of 40 major genes in 516 patients with colorectal cancer. TMB was defined as total number of nonsynonymous mutations per tumor. Cutoff value for TMB-high was chosen by which best discriminated relapse-free survival (RFS) using the Contal and O'Quigley method.Results:In the TCGA data, mutation count of the selected 40 genes reflected the whole exome mutation burden (Pearson correlation = 0.873, P < 0.001). In our patient cohort, 8 or more mutations in the 40 genes was defined as TMB-high, which best discriminated RFS. A total of 55 patients (10.7%) had TMB-high. TMB-high tumors were more frequently found in a proximal location (63.6%) and had a higher proportion of N0 disease (30.9%) and MSI-H (49.1%) compared with TMB-low. Most importantly, TMB-high was associated with better 5-year RFS compared with TMB-low (96.3% vs. 79.8%, P = 0.005). Although there was significant overlap between TMB-high and MSI-H, MSI-H status was not significantly associated with RFS. Multivariate analysis revealed TMB-high as an independent positive prognostic factor for RFS [adjusted HR, 0.16 (95% confidence interval, 0.04–0.66), P = 0.011].Conclusions:TMB-high is associated with better prognosis in patients with colorectal cancer treated with curative surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy.

Purpose and Experimental Design: The molecular events in the malignant progression of colon adenoma after loss of adenomatous polyposis coli (APC) are not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin) increases the invasiveness of colorectal cancer cells, and we identified a novel EGFR-independent oncogenic signal of EGF that works under coexpressed KITENIN and ErbB4. Here we tested whether elevated KITENIN and ErbB4 contribute to further progression of intestinal adenoma following APC loss.Results: The intestinal tissues of villin-KITENIN transgenic mice in which villin-driven KITENIN expression induces increased c-Jun expression exhibit mild epithelial cell proliferation but no epithelial lineage changes compared with those of nontransgenic mice. Among the four ErbB4 isoforms, JM-a/CYT-2 and JM-b/CYT-2 exhibited the highest AP-1 activity when cells coexpressing KITENIN and each isoform were stimulated by EGF. Interestingly, predominant overexpression of the ErB4-CYT-2 mRNA as well as increased EGFR expression were observed in intestinal adenoma of APCmin/+ mice, which makes the microenvironment of activated EGF signaling. When we crossed villin-KITENIN mice with APCmin/+ mice, intestinal tumor tissues in the crossed mice showed the characteristics of early-stage invading adenocarcinoma. In patients with colorectal cancer, ErbB4-CYT-2 mRNA expression was significantly greater in tumor tissues than in normal adjacent tissues, but no significant differences in tumor tissue expression were found between different colorectal cancer stages. Furthermore, the mRNA expression of KITENIN and that of ErbB4-CYT-2 were positively correlated in human colorectal cancer tissue.Conclusions: Elevated coexpression of KITENIN and ErbB4-CYT-2 promotes the transition of colon adenoma to adenocarcinoma within an APC loss–associated tumor microenvironment. Clin Cancer Res; 22(5); 1284–94. ©2015 AACR.

PDF file - 177K, Figure 2. Effects of functional blockade of EGFR via treatment of an anti-EGFR monoclonal antibody on enhanced cell invasion by EGF.

PDF file - 135K, Figure 3. EGF-induced AP-1 synergy under elevated KITENIN conditions does not require activated JNK.

PDF file - 662K, Figure 8. Higher expression levels of KITENIN affect the survival of CRC cells to the anti-proliferative effects of cetuximab.

Supplementary data

Characterization of intestinal epithelial tissues from non-TG and KIT-TG mice.

PDF file - 143K, Figure 5. Dvl2-knockdown does not increase transcription of c-Jun and Dvl2 does not interact with c-Jun.

Schematic showing the underlying mechanism of KITENIN/ErbB4-CYT-2 in colon tumor progression.

Effects of elevated KITENIN on cell proliferation and anoikis of intestinal epithelial cells.

Tumor mutation burden in MSI-H patients

Generation of villin-KITENIN transgenic (KIT-TG) mice.

Increased expression of ErbB4-CYT-2, but not ErbB4-CYT-1, isoform in human CRC mucosa.

Immunohistochemical analyses of nontumor intestinal tissues from the APCmin/+ mouse and the compound KIT-TG/APCmin/+ mouse.

