5-FU promotes stemness of colorectal cancer via p53-mediated WNT/ß-catenin pathway activation.

5-Fluorouracil (5-FU) remains the first-line treatment for colorectal cancer (CRC). Although 5-FU initially de-bulks the tumor mass, recurrence after chemotherapy is the barrier to effective clinical outcomes for CRC patients. Here, we demonstrate that p53 promotes WNT3 transcription, leading to activation of the WNT/ß-catenin pathway in ApcMin/+/Lgr5EGFP mice, CRC patient-derived tumor organoids (PDTOs) and patient-derived tumor cells (PDCs). Through this regulation, 5-FU induces activation and enrichment of cancer stem cells (CSCs) in the residual tumors, contributing to recurrence after treatment. Combinatorial treatment of a WNT inhibitor and 5-FU effectively suppresses the CSCs and reduces tumor regrowth after discontinuation of treatment. These findings indicate p53 as a critical mediator of 5-FU-induced CSC activation via the WNT/ß-catenin signaling pathway and highlight the significance of combinatorial treatment of WNT inhibitor and 5-FU as a compelling therapeutic strategy to improve the poor outcomes of current 5-FU-based therapies for CRC patients.

PIBF1 suppresses the ATR/CHK1 signaling pathway and promotes proliferation and motility of triple-negative breast cancer cells.

PURPOSE: This study evaluates the oncogenic role of PIBF1 in triple-negative breast cancer (TNBC). TNBC is considered to have a poorer prognosis than other types of breast cancer and is associated with high risk of recurrence and distant metastasis. Currently, there are no effective therapies for the TNBC patients with distant metastasis due to the lack of targeted therapeutic options. METHODS: The effects of PIBF1 knockdown on the cell viability and motility of TNBC cell lines were investigated. Effects of PIBF1 overexpression on tumorigenicity and cell motility were confirmed using Ba/F3 cell line and xenograft study on BALB/c nude mice. RESULTS: In TNBC cell lines that highly express PIBF1, knockdown of PIBF1 induces apoptosis and suppresses cell viability and motility with activation of the ATR/CHK1 signaling pathway. Moreover, the oncogenic function of PIBF1 was confirmed using the Ba/F3 cell line. CONCLUSION: For the first time, these findings clarify the role of PIBF1 in regulating ATR/CHK1 signaling pathway and inhibiting the proliferation and migration of TNBC cell lines. These results demonstrate the oncogenic roles of PIBF1 and provide new insights into the function and the molecular mechanism of PIBF1 in malignant TNBC.

Small molecule-induced simultaneous destabilization of ß-catenin and RAS is an effective molecular strategy to suppress stemness of colorectal cancer cells.

BACKGROUND: Cancer stem cells (CSCs), the major driver of tumorigenesis, is a sub-population of tumor cells responsible for poor clinical outcomes. However, molecular mechanism to identify targets for controlling CSCs is poorly understood. METHODS: Gene Set Enrichment Analyses (GSEA) of Wnt/ß-catenin and RAS signaling pathways in stem-like subtype of colorectal cancer (CRC) patients were performed using two gene expression data set. The therapeutic effects of destabilization of ß-catenin and RAS were tested by treatment of small molecule KYA1797K using CRC patient derived cells. RESULTS: Treatment with KYA1797K, a small molecule that destabilizes both ß-catenin and RAS via Axin binding, effectively suppresses the stemness of CSCs as shown in CRC spheroids and small intestinal tumors of ApcMin/+/K-RasG12DLA2 mice. Moreover, KYA1797K also suppresses the stemness of cells in CRC patient avatar model systems, such as patient-derived tumor organoids (PDTOs) and patient-derived tumor xenograft (PDTX). CONCLUSION: Our results suggest that destabilization of both ß-catenin and RAS is a potential therapeutic strategy for controlling stemness of CRC cells. Video abstract.

WDR76 degrades RAS and suppresses cancer stem cell activation in colorectal cancer.

