Wnt Signaling and Drug Resistance in Cancer

Zheng Zhong; David M. Virshup

doi:10.1124/mol.119.117978

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Fig. 1.
Wnts are secreted proteins with conserved domains and residues. (A) The consensus modeling of 19 human Wnts. The amino acid sequences of 19 human Wnts were aligned, and the amino acid conservation scores were calculated using The ConSurf Server website (http://consurf.tau.ac.il). The conservation scores were then mapped on the human Wnt3 crystal structure (PDB 6AHY chain B). (B) The consensus modeling of Wnt homologs. The palmitoleation site and Frizzled interaction sites are conserved in all Wnts. 2635 amino acid sequences that are homologs of human Wnt3 were collected from UNIREF90 using the HMMER algorithm. The conservation scores were calculated from 150 amino acid sequences representative of the 2635 sequences using The ConSurf Server website and mapped on the human Wnt3 crystal structure. (C) The crystal structure of human Wnt3 in complex with mouse Frizzled 8 CRD (PDB 6AHY). (D) The Wnt secretion pathway. Wnts are palmitoleated by PORCN in the ER. The palmitoleic moiety (red line) facilitates the interaction of Wnts with the cargo receptor WLS that transports Wnts to the plasma membrane. Multiple routes of Wnt release and extracellular transport including diffusion, exovesicles, and cytoneme-mediated transport have been proposed.
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Fig. 2.
The abundance of cell surface Wnt receptors is tightly regulated by RNF43/ZNRF3, USP6, and R-Spondins. (A) RNF43 and ZNRF3 are Wnt/β-catenin target genes. (B) RNF43 and ZNRF3 ubiquitinate the cytosolic domain of Frizzleds causing the internalization and degradation of Frizzleds. (C) USP6 reverses the effects of RNF43/ZNRF3 by deubiquitinating Frizzleds. (D) R-Spondins bind to the extracellular domains of both RNF43/ZNRF3 and LGR4/5 leading to their membrane clearance. Ub, ubiquitin.
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Fig. 3.
The canonical Wnt/β-catenin signaling cascade. (Left panel) In the absence of Wnt ligands, β-catenin is phosphorylated, ubiquitinated, and degraded by the β-catenin destruction complex. (Right panel) Binding of Wnt ligands to the receptors relocalizes the destruction complex to the membrane and interferes with its activity. Subsequently, newly synthesized β-catenin accumulates in the cytoplasm and enters the nucleus to regulate gene transcription. Refer to the text for detailed description. DVL, dishevelled.

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TABLE 1

Common genetic alterations of Wnt pathway components in human cancers

Gene	Type of Mutation	Primary Tissues	% Mutated	Reference
APC	Mainly frameshift and nonsense mutations that lead to truncated APC proteins with compromised ability to degrade β-catenin; LOH	Large intestine	70	^a
		Stomach	13	^a
		Endometrium	7	^a
		Liver	2.7	^a
AXIN1	Mainly missense mutations, truncating mutations, and deep deletions	Liver	7	^a
		Stomach	3	^a
		Large intestine	2.5	^a
CTNNB1	Mainly missense mutations in the N-terminal Ser/Thr phosphorylation sites of β-catenin that prevent its degradation	Liver	29	^a
		Endometrium	18	^a
		Adrenal cortex	16	^a
		Large intestine	6	^a
		Stomach	6	^a
		Pancreas	2.7	^a
RNF43	Mainly missense mutations and truncating mutations due to frameshift or nonsense mutations, LOH, and homozygous deletion	Ovary (mucinous carcinoma/mucinous borderline tumor)	21/9	Ryland et al., 2013
		Stomach	13	^a
		Biliary tract (liver fluke-associated cholangiocarcinoma)	9.3	Ong et al., 2012
		Large intestine	9	^a
		Pancreas	7	^a
		Endometrium	4	^a
ZNRF3	Mainly missense mutations and truncating mutations due to frameshift or nonsense mutations, LOH, and homozygous deletion	Adrenal cortex	20	^a
		Large intestine	4	^a
		Stomach	2.1	^a
RSPO2	Chromosome rearrangement leading to the recurrent EIF3E-RSPO2 gene fusions	Large intestine	2.9	Seshagiri et al., 2012
RSPO2		Others (lung, head and neck, esophagus, stomach, ovary, and breast)	1–2	Cardona et al., 2014; Li et al., 2018
RSPO3	Chromosome rearrangement leading to the recurrent PTPRK-RSPO3 gene fusions	Large intestine	7.4	Seshagiri et al., 2012
RSPO3		Others (lung, head and neck, esophagus, ovary, and breast)	1–11	Cardona et al., 2014

LOH, loss of heterozygosity.
↵^a Curated from the cBioPortal database (https://www.cbioportal.org) in July 2019.