Purpose: EGF-stimulated signaling via EGF receptor (EGFR) is important in colorectal tumorigenesis and drug targeting. However, anti-EGFR therapy is not effective in a subset of patients with colorectal cancer, suggesting that unidentified EGF-stimulated pathways might play roles in colorectal cancer. Previously, we identified KAI1 C-terminal interacting tetraspanin (KITENIN) as a metastasis-enhancing gene and found it to be highly expressed in sporadic colorectal cancer tissues. We recently found that EGF further increases KITENIN-induced elevated AP-1 activity. Here we attempted to clarify this novel EGF-stimulated molecular pathway and its roles in colorectal cancer.Experimental Design: We analyzed how EGF modulates the downstream signaling pathway of oncogenic KITENIN in colorectal cancer cells. Biological alterations following EGF treatment were identified in KITENIN-overexpressed colorectal cancer cells with or without alteration of EGFR activity.Results: We identified the KITENIN/ErbB4–Dvl2–c-Jun axis as a novel downstream signal of EGF that is switched on under elevated KITENIN conditions in an EGFR-independent manner. This unconventional EGF signal upregulates c-Jun and enhances invasion and anchorage-independent growth of colorectal cancer cells. In addition, tumor tissues from metastatic patients with colorectal cancer who showed initial poor responses to cetuximab/chemotherapy expressed higher levels of KITENIN than did responders to therapy.Conclusions: Our results highlight the role of an EGFR-independent EGF signal in mediating the invasiveness and tumorigenesis of colorectal cancer cells. This unconventional pathway might be related to the limited clinical efficacy of anti-EGFR agents in a subset of patients with colorectal cancer. Clin Cancer Res; 20(15); 4115–28. ©2014 AACR.

Background: Next-generation sequencing (NGS) technology is gradually expanding in cancer diagnosis and therapeutic decision-making. However, reports on the clinical performance and utility of pan-cancer NGS panel in pediatric patients are still lacking. Methods: We analyzed the patients who underwent brain tumor or pan-cancer NGS panel sequencing at Seoul National University Children’s Hospital. DNA-based NGS panel testing was started in April 2018, and RNA panel was added from January 2022. Single nucleotide variants/small insertions and deletions (SNV/INDEL), fusion, copy number alteration (CNA), microsatellite instability (MSI) and tumor mutational burden (TMB) were evaluated. Results: A total of 140 patients were analyzed between August 2018 and March 2022. The median age at diagnosis was 8.5 (range 0.0-31.8) years. The diagnoses were CNS tumors in 56 (40.0%), sarcoma in 45 (32.1%), other solid tumors in 36 (25.7%) and histiocytosis in 3 (2.1%) patients. At the time of NGS testing, 115 patients (75.2%) were newly diagnosed and 38 patients (24.8%) were in relapsed or refractory status. In all patients, 85.7% had at least one pathogenic or likely pathogenic variant. Diagnosis was refined or changed in five patients (3.6%) after the NGS panel results were reported. Oncogenic gene fusions were discovered in 45 (32.1%) patients. EWSR1, BRAF, NTRK and RET-related fusions were the most common. Frequent SNV/INDEL included BRAF, CTNNB1 and TP53 mutations. Common CNA were CDKN2A and CDKN2B loss, MYCN amplification, CCND3 amplification and PTEN loss. Nine (6.4%) patients were identified with germline alteration, additionally. No patient had MSI, and two patients had high TMB. Fifteen patients (10.7%) were enrolled in clinical trials or compassionate use program according to molecular profiling (DAY101 [BRAF inhibitor] in 3, selpercatinib in 3, palbociclib in 2, olaparib in 2, alectinib in 1, repotrectinib in 1, larotrectinib in 1, atezolizumab in 1 and erdafitinib in 1). Two (1.4%), one (0.7%) and one (0.7%) patients received vemurafenib, larotrectinib, and pembrolizumab with non-reimbursement. Best responses included two complete response (1 high grade glioma [HGG] and 1 renal cell carcinoma), five partial responses (2 low grade glioma, 1 HGG, 1 Langerhans cell histiocytosis, and 1 papillary thyroid carcinoma), two stable disease and six progressive diseases. Four patients were not evaluable due to short duration of drug administration. One-year progression free survival and overall survival was 54.5% and 52.7%, respectively. Conclusions: Application of NGS panel in pediatric cancer aid in diagnosis, treatment decision, clinical trial enrollment and germline risk determination. Although there are not many cases linked with molecular target-based therapy, it is leading to clinical benefits in pediatric patients and more understanding genomic profiling of pediatric cancer. Citation Format: Jung Yoon Choi, Hyun Jin Park, Bo Kyung Kim, Kyung Taek Hong, Jaemoon Koh, Sung-Hye Park, Jeong Mo Bae, Hongseok Yun, Hyoung Jin Kang. Clinical use of next-generation sequencing panel in pediatric oncology patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3398.