BACKGROUND: Stabilization of RAS is a key event for the hyper-activation of Wnt/ß-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC). WD Repeat protein 76 (WDR76) mediates the polyubiquitination-dependent degradation of RAS in hepatocellular carcinoma (HCC). We investigated whether WDR76 destabilizes RAS and acts as a tumor suppressor inhibiting CSC activation in CRC. METHODS: We generated mice with deletion of Wdr76 (Wdr76-/-) and crosses of Wdr76-/- with ApcMin/+ (Wdr76-/-; ApcMin/+) and compared them with wildtype mice (Wdr76+/+) and ApcMin/+ mice (Wdr76+/+; ApcMin/+), respectively. Intestinal crypt lengthening, tumorigenesis and CSC activation were analyzed by histology, immunohistochemistry, and immunoblotting. CRC cell line was engineered to stably express or knockdown WDR76 or control vector and was analyzed after spheroid culture. RESULTS: Wdr76-/- mice, with increased Ras level, displayed crypt elongation and hyper-proliferation. Wdr76-/-; ApcMin/+ mice developed more tumors with bigger sizes than ApcMin/+ mice and their tumors showed increased proliferation and CSC activation with elevated RAS and ß-catenin levels. In CRC cells, overexpression or knockdown of WDR76 decreased or increased the numbers and sizes of CRC spheroids with inhibition or activation of CSC markers, respectively. In human CRC, lower level of WDR76 was associated with poor patient survival. CONCLUSIONS: In analyses of mice with deletion of Wdr76 and CRC spheroids, we found that RAS stability plays important roles in tumorigenesis by affecting proliferation and CSC activation. Our results suggest that destabilization of RAS by WDR76 is a potential strategy for targeting malignant CRC involving CSC activation.

WDR76 is a RAS binding protein that functions as a tumor suppressor via RAS degradation.

Stability regulation of RAS that can affect its activity, in addition to the oncogenic mutations, occurs in human cancer. However, the mechanisms for stability regulation of RAS involved in their activity and its roles in tumorigenesis are poorly explored. Here, we identify WD40-repeat protein 76 (WDR76) as one of the HRAS binding proteins using proteomic analyses of hepatocellular carcinomas (HCC) tissue. WDR76 plays a role as an E3 linker protein and mediates the polyubiquitination-dependent degradation of RAS. WDR76-mediated RAS destabilization results in the inhibition of proliferation, transformation, and invasion of liver cancer cells. WDR76-/- mice are more susceptible to diethylnitrosamine-induced liver carcinogenesis. Liver-specific WDR76 induction destabilizes Ras and markedly reduces tumorigenesis in HRasG12V mouse livers. The clinical relevance of RAS regulation by WDR76 is indicated by the inverse correlation of their expressions in HCC tissues. Our study demonstrates that WDR76 functions as a tumor suppressor via RAS degradation.

A small molecule approach to degrade RAS with EGFR repression is a potential therapy for KRAS mutation-driven colorectal cancer resistance to cetuximab.

Drugs targeting the epidermal growth factor receptor (EGFR), such as cetuximab and panitumumab, have been prescribed for metastatic colorectal cancer (CRC), but patients harboring KRAS mutations are insensitive to them and do not have an alternative drug to overcome the problem. The levels of ß-catenin, EGFR, and RAS, especially mutant KRAS, are increased in CRC patient tissues due to mutations of adenomatous polyposis coli (APC), which occur in 90% of human CRCs. The increases in these proteins by APC loss synergistically promote tumorigenesis. Therefore, we tested KYA1797K, a recently identified small molecule that degrades both ß-catenin and Ras via GSK3ß activation, and its capability to suppress the cetuximab resistance of KRAS-mutated CRC cells. KYA1797K suppressed the growth of tumor xenografts induced by CRC cells as well as tumor organoids derived from CRC patients having both APC and KRAS mutations. Lowering the levels of both ß-catenin and RAS as well as EGFR via targeting the Wnt/ß-catenin pathway is a therapeutic strategy for controlling CRC and other types of cancer with aberrantly activated the Wnt/ß-catenin and EGFR-RAS pathways, including those with resistance to EGFR-targeting drugs attributed to KRAS mutations.

ß-Catenin-RAS interaction serves as a molecular switch for RAS degradation via GSK3ß.