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TABLE 2

Inhibitors of the Wnt signaling and their effects in cancer

Targets and Functions	Agent Name	Functional Effects in Cancer	Development Stage	Reference
Small molecule tankyrase (TNKS1/2) inhibitors, stabilizing AXIN1/2	IWRs		Discovery	Chen et al., 2009
	XAV939	Inhibited colony formation of colorectal cancer cell line DLD-1.		Huang et al., 2009
	WIKI4			James et al., 2012
	NVP-TNKS656			Shultz et al., 2013
	JW67, JW74	Both compounds suppressed in vitro proliferation of colorectal cancer cell line SW480; JW74 reduced SW480 in vivo tumor growth and adenoma formation in Apc^Min mice.	Preclinical	Waaler et al., 2011
	JW55	Suppressed in vitro proliferation of SW480; decreased adenoma formation in conditional Apc knockout mice.		Waaler et al., 2012
	G007-LK	Suppressed in vitro colony formation and in vivo tumor growth of colorectal cancer cell lines COLO-320DM and SW403 and spheroid formation of Apc^Min mouse intestinal adenoma.		Lau et al., 2013
	K-756	Suppressed in vitro proliferation of COLO-320DM and SW403; showed dose-dependent inhibition of Wnt signaling in DLD-1 xenografts.		Okada-Iwasaki et al., 2016
Small molecule CK1α activators, promoting β-catenin degradation	Pyrvinium	Suppressed in vitro proliferation of colorectal cancer cell lines SW480, DLD-1, SW620, HCT116, and HT29; reduced adenoma formation in Apc^Min mice.	Preclinical	Thorne et al., 2010; Li et al., 2014;
	SSTC3	Suppressed intestinal organoid formation from Apc^Min mice and Apc knockout mice; reduced adenoma formation in Apc^Min mice; suppressed in vitro colony formation of HT29, SW403, and HCT116 and in vivo tumor growth of HCT116; suppressed tumor organoid formation from three patient colorectal tumors and in vivo growth of xenografts.	Preclinical	Li et al., 2017
Small molecule inhibitors blocking β-catenin/TCF interaction	iCRT3, iCRT5, and iCRT14	iCRTs led to cell cycle arrest in HCT116 and HT29; iCRT3 suppressed in vitro growth of primary human colon cancer specimens; iCRT14 reduced HCT116 and HT29 xenograft growth.	Preclinical	Gonsalves et al., 2011
Small molecule inhibitors blocking β-catenin/CBP interaction	ICG-001	Suppressed in vitro growth and led to apoptosis in SW480 and HCT116; suppressed in vivo growth of SW620 xenografts; reduced adenoma formation in Apc^Min mice; suppressed in vitro growth of pancreatic cancer cell lines AsPC-1, L3.6pl, MIA PaCa-2, and PANC-1; improved survival of AsPC-1 orthotopic xenograft-bearing mice.	Preclinical	Emami et al., 2004; Arensman et al., 2014
	PRI-724	The active enantiomer of ICG-001; entered Phase 1 and 2 clinical trials for treating advanced cancers.	Phase 1 and 2 clinical trials	ClinicalTrials.gov NCT01764477, NCT01302405, NCT01606579, NCT02413853
Small molecule inhibitor blocking β-catenin/p300 interaction	Windorphen	Suppressed in vitro proliferation of colorectal cancer cell lines SW480 and RKO and prostate cancer cell lines DU145 and PC3.	Discovery	Hao et al., 2013
Small molecule PORCN inhibitors, blocking Wnt secretion	IWPs		Discovery	Chen et al., 2009
	C59	Inhibited MMTV-Wnt1 tumor growth in vivo.	Preclinical	Proffitt et al., 2013