Distant metastasis is a major reason for cancer-related death. We previously identified KITENIN (KAI1 C-terminal interacting tetraspanin, Vangl1), a gene encoding a membrane-associated protein, as a metastasis-enhancing gene. The oncogenic KITENIN complex is found to be involved in metastatic dissemination of KITENIN-overexpressing colorectal cancer (CRC) cells and also plays an important role in colorectal carcinogenesis within an APC-loss environment, all of which proposing that the KITENIN complex represents a molecular target for therapeutics aimed at blocking the malignant progression of CRC. To develop novel anti-metastatic agents, we sought to identify therapeutics capable of breaking down the oncogenic KITENIN complex in CRC cells and searched for low-molecular weight compounds that break down the complex, thereby shutting off its oncogenic signals. We then tested the effectiveness of the identified substances in suppressing colorectal liver metastasis (CLM). We found a compound that specifically blocked oncogenic signals from the functional KITENIN complex in CRC cells and renamed it as DKC (Disintegrator of KITENIN Complex) compound. DKC compound bound the KH-type splicing regulatory protein (KSRP), a downstream factor and stabilizer of the functional KITENIN complex, and specifically suppressed invasiveness of CRC cells expressing higher levels of KITENIN. After treatment with DKC compound, RACK1 and microRNA-124 were recruited, resulting in removal of KITENIN from the complex followed by its degradation. DKC suppressed hepatic metastasis effectively in a mouse model of CLM with higher KITENIN. Intriguingly, DKC compound combined with 5-fluorouracil resulted in an enhanced therapeutic effect. Thus, disintegration of the functional KITENIN complex following DKC treatment led to alteration of the specific cellular context induced by the KITENIN complex. Among the synthetic DKC analogues, we selected the one as the optimized leading compound. A docking model study suggested that the interaction between the optimized leading compound with KSRP occurs via insertion of the compound into the binding pocket of the fourth KH-domain and pharmacokinectic studies showed that the favorable blood level was maintained after oral or intravenous administration in rat. Overall, DKC compound specifically blocked oncogenic signals from the functional KITENIN complex in CRC cells with higher KITENIN and suppressed CLM by targeting the KITENIN complex. Our results suggest that a combination regimen with DKC compound could be used more effectively to treat distant metastasis and chemoresistance in CRC patients with high KITENIN expression. Citation Format: Sung Jin Kim, Jeong A. Bae, Yoon Gyoon Kim, Eun Ae Kim, Hyung-Ho Ha, Hangun Kim, Kyung Keun Kim. Suppression of distant metastasis of colorectal cancer by small molecule compound through targeting the oncogenic KITENIN complex [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B018.

Purpose: Recent sequencing studies revealed that a subset of colorectal cancer harbors a significantly higher number of somatic mutations. These hypermutated tumors show distinct clinicopathologic features. However, the prognostic impact of the hypermutated tumors is not clearly established. Experimental Design: We analyzed tumor mutation burden (TMB) from targeted next-generation sequencing data of 40 major genes in 516 patients with colorectal cancer. TMB was defined as total number of nonsynonymous mutations per tumor. Cutoff value for TMB-high was chosen by which best discriminated relapse-free survival (RFS) using the Contal and O'Quigley method. Results: In the TCGA data, mutation count of the selected 40 genes reflected the whole exome mutation burden (Pearson correlation = 0.873, P < 0.001). In our patient cohort, 8 or more mutations in the 40 genes was defined as TMB-high, which best discriminated RFS. A total of 55 patients (10.7%) had TMB-high. TMB-high tumors were more frequently found in a proximal location (63.6%) and had a higher proportion of N0 disease (30.9%) and MSI-H (49.1%) compared with TMB-low. Most importantly, TMB-high was associated with better 5-year RFS compared with TMB-low (96.3% vs. 79.8%, P = 0.005). Although there was significant overlap between TMB-high and MSI-H, MSI-H status was not significantly associated with RFS. Multivariate analysis revealed TMB-high as an independent positive prognostic factor for RFS [adjusted HR, 0.16 (95% confidence interval, 0.04–0.66), P = 0.011]. Conclusions: TMB-high is associated with better prognosis in patients with colorectal cancer treated with curative surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy.

Purpose and Experimental Design: The molecular events in the malignant progression of colon adenoma after loss of adenomatous polyposis coli (APC) are not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin) increases the invasiveness of colorectal cancer cells, and we identified a novel EGFR-independent oncogenic signal of EGF that works under coexpressed KITENIN and ErbB4. Here we tested whether elevated KITENIN and ErbB4 contribute to further progression of intestinal adenoma following APC loss. Results: The intestinal tissues of villin-KITENIN transgenic mice in which villin-driven KITENIN expression induces increased c-Jun expression exhibit mild epithelial cell proliferation but no epithelial lineage changes compared with those of nontransgenic mice. Among the four ErbB4 isoforms, JM-a/CYT-2 and JM-b/CYT-2 exhibited the highest AP-1 activity when cells coexpressing KITENIN and each isoform were stimulated by EGF. Interestingly, predominant overexpression of the ErB4-CYT-2 mRNA as well as increased EGFR expression were observed in intestinal adenoma of APCmin/+ mice, which makes the microenvironment of activated EGF signaling. When we crossed villin-KITENIN mice with APCmin/+ mice, intestinal tumor tissues in the crossed mice showed the characteristics of early-stage invading adenocarcinoma. In patients with colorectal cancer, ErbB4-CYT-2 mRNA expression was significantly greater in tumor tissues than in normal adjacent tissues, but no significant differences in tumor tissue expression were found between different colorectal cancer stages. Furthermore, the mRNA expression of KITENIN and that of ErbB4-CYT-2 were positively correlated in human colorectal cancer tissue. Conclusions: Elevated coexpression of KITENIN and ErbB4-CYT-2 promotes the transition of colon adenoma to adenocarcinoma within an APC loss–associated tumor microenvironment. Clin Cancer Res; 22(5); 1284–94. ©2015 AACR.

