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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.

Usnic acid (UA), a lichen secondary substance, has considerable anticancer activity in vitro, whereas its effect in vivo is limited. Here, potassium usnate (KU) was prepared by the salinization of UA to enhance its water solubility. KU showed increased bioavailability compared with UA in the tumor, liver, and plasma of a CT26 syngeneic mouse tumor xenograft model after oral administration, as determined by LC-MS/MS analysis. KU exhibited potent anticancer effects on colorectal cancer cells and inhibited liver metastasis in an orthotopic murine colorectal cancer model. KU treatment downregulated the epithelial-mesenchymal markers Twist, Snail, and Slug and the metastasis-related genes CAPN1, CDC42, CFL1, IGF1, WASF1, and WASL in cells and tumor tissues. The present results suggest the potential application of the water-soluble form of UA, KU, in anticancer therapy. Citation Format: Yi Yang, Woo Kyun Bae, Kyung Hwa Lee, Kyung-Sub Moon, So-Yeon Park, Rui Zhou, Isa Taş, Chathurika Gamage, Sueun Lee, Chaeyong Jung, Kyung Keun Kim, Hyung-Ho Ha, Hangun Kim. Potassium usnate inhibits invasion and metastasis in colorectal cancer: Implication of clinical application of a water-soluble form of usnic acid in anticancer therapy [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 2011.

Lichens produce various unique chemicals that can be used for pharmaceutical purposes. To screen for novel lichen secondary metabolites showing inhibitory activity against lung cancer cell motility, we tested acetone extracts of 13 lichen samples collected in Chile. Physciosporin, isolated from Pseudocyphellaria coriacea (Hook f. & Taylor) D.J. Galloway & P. James, was identified as an effective compound and showed significant inhibitory activity in migration and invasion assays against human lung cancer cells. Physciosporin treatment reduced both protein and mRNA levels of N-cadherin with concomitant decreases in the levels of epithelial-mesenchymal transition markers such as snail and twist. Physciosporin also suppressed KITENIN (KAI1 C-terminal interacting tetraspanin)-mediated AP-1 activity in both the absence and presence of epidermal growth factor stimulation. Quantitative real-time PCR analysis showed that the expression of the metastasis suppressor gene, KAI1, was increased while that of the metastasis enhancer gene, KITENIN, was dramatically decreased by physciosporin. Particularly, the activity of 3'-untranslated region of KITENIN was decreased by physciosporin. Moreover, Cdc42 and Rac1 activities were decreased by physciosporin. These results demonstrated that the lichen secondary metabolite, physciosporin, inhibits lung cancer cell motility through novel mechanisms of action. Citation Format: Yi Yang, Young Hyun Yu, Hyung-Ho Ha, Kyung-Hwa Lee, Kyung-Sub Moon, Kyung Keun Kim, Hangun Kim. Inhibitory activity of lichen secondary metabolite, physciosporin, against lung cancer cell motility [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 4209. doi:10.1158/1538-7445.AM2017-4209

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.