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. 2016 Apr 28;35(17):2197-207.
doi: 10.1038/onc.2015.280. Epub 2015 Aug 10.

Wnt addiction of genetically defined cancers reversed by PORCN inhibition

B Madan  1 , Z Ke  2 , N Harmston  3 , S Y Ho  2 , A O Frois  1 , J Alam  2 , D A Jeyaraj  2 , V Pendharkar  2 , K Ghosh  1 , I H Virshup  1 , V Manoharan  2 , E H Q Ong  2 , K Sangthongpitag  2 , J Hill  2 , E Petretto  3 , T H Keller  2 , M A Lee  2 , A Matter  2 , D M Virshup  1   4
Affiliations

Wnt addiction of genetically defined cancers reversed by PORCN inhibition (V体育ios版)

B Madan et al. Oncogene. .

Abstract

Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors VSports手机版. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient-derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers. .

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Conflict of interest statement

Many of the authors are named in the patent regarding ETC-159.

VSports最新版本 - Figures

Figure 1
Figure 1
Development of novel PORCN inhibitors. (a) ETC-159 and ETC-131 inhibit Wnt/β-catenin reporter activity and secretion of Wnt3a (i) STF3A cells were treated with the indicated compounds and luciferase activity was measured after 24 h. Data represents mean±s.d. Inset: structure of ETC-159 and ETC-131. (ii) STF3A cells were treated with 100 nM of the indicated compounds and Wnt secretion was assessed by immunoblot of Wnt3A protein in culture supernatants. (b) ETC-159 and ETC-131 inhibit Wnt3A palmitoleation: HeLa cells transiently expressing Wnt3A-V5 were metabolically labeled for 16 h with alkyne-palmitate (Alk-C16) in the presence of 100 nM of the indicated compounds. Biotin-azide-clicked palmitate (upper panel), Wnt3a-V5 (lower panel). (c) PORCN overexpression rescues the inhibition of β-catenin reporter activity: HT1080 cells transfected with Wnt3a, PORCN expression plasmids, and Super 8xTOPFLASH reporter were treated as indicated for 16 h. The cells were harvested and luciferase activity was measured. Data represents mean±s.d. Data were analyzed using unpaired t-test and corrected for multiple comparisons. **P⩽0.01, ****P⩽0.0001 (d) ETC-159 and ETC-131 prevent interaction of Wnt with Wntless: HeLa cells transiently expressing Wnt3A-V5 were treated overnight with 100 nM of the indicated compounds before immunoprecipitation of endogenous Wntless. (e) ETC-159 promotes β-catenin degradation: mouse L cells stably expressing Wnt3a were trypsinizised and treated with DMSO or 100 nM ETC-159 before plating in cell culture dishes. The cells were harvested at indicated time points and total β-catenin levels were assessed by immunoblot. (f) ETC-159 inhibits Wnt/β-catenin reporter activity induced by diverse Wnts: HT1080 cells were transfected with the Super 8xTOPFLASH and indicated Wnt expression plasmid, and then treated with 100 nM ETC-159 for 24 h before luciferase assay. Bars represent the mean±s.d. Data were analyzed using unpaired t-test. *P⩽0.05, **P⩽0.01, ***P⩽0.001 (g) ETC-159 is a more potent inhibitor of mammalian PORCN: porcupine null HT1080 cells transfected with either a murine (1 ng) or Xenopus porcn (0.75 ng) expression plasmid and Super 8xTOPFLASH reporter were treated with indicated concentrations of ETC-159. The β-catenin reporter activity is shown as percentage of the DMSO treated control. Data represent the mean±s.d.
Figure 2
Figure 2
ETC-159 is orally bioavailable and effectively inhibits the growth of mouse mammary tumor virus (MMTV)-Wnt1 tumors.(a) ETC-159 is orally bioavailable with a dose proportional increase in mouse plasma: Plasma levels of ETC-159 were measured following a single oral dose of 5, 30 or 100 mg/kg. The data is presented as mean±s.d. (b) Anti-tumor efficacy of ETC-159 in the MMTV-Wnt1 orthotopic-mouse model: BALB/c nude mice were implanted in the 4th fat-pad with a fragment of MMTV-Wnt1 tumor from a transgenic mouse. Following the development of palpable tumors, mice were randomized into four groups matched for tumor size and treated daily unblinded either with vehicle or with indicated doses of ETC-159. Data are presented as mean±s.e.m. n=8/group. (c) PORCN inhibition promotes nuclear and cytoplasmic exclusion of β-catenin: Tumors harvested at the end of the study were stained for β-catenin. Representative sections from three independent tumors of vehicle and ETC-159 treatment group are shown. Scale bar=50 μm. (d) Downregulation of Wnt target gene expression: Transcript abundance of select Wnt/β-catenin target genes was assessed in the vehicle and ETC-159 treated tumors harvested 6 h after the last dose. n=8/group. Data were analyzed using unpaired t-test. **P⩽0