Purpose: EGF-stimulated signaling via EGF receptor (EGFR) is important in colorectal tumorigenesis and drug targeting. However, anti-EGFR therapy is not effective in a subset of patients with colorectal cancer, suggesting that unidentified EGF-stimulated pathways might play roles in colorectal cancer. Previously, we identified KAI1 C-terminal interacting tetraspanin (KITENIN) as a metastasis-enhancing gene and found it to be highly expressed in sporadic colorectal cancer tissues. We recently found that EGF further increases KITENIN-induced elevated AP-1 activity. Here we attempted to clarify this novel EGF-stimulated molecular pathway and its roles in colorectal cancer. Experimental Design: We analyzed how EGF modulates the downstream signaling pathway of oncogenic KITENIN in colorectal cancer cells. Biological alterations following EGF treatment were identified in KITENIN-overexpressed colorectal cancer cells with or without alteration of EGFR activity. Results: We identified the KITENIN/ErbB4–Dvl2–c-Jun axis as a novel downstream signal of EGF that is switched on under elevated KITENIN conditions in an EGFR-independent manner. This unconventional EGF signal upregulates c-Jun and enhances invasion and anchorage-independent growth of colorectal cancer cells. In addition, tumor tissues from metastatic patients with colorectal cancer who showed initial poor responses to cetuximab/chemotherapy expressed higher levels of KITENIN than did responders to therapy. Conclusions: Our results highlight the role of an EGFR-independent EGF signal in mediating the invasiveness and tumorigenesis of colorectal cancer cells. This unconventional pathway might be related to the limited clinical efficacy of anti-EGFR agents in a subset of patients with colorectal cancer. Clin Cancer Res; 20(15); 4115–28. ©2014 AACR.

Introduction: Determining immunogenicity of tumors is important in predicting response to cancer immunotherapy. Tumor mutation burden, the degree of copy number variation expressed as chromosomal instability score and gene expression profiles (GEP) of tumors, such as cytolytic activity score, interferon-gamma signature and immune signature, are known biomarkers of immunogenicity. However, correlation between methylation burden and signature of tumor immunogenicity is unknown. Methods: We used The Cancer Genome Atlas (TCGA) pan-cancer database to investigate association of RNA sequencing data and methylation signature of tumors generated by HumanMethylation450K BeadChip. The estimated methylation levels in the CpG sites were expressed in β-scores. To determine immunogenicity in a sample, we used cytolytic activity (CytAct) score defined by the summation of RNA transcript levels of granzyme A (GZMA) and perforin 1 (PRF1). In each CpG site, β-scores of top 5% and bottom 5% were defined as “hypermethylated” and “hypomethylated”, respectively. Methylation burden of a sample was defined as number of CpG sites having hypermethylated or hypomethylated β-scores. Results: In TCGA, total of 7,914 pan-cancer samples had both RNA sequencing data and methylation array data. Methylation burdens of pan-cancer samples were negatively correlated with CytAct scores. (Spearman’s correlation rho value -0.37, p < 2.2e-16). This negative trend was consistently observed in most of cancer types and methylation subtypes of individual cancer including breast cancer. Using multivariate linear regression model, methylation burden predicted CytAct score independently along with mutation burden and chromosomal instability score. Hypermethylations in CpG sites of genes related to interferon gamma response(CXCL10, STAT1, IFI6and IFI27), lymphocyte infiltration(CD8A, CD3, CD79, LCK and CCL5) and tumor antigen recognition were associated with decreased CytAct scores whereas hypomethylation in CpG sites of genes related to TGF-β(CTNNB1, COL1A2, IGF2R, ITGB2, MMP17, SPARC and SMO) and fibroblast response(PLAUR, PLOD2, LOXL2 and MET) were associated with decreased CytAct scores in pan-cancer analysis. Conclusions: The methylation burden of tumor has negative correlation with the immunogenicity of tumors in general pan-cancer. Correlation of the specific methylation pattern with the response of immunotherapy is warranted in further clinical study. Citation Format: Changhee Park, Kyeonghun Jeong, Joon-Hyeong Park, Jeong Mo Bae, Kwangsoo Kim, Chan-Young Ock, Miso Kim, Bhumsuk Keam, Tae Min Kim, Yoon Kyung Jeon, Dong-Wan Kim, Gyeong Hoon Kang, Doo Hyun Chung, Dae Seog Heo. Methylation landscape of cancers associated with immunogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1498.