	LGK974	Induced regression of MMTV-WNT1 tumors; inhibited in vitro colony formation of head and neck cancer cell line HN30 and RNF43-mutant pancreatic cancer cell lines Patu8988S and HPAF-II; inhibited in vivo tumor growth in xenografts of HN30 and RNF43-mutant pancreatic cancer cell lines Capan2 and HPAF-II; entered Phase 1 and 2 clinical trials for treating cancers.	Phase 1 and 2 clinical trials	Jiang et al., 2013; Liu et al., 2013; ClinicalTrials.gov NCT01351103, NCT02278133, NCT02649530
	ETC-159	Inhibited in vitro colony formation of teratocarcinoma cell lines PA-1 and RNF43-mutant pancreatic and ovarian cancer cell lines HPAF-II, AsPC-1, and MCAS; suppressed in vivo tumor growth in xenografts of HPAF-II, AsPC-1, teratocarcinoma cell lines PA-1 and NCCIT, and patient-derived colorectal tumor xenografts with RSPO3 fusions; entered Phase 1 clinical trial for treating advanced cancers.	Phase 1 clinical trial	Madan et al., 2016a; ClinicalTrials.gov NCT02521844
	RXC004	Suppressed in vivo tumor growth of Capan2 xenografts; entered Phase 1 clinical trial for treating advanced cancers.	Phase 1 clinical trial	Bhamra et al., 2017; ClinicalTrials.gov NCT03447470
	CGX1321	Suppressed in vivo tumor growth of patient-derived gastric and colorectal tumor xenografts with RSPO2 fusions; entered Phase 1 clinical trial for treating advanced cancers.	Phase 1 clinical trial	Li et al., 2018; ClinicalTrials.gov NCT03507998, NCT02675946
Decoy Wnt receptor	Ipafricept (OMP-54F28)	Suppressed in vivo tumor growth of patient-derived hepatocellular carcinoma, pancreatic and ovarian cancer xenografts, and decreased the cancer stem cell frequency; entered Phase 1 clinical trials for treating advanced cancers; in a Phase 1 clinical trial, two desmoid tumor, and a patient with germ cell cancer treated with Ipafricept experienced stable disease for >6 mo.	Phase 1 clinical trials	Jimeno et al., 2017; ClinicalTrials.gov NCT02069145, NCT02092363, NCT02050178, NCT01608867
Anti-Frizzled1,2,5,7,8 antibody	Vantictumab (OMP-18R5)	Suppressed in vivo tumor growth of patient-derived colorectal, breast, lung, and pancreatic tumor xenografts and PA-1 xenografts; decreased the cancer stem cell frequency; entered Phase 1 clinical trials for treating cancers.	Phase 1 clinical trials	Gurney et al., 2012; ClinicalTrials.gov NCT01345201, NCT02005315, NCT01957007, NCT01973309
Anti-Frizzled5,8 antibodies	IgG-2919, IgG-2921	Both antibodies suppressed in vitro proliferation of RNF43-mutant pancreatic cancer cell lines HPAF-II, Patu8988S, and AsPC-1, and led to cell cycle arrest; IgG-2912 suppressed in vivo tumor growth of HPAF-II and AsPC-1 xenografts and organoid formation of RNF43-mutant colorectal cancers.	Preclinical	Steinhart et al., 2017
Anti-LRP6 antibodies	A7-IgG, B2-IgG	A7-IgG specifically inhibited tumor growth of MMTV-Wnt1 xenografts; B2-IgG specifically inhibited tumor growth of MMTV-Wnt3 xenografts.	Preclinical	Ettenberg et al., 2010
Anti-RSPO antibodies	Anti-RSPO1 antibody, anti-RSPO2 antibody, anti-RSPO3 antibodies	Suppressed in vivo tumor growth of patient-derived colorectal, lung, and ovarian tumor xenografts with corresponding RSPO overexpression; Anti-RSPO3 antibody (OMP-131R10) has entered Phase 1 clinical trial for treating advanced solid tumors and metastatic colorectal cancers.	Preclinical, phase 1 clinical trial (OMP-131R10)	Chartier et al., 2016; Storm et al., 2016; ClinicalTrials.gov NCT02482441