RAS proteins play critical roles in various cellular processes, including growth and transformation. RAS proteins are subjected to protein stability regulation via the Wnt/ß-catenin pathway, and glycogen synthase kinase 3 beta (GSK3ß) is a key player for the phosphorylation-dependent RAS degradation through proteasomes. GSK3ß-mediated RAS degradation does not occur in cells that express a nondegradable mutant (MT) ß-catenin. Here, we show that ß-catenin directly interacts with RAS at the α-interface region that contains the GSK3ß phosphorylation sites, threonine 144 and threonine 148 residues. Exposure of these sites by prior ß-catenin degradation is required for RAS degradation. The introduction of a peptide that blocks the ß-catenin-RAS interaction by binding to ß-catenin rescues the GSK3ß-mediated RAS degradation in colorectal cancer (CRC) cells that express MT ß-catenin. The coregulation of ß-catenin and RAS stabilities by the modulation of their interaction provides a mechanism for Wnt/ß-catenin and RAS-ERK pathway cross-talk and the synergistic transformation of CRC by both APC and KRAS mutations.

Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of ß-catenin signaling.

Although the development of drugs that control Ras is an emerging topic in cancer therapy, no clinically applicable drug is currently available. We have previously utilized knowledge of the Wnt/ß-catenin signaling-dependent mechanism of Ras protein stability regulation to identify small molecules that inhibit the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both ß-catenin and Ras. Due to the absence of Ras degradation in cells expressing a nondegradable mutant form of ß-catenin and the need to determine an alternative mechanism of Ras degradation, we designed a cell-based system to screen compounds that degrade Ras independent of the Wnt/ß-catenin signaling pathway. A cell-based high-content screening (HCS) system that monitors the levels of EGFP-K-RasG12V was established using HCT-116 cells harboring a nondegradable mutant CTNNB1 (ΔS45). Through HCS of a chemical library composed of 10,000 compounds and subsequent characterization of hits, we identified several compounds that degrade Ras without affecting the ß-catenin levels. KY7749, one of the most effective compounds, inhibited the proliferation and transformation of CRC cells, especially KRAS-mutant cells that are resistant to the EGFR monoclonal antibody cetuximab. Small molecules that degrade Ras independent of ß-catenin may able to be used in treatments for cancers caused by aberrant EGFR and Ras.

Interaction between Wnt/ß-catenin and RAS-ERK pathways and an anti-cancer strategy via degradations of ß-catenin and RAS by targeting the Wnt/ß-catenin pathway.

Aberrant activation of the Wnt/ß-catenin and RAS-extracellular signal-regulated kinase (ERK) pathways play important roles in the tumorigenesis of many different types of cancer, most notably colorectal cancer (CRC). Genes for these two pathways, such as adenomatous polyposis coli (APC) and KRAS are frequently mutated in human CRC, and involved in the initiation and progression of the tumorigenesis, respectively. Moreover, recent studies revealed interaction of APC and KRAS mutations in the various stages of colorectal tumorigenesis and even in metastasis accompanying activation of the cancer stem cells (CSCs). A key event in the synergistic cooperation between Wnt/ß-catenin and RAS-ERK pathways is a stabilization of both ß-catenin and RAS especially mutant KRAS by APC loss, and pathological significance of this was indicated by correlation of increased ß-catenin and RAS levels in human CRC where APC mutations occur as high as 90% of CRC patients. Together with the notion of the protein activity reduction by lowering its level, inhibition of both ß-catenin and RAS especially by degradation could be a new ideal strategy for development of anti-cancer drugs for CRC. In this review, we will discuss interaction between the Wnt/ß-catenin and RAS-ERK pathways in the colorectal tumorigenesis by providing the mechanism of RAS stabilization by aberrant activation of Wnt/ß-catenin. We will also discuss our small molecular anti-cancer approach controlling CRC by induction of specific degradations of both ß-catenin and RAS via targeting Wnt/ß-catenin pathway especially for the KYA1797K, a small molecule specifically binding at the regulator of G-protein signaling (RGS)-domain of Axin.