The most reliable predictive biomarker of cancer immunotherapy is gene expression profile (GEP) of tumor microenvironment. GEPs such as local immune cytolytic activity, interferon-gamma signature, and immune signature score have been reported to represent anti-tumor immune signature. Previously, we reported that immune signature score was positively correlated with tumor mutational burden, but negatively correlated with chromosomal instability (CIN) score, since tumors with high CIN score had significantly low neoantigen burden. However, methylation signature or burden of tumor would also affect antitumor immunogenicity, there has been no analysis reported so far. In the current study, we investigated if methylation landscape of tumor would be associated with GEPs of anti-tumor immune signature using The Cancer Genome Atlas (TCGA) pan-cancer database. In TCGA, 8269 pan-cancer samples had both RNA sequencing data and methylation data using Infinium HumanMethylation450K BeadChip, which were included in the main analysis. Although tumors with high mutational burden (Mu-type) and high CIN burden (C-type) were exclusively classified with negative correlation, methylation burden was not correlated with mutational burden or CIN burden in any pattern. Interestingly, antitumor immune signature measured by local immune cytolytic activity (CytAct) was clearly decreased with high methylational burden, as seen in high CIN burden. Hypermethylation of promoter of genes related to tumor antigen recognition by T-cell such as HLA family, B2M, CD74, and CD274 (PD-L1) were negatively associated with CytAct in pan-cancer analysis. In conclusion, methylation signature of tumor is also associated with antitumor immunogenicity with a negative correlation in general. Further study of whether specific methylation pattern would be associated with anti-PD-1/PD-L1 inhibitors in clinical study would be warranted. Citation Format: Chan-Young Ock, Changhee Park, Kyeonghun Jeong, Sohee Jung, Jeong Mo Bae, Kwangsoo Kim. Methylation landscape of tumors associated with antitumor immune signature [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B084.

Purpose: Understanding the complex biological functions of E3-ubiquitin ligases may facilitate the modulation of E3-ubiquitin ligases as a promising approach for the development of novel anticancer drugs. We recently identified that the KITENIN/ErbB4-Dvl2-c-Jun axis works as a novel unconventional downstream signal of epidermal growth factor in colorectal cancer (CRC) tissues and that the immunohistochemical expression of KITENIN/ErbB4 was highly expressed in tumor tissues from advanced CRC stage. However, the detailed mechanisms that explain the higher levels of ErbB4 in colon cancer tissues are largely unknown. Here we investigated whether E3-ubiquitin ligases participate in the operation of the KITENIN/ErbB4-Dvl2-c-Jun axis and in the maintenance of elevated KITENIN/ErbB4 complex in CRC. Results & Discussion: We found that Nrdp1, an E3-ligase for ErbB3/ErbB4, interacted with KITENIN (KAI1 C-terminal interacting tetraspanin) to form a functional KITENIN/ErbB4/Nrdp1 complex and is responsible for down-regulating Dvl2 within this complex. Interestingly, ErbB4 was resistant to degradation by Nrdp1 in KITENIN/Nrdp1-co-transfected CRC cells, and KITENIN bound to the C-terminal coiled-coil domain of Nrdp1. Chemical blockade of ErbB kinase did not block the action of EGF to increase in total/phospho-ErbB4 and phospho-ERK in KITENIN/ErbB4-cotransfected cells, whereas it blocked the action of EGF in ErbB4 alone-transfected CRC cells. In human CRC tissues, higher expressions of ErbB4 and KITENIN and lower expression of Dvl2 was observed in stage IV samples than in stage I, but a low level of Nrdp1 was expressed in both stages and it did not differ significantly by stage. These results indicated that Nrdp1 is necessary for the reduction in Dvl2 to generate c-Jun in the EGF-KITENIN/ErbB4-c-Jun axis, but more importantly, elevated KITENIN protects KITENIN-bound ErbB4 from Nrdp1-mediated degradation via physical collaboration between the KITENIN/ErbB4 complex and Nrdp1, but not via modulation of ErbB kinase activity. Thus, KITENIN functions in the maintenance of a higher expression level of ErbB4 in advanced CRC tissues, independent of ubiquitin-mediated degradation via Nrdp1. Conclusion: Our present findings add a new component to our understanding of the molecular events underlying the regulation of ErbB4 expression level in CRC tissues: KITENIN is also a fine regulator of ErbB4 expression in addition to E3-ubiquitin ligase Nrdp1. Citation Format: Jeong A Bae, Eun Gene Sun, Yoo-Seung Ko, Hui Jeong Choi, Chaeyong Jung, Kyung-Hwa Lee, Ik Joo Chung, Kyung-Sub Moon, Young Hyun Yu, Hyung-Ho Ha, Hangun Kim, Kyung Keun Kim. KITENIN works as a fine regulator of ErbB4 expression in colorectal cancer tissues in addition to E3-ubiquitin ligase Nrdp1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3530. doi:10.1158/1538-7445.AM2017-3530