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TABLE 3

Anti-Wnt signaling-based drug combinations in cancer therapy

Drug A	Drug B (Wnt Pathway Inhibitor)	Drug Combination Rationales/Preclinical Results	Clinical Trials
Paclitaxel and carboplatin (chemo agents)	Ipafricept/OMP-54F28 (decoy Wnt recptor)	The drug combinations showed synergistic and potent tumor growth inhibition in patient-derived ovarian, pancreatic, hepatocellular, breast, lung, and/or colorectal cancer xenografts (Chartier et al., 2016; Fischer et al., 2017a,b; Jimeno et al., 2017).	Phase 1 clinical trial (NCT02092363) in patients with recurrent platinum-sensitive ovarian cancer
Nab-paclitaxel and gemcitabine (chemo agents)			Phase 1 clinical trial (NCT02050178) in patients with previously untreated stage IV pancreatic cancer
Sorafenib (RAF, VEGFR, and PDGFR inhibitor)			Phase 1 clinical trial (NCT02069145) in patients with hepatocellular cancer
Paclitaxel (chemo agent)	Vantictumab/OMP-18R5 (anti-Frizzleds antibody)		Phase 1 clinical trial (NCT01973309) in patients with locally recurrent or metastatic breast cancer
Docetaxel (chemo agents)			Phase 1 clinical trial (NCT01957007) in patients with previously treated non-small-cell lung carcinoma
Nab-paclitaxel and gemcitabine (chemo agents)			Phase 1 clinical trial (NCT02005315) in patients with previously untreated stage IV pancreatic cancer
Taxol, gemcitabine, or irinotecan (chemo agents)	Anti-RSPO1, anti-RSPO2, and anti-RSPO3 antibodies
FOLFIRI (chemo agents)	OMP-131R10 (anti-RSPO3 antibody)		Phase 1 clinical trial (NCT02482441) in patients with metastatic colorectal cancer
API2 (AKT inhibitor)	NVP-TNKS656 (tankyrase inhibitor)	Tankyrase inhibition reverted resistance to AKT inhibition in patient-derived colorectal cancer xenografts (Arques et al., 2016).
Gefitinib or AZD9291 (EGFR inhibitors)	AZ1366 (tankyrase inhibitor)	The drug combination significantly slowed down growth of non–small cell lung cancer orthotopic xenografts and improved survival of tumor-bearing mice (Scarborough et al., 2017).
Vemurafenib (BRAF inhibitor)	ICG-001 (blocking β-catenin/TCF interaction)	BRAF inhibition upregulated β-catenin signaling in colorectal cancer cell lines in vitro. The drug combination led to synergistic tumor growth inhibition in HT29 xenografts (Chen et al., 2018).
LGX818 (BRAF inhibitor) and Cetuximab (EGFR inhibitor)	LGK974 (PORCN inhibitor)	A subset of colorectal cancers harbor concurrent mutations in BRAF and RNF43 or RSPOs (Yan et al., 2017).	Phase 1/2 clinical trial (NCT02278133) in patients with BRAF-mutant metastatic colorectal cancer with Wnt pathway mutations
Buparlisib/BKM120 (pan-PI3K inhibitor)	LGK974 (PORCN inhibitor)	Burparlisib treatment upregulated Wnt signaling in triple negative breast cancer (TNBC) cell lines in vitro. The drug combination led to synergistic tumor growth inhibition in xenografts of TNBC cell line TMD231 (Solzak et al., 2017).
Multiple PI3K/mTOR inhibitors including GDC-0941, ZSTK474, LY-294002, AS252424, and Ku0063794	ETC-159 (PORCN inhibitor)	ETC-159 in combination with one of the five PI3K/mTOR inhibitors synergistically suppressed 3D colony formation of several Wnt-addicted pancreatic and cholangiocarcinoma cell lines. ETC-159 in combination with GDC-0941 led to synergistic tumor growth inhibition in HPAF-II and AsPC-1 xenografts (Zhong et al., 2019).
Olaparib (PARP inhibitor)	Pyrvinium (CK1α activator) or XAV939 (tankyrase inhibitor)	Wnt/β-catenin signaling mediated resistance to PARP inhibition in ovarian cancer due in part to upregulation of DNA damage repair. Inhibition of β-catenin signaling reverted resistance to olaparib in ovarian cancer xenografts (Fukumoto et al., 2019; Yamamoto et al., 2019).
Vismodegib (Hedgehog signaling inhibitor)	LGK974 (PORCN inhibitor) or anti-LRP6 antibody	A LGR5⁺ tumor cell population maintained by active Wnt signaling persists vismodegib treatment in human and mouse basal cell carcinoma and mediates relapse after treatment discontinuation. The drug combination reduced tumor burden (vismodegib + anti-LRP6 antibody) or eliminated resistant tumor cells (vismodegib + LGK974) in mouse models (Biehs et al., 2018; SanchezDanes et al., 2018).
PDR001 (anti-PD1 antibody)	LGK974 (PORCN inhibitor)	Preclinical studies showed that Wnt signaling mediated resistance to immunotherapies, and concurrent inhibition of Wnt/β-catenin signaling enhanced the efficacy of immune checkpoint inhibitors in melanoma mouse models (Holtzhausen et al., 2015; Spranger et al., 2015; Zhao et al., 2018).	Phase 1 clinical trial (NCT01351103) in patients with malignancies dependent on Wnt ligands
Pembrolizumab (anti-PD1 antibody)	ETC-159 (PORCN inhibitor)		Phase 1 clinical trial (NCT02521844) in patients with advanced solid tumor
Pembrolizumab (anti-PD1 antibody)	CGX1321 (PORCN inhibitor)		Phase 1 clinical trial (NCT02675946) in patients with advanced gastrointestinal tumor

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Supplementary Data - PDB 6ahy 19seq

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Supplementary Data - PY 6ahy 19seq

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Supplementary Data - PDB 6ahy Global

Supplemental Data -
Supplementary Data - PY 6ahy Global