KY1022, a small molecule destabilizing Ras via targeting the Wnt/ß-catenin pathway, inhibits development of metastatic colorectal cancer.

APC (80-90%) and K-Ras (40-50%) mutations frequently occur in human colorectal cancer (CRC) and these mutations cooperatively accelerate tumorigenesis including metastasis. In addition, both ß-catenin and Ras levels are highly increased in CRC, especially in metastatic CRC (mCRC). Therefore, targeting both the Wnt/ß-catenin and Ras pathways could be an ideal therapeutic approach for treating mCRC patients. In this study, we characterized the roles of KY1022, a small molecule that destabilizes both ß-catenin and Ras via targeting the Wnt/ß-catenin pathway, in inhibiting the cellular events, including EMT, an initial process of metastasis, and apoptosis. As shown by in vitro and in vivo studies using APCMin/+/K-RasG12DLA2 mice, KY1022 effectively suppressed the development of mCRC at an early stage of tumorigenesis. A small molecular approach degrading both ß-catenin and Ras via inhibition of the Wnt/ß-catenin signaling would be an ideal strategy for treatment of mCRC.

Small molecule inhibitors of the Dishevelled-CXXC5 interaction are new drug candidates for bone anabolic osteoporosis therapy.

Bone anabolic agents promoting bone formation and rebuilding damaged bones would ideally overcome the limitations of anti-resorptive therapy, the current standard prescription for osteoporosis. However, the currently prescribed parathyroid hormone (PTH)-based anabolic drugs present limitations and adverse effects including osteosarcoma during long-term use. Also, the antibody-based anabolic drugs that are currently being developed present the potential limits in clinical application typical of macromolecule drugs. We previously identified that CXXC5 is a negative feedback regulator of the Wnt/ß-catenin pathway via its interaction with Dishevelled (Dvl) and suggested the Dvl-CXXC5 interaction as a potential target for anabolic therapy of osteoporosis. Here, we screened small-molecule inhibitors of the Dvl-CXXC5 interaction via a newly established in vitro assay system. The screened compounds were found to activate the Wnt/ß-catenin pathway and enhance osteoblast differentiation in primary osteoblasts. The bone anabolic effects of the compounds were shown using ex vivo-cultured calvaria. Nuclear magnetic resonance (NMR) titration analysis confirmed interaction between Dvl PDZ domain and KY-02061, a representative of the screened compounds. Oral administration of KY-02327, one of 55 newly synthesized KY-02061 analogs, successfully rescued bone loss in the ovariectomized (OVX) mouse model. In conclusion, small-molecule inhibitors of the Dvl-CXXC5 interaction that block negative feedback regulation of Wnt/ß-catenin signaling are potential candidates for the development of bone anabolic anti-osteoporosis drugs.

House dust mite allergen Der f 2 induces interleukin-13 expression by activating the PI3K/Akt pathway.

House dust mites (HDMs) are a common cause of allergic asthma. The group 2 allergen from Dermatophagoides farinae, Der f 2, is one of the major HDM allergens. Elevated Der f 2 immunoglobulin E (IgE) levels are observed in most of the allergic patients. Interleukin-13 (IL-13), a gene associated with asthma pathology, was induced by Der f 2 in BEAS-2B human airway epithelial cells; however, the signaling pathways associated with Der f 2 are not fully understood. In this study, we identified a role of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, a well-known potential target for anti-asthma drugs, in the IL-13 induction by Der f 2. First, Der f 2 activated the PI3K/Akt pathway, which subsequently activated the nuclear factor-kappa B (NF-κB) pathway and induced IL-13 expression in BEAS-2B cells. Treatment with the PI3K inhibitor LY294002 abolished Der f 2-induced activation of Akt and NF-κB and the expression of IL-13. Furthermore, Der f 2-induced activation of the PI3K/Akt and NF-κB pathways, expression of IL-13, and the blockade of these effects with a PI3K inhibitor were confirmed in the lungs of mice that were intranasally exposed to Der f 2. Taken together, these results indicate that the PI3K/Akt pathway regulates Der f 2-induced IL-13 expression via activation of the NF-κB pathway.