Purpose: The molecular events in the malignant progression of colon adenoma after loss of adenomatous polyposis coli (APC) are not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin) increases the invasiveness of colorectal cancer (CRC) cells and we recently identified a novel EGFR-independent oncogenic signal of EGF that works under co-expressed KITENIN and ErbB4. Here we tested whether elevated KITENIN and ErbB4 contributes to further progression of intestinal adenoma following APC loss. Results & Discussion: The intestinal tissues of villin-KITENIN transgenic mice in which villin-driven elevated KITENIN induced increased c-Jun expression exhibit mild epithelial cell proliferation but no epithelial lineage changes, compared with those of non-transgenic mice. Among the 4 ErbB4 isoforms, JM-a/CYT-2 and JM-b/CYT-2 exhibited the highest AP-1 activity when cells co-expressing KITENIN and each isoform were stimulated by EGF. Interestingly, predominant overexpression of ErbB4 CYT-2 isoform mRNA as well as increased EGFR expression were observed in intestinal adenoma of APCmin/+ mice making the microenvironment of activated EGF signaling. When we crossed villin-KITENIN mice with APCmin/+ mice, intestinal tumor tissues in the crossed mice showed the characteristics of early stage invading adenocarcinoma. In CRC patients, ErbB4-CYT-2 mRNA expression was significantly greater in tumor tissues than in normal adjacent tissues, but no differences in tumoral expression between the stages, and a positive correlation exists between KITENIN and ErbB4-CYT-2 mRNA expression. Thus, elevated expression of KITENIN and ErbB4-CYT-2 is newly identified as a factor that promotes the transition of colon adenoma to adenocarcinoma within an APC-loss-associated tumor microenvironment. Conclusion: Higher mRNA expression of both KITENIN and ErbB4-CYT-2 in colon adenoma tissues can be used as a molecular marker of the possible development of malignant transformation. Citation Format: Jeong A Bae, Eun Gene Sun, Yoo-Seung Ko, Hui Jeong Choi, Woo-Kyun Bae, Sang-Hee Cho, Ik Joo Chung, Kyung-Sub Moon, Young-Hyun Yu, Hyung-Ho Ha, Hangun Kim, Kyung Keun Kim. Identification of higher mRNA expression of both Kitenin and ErbB4 CYT-2 isoform as a molecular marker for the prediction of transition from colon adenoma to carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5150.

Background Although genomic data of CRC continue to accumulate, only limited information on prognostic role of the alterations have been reported. We have analyzed the prognostic impact of the key mutations in CRC using next generation sequencing technology. Methods We selected 40 genes from 5 critical pathways (WNT, TGF-B, PI3K, RTK-RAS and P53) of CRC based on TCGA data. Homogenous population of CRC patients were used to investigate the prognostic implication: 188 stage III or high-risk stage II CRC patients treated with curative surgery followed by adjuvant 5-fluorouracil/ leucovorin/oxaliplatin (FOLFOX) chemotherapy. Archival tissue from Seoul National University Hospital Tumor Bank was used. The target (size 109kb) enrichment process was proceeded base on in-solution hybridization with biotinylated probes. The captured library was amplified and sequenced using Hiseq 2500 (Illumina, USA). Sequencing data was aligned to GRCh37 and variant call and somatic analysis processes was performed by VarScan2 and were annotated with ANNOVAR. Using somatic nonsynonymous mutation data, we selected mutant gene signature which is associated with prognosis. We used binary collapsing method in a forward stepwise selection method to combine mutated genes. Test statistics were obtained from Cox-proportional hazard model. Two-sided p-values of less than 0.05 were considered statistically significant. Results Among a total of 188 patients, 63 had tumor in proximal location and 125 had tumor in distal location. Tumor stage was high-risk stage II in 21 patients and III in 167 patients. Average coverage of the total samples was 417X (414X tumor and 420X normal mucosa). Mutation frequencies were similar with the TCGA data except for NRAS and DKK2, having lower frequency in the present study. During a median follow-up duration of 58 months, 45 relapse and 23 death events have occurred. Alteration in individual gene or pathway did not have prognostic significance in terms of disease free survival (DFS) and overall survival (OS). By using binary collapsing method, we identified a 4 gene signature comprising ACVR1B, ERBB2, LRP5, and KRAS. 89 patients (47.3%) had 1 or more mutation in these 4 genes. 4 gene mutant group had worse DFS and OS compared with 4 gene wild type group. 3-year DFS and 5-year OS was 72.8% and 82.5% in mutant group compared with 88.7% and 96.0% in wild type group (p-values 0.004 and 0.001, respectively). Multivariate analysis revealed 4 gene signature as an independent negative prognostic factor of DFS and OS (adjusted hazard ratios 2.48 and 4.26, respectively). Conclusion Mutations in the 4 genes (ACVR1B, ERBB2, LRP5, and KRAS) were associated with poor prognosis in CRC patients treated with surgery followed by adjuvant FOLFOX chemotherapy. Validation study in an independent cohort is currently underway. Citation Format: Dae-Won Lee, Yongjun Cha, Sae-Won Han, Si-Hyun Lee, Hwang-Phill Kim, Jaemyun Lyu, Hyojun Han, Hyoki Kim, Hoon Jang, Duhee Bang, Iksoo Huh, Taesung Park, Jeong Mo Bae, Jae-Kyung Won, Seung-Yong Jeong, Kyu Joo Park, Gyeong Hoon Kang, Tae-You Kim. Mutation signature associated with colorectal cancer prognosis identified by targeted next-generation sequencing. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A72.

β-catenin is a major transducer in Wnt signaling pathway, which is highly expressed in colorectal and other cancers. Previously, we have shown that β-catenin is down-regulated in colorectal cancer cells by glycoprotein, 90K, through ISGylation-dependent ubiquitination. The aim of this study is to further clarify a novel mechanism of β-catenin down-regulation pathway by 90K glycoprotein. To identify ISGylation site of β-catenin, deletion mutants of β-catenin lacking N- or C-terminal domain were employed. As only two lysine residues, K19 and K49, are reside in N-terminus of β-catenin, we sequenced cDNA of β- catenin from paired human colorectal cancer tissues to find the mutation and generated arginine point-mutation. As a result, there is no mutation on K19 and/or K49 in examined specimens and did not affect the 90K-mediated ISGylational degradation. To rule out the possibility of involving non-lysine residue in the ISG15 conjugation, such as serine or threonine, we constructed several deletion mutants and found that none are responsible for the ISG15 conjugation. Now, N-terminal portion of β-catenin is under screening for possible interactions with putative E3 ligases, and further detailed studies delineating the complex formation among ISG15 and β-catenin/E3 ligases are under way. Citation Format: Somy Yoon, Jeong A Bae, Hangun Kim, Kyung-Sub Moon, Kyung Keun Kim. N-terminal portion of β-catenin is important in ISGylation of β-catenin by 90K glycoprotein in colorectal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4431. doi:10.1158/1538-7445.AM2014-4431

MicroRNAs (miRNAs) are increasingly implicated in modulating the progression of various cancers. Although there is emerging evidences that some miRNAs can function as oncogenes or tumour suppressors, the regulation of colorectal cancer (CRC) progression by miRNA is not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin, Vangl1) is not only a major component of PCP pathway but also a functional protein promoting CRC cell motility and invasiveness. Previously, we observed that KITENIN acts as a metastasis-enhancing protein in a mouse model of colon cancer and that KITENIN is highly expressed in sporadic human CRC tissues. But the underlying regulation mechanisms of aberrant expression of KITENIN in CRC are not clearly understood. Here we tried to identify the miRNAs, which modulate the expression of KITENIN, using luciferase assay and with computational prediction of miRNA targeting KITENIN. We identified several miRNAs and one of them is miR-124. miR-124 negatively regulated KITENIN expression through binding to the 3′-untranslated region of KITENIN. We further examined whether increased expression of miR-124 affects colorectal tumor progression via targeting KITENIN. Ectopic expression of miR-124 was found to suppress the migration and invasiveness of various CRC cells. miR-124 also inhibited tumor growth in a mouse xenograft model. These findings provide the experimental evidence to a possible therapeutic effect of miR-124 on suppressing colorectal tumor progression and identification of miR-124-mediated regulation of KITENIN might provide a promising therapeutic target in treating CRC. Citation Format: So-Yeon Park, Jeong A Bae, Somy Yoon, Hangun Kim, Kyung Keun Kim. miR-124 suppresses colorectal tumor progression in a mouse xenograft model via targeting KITENIN. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4201. doi:10.1158/1538-7445.AM2013-4201

Background & aims: KITENIN (KAI1 C-terminal interacting tetraspanin) is not only a membrane-associated protein but also a functional protein promoting the invasiveness of colorectal cancer (CRC) cells. Recent studies about the mechanism of KITENIN function in the CRC cells indicate that KITENIN plays a pivotal role in tumor initiation and promotion through activating the activator protein-1 (AP-1) signaling. However, the contribution of each functional domain of KITENIN to AP-1 signaling is still unknown and the biochemical properties of C-terminal region of KITENIN were investigated. Methods: To compare the biochemical and functional properties of KITENIN deletion mutants with wild-type KITENIN, the various mutant constructs of C-terminal region (238-512 aa) were generated and being expressed in the 293T and Caco2 cells. AP-1 activity was measured via AP-1 luciferase reporter assays, and the cellular phenotypes of these cells expressing KITENIN mutants were examined via in vitro cell invasion and wound-healing assays. Results: KITENIN mutant with deletion of C-terminal 63 amino acids (DCKIT) markedly increased the AP-1 activity in contrast to other deletion mutants and wild-type KITENIN. Also, DCKIT increased the degradation of Dvl2 and stability of c-Jun, which are responsible for AP-1 activation by KITENIN. The C-terminal fragment of KITENIN (KICD, roughly 30 kDa) was generated and preferentially translocated to the nucleus after PMA treatment, but its functional significance is under investigation. A mutant construct of 408ALRA411-KITENIN was obtained by mutation of YXXΨ motifs, which directs the clathrin-dependent endocytosis of membrane-spanning proteins. This mutation did not influence the AP-1 activity, cell invasiveness, and cell migration by KITENIN but disrupted the interaction of KITENIN with Dvl2. Moreover, co-expression of this mutant with ErbB4 exhibited more attenuated AP-1 activity than that of forced expression of wild-type KITENIN and ErbB4. These results indicate that endocytosis of KITENIN and subsequent interaction with Dvl2 is essential to activating AP-1 signal and modulating cell motility by KITENIN. Conclusion: These biochemical properties of deletion mutants of KITENIN suggest that the C-terminal region of KITENIN is an important component for modulating AP-1 signal and CRC cell motility by KITENIN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4006. doi:1538-7445.AM2012-4006

KITENIN (KAI1 C-terminal interacting tetraspanin, Vangl1) is not only a major component of PCP pathway but also a functional protein promoting colorectal cancer (CRC) cell motility and invasiveness. Here, we report the mechanism of which KITENIN leads further malignant changes in CRC progression after loss of tumor suppressor Adenomatous polyposis coli (APC). KITENIN increases the level of AP-1 activity through interaction with Dvl2, which is subsequently being degraded. Decreasing Dvl2 via interaction with KITENIN or via treatment of si-RNA to Dvl2 significantly increases the AP-1 activity through elevating the level of c-jun and c-jun phosphorylation. EGF acts upstream of KITENIN/AP-1 signaling and induces synergic AP-1 activation in the presence of elevated KITENIN, which requires the interaction of KITENIN with ErbB4. The morphological characteristics of adenocarcinoma were observed in intestinal tumor tissues from compound villin-KITENIN transgenic/APCmin/+ mice but not from APCmin/+ mice, both of which express the elevated level of ErbB4. This result demonstrates that further malignant phenotype of intestinal tumor can be induced by elevated KITENIN and ErbB4 via promoting c-Jun/AP-1 activation within an APC loss-associated tumor microenvironment. Our present results suggest the possibility of the addition of anti-KITENIN therapy to the use of EGFR inhibitors in the treatment of CRC patients with APC mutation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2733. doi:10.1158/1538-7445.AM2011-2733

Backgrounds: Previously, we have observed that a tumor-associated 90K glycoprotein plays a potential tumor suppressor role in colorectal cancer (CRC) cells via antagonizing the canonical Wnt signaling pathway, which is functionally masked via interaction with extracellular galectin. Here, we investigated the signaling pathways of 90K action on CRC cells and its expression in human CRC tissues. Results: We identify a novel pathway comprising a secretory 90K and a CD9/CD82 tetraspanin web; in this pathway, 90K interacts with CD9/CD82, suppresses the Wnt/β-catenin signal via a novel proteasomal-ubiquitination mechanism of β-catenin that is dependent of ISG15 modification (ISGylation). In colon tissues from stage IV human CRC and invading cancer cells of corresponding metastatic liver tissues, in which β-catenin and galectin expression was higher, immunostained 90K and CD9/CD82 were lower than in adjacent hepatic tissues or colon tissues from stage I. Conclusions: 90K itself has antitumor activity in CRC cells via suppression of Wnt signaling with a novel mechanism of ISGylation-dependent ubiquitination of β-catenin when it interacts with CD9/CD82, but is downregulated in advanced CRC tissues. Our data suggest a strategy of strengthening this novel pathway with concomitant knockdown of galectins as a potential therapeutic approach to CRC progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1471.

Background: Currently, primary treatments for stage IV breast cancer are radiation and systemic therapy which include chemotherapy, endocrine therapy and targeted therapy. Surgical resection of primary tumor is usually done for tumor-related complications. A recent review suggested that surgery may improve long-term survival in stageIV breast cancer patients. We evaluated the impact of primary site surgical resection on survival in such patients.Material and Methods: We reviewed the records of stage IV breast cancer patients treated at Seoul National University Hospital between 1992 and 2008. Clinical and tumor characteristics, systemic and local treatments were compared for the surgically versus nonsurgically treated patients.Results: Of 199 patients identified, 111(55.8%) recevied surgical excision of their primary tumor and 88(44.2%) did not. The mean survival of surgically treated patients was 67 months versus 52 months for patients those who did not(p=0.0276). In multivariate analysis, after adjustment for ER status, visceral metastasis, number of metastatic sites and herceptin treatment, surgery remained an independent factor associated with improved survival(HR 0.547[95%CI 0.359-0.971] p=0.001).Discussion: Surgical resection of the primary tumor in stage IV breast cancer patient was independently associated with improved survival, even after adjustment for other factors associated with survival. Randomized prospective trials are needed to validate these findings. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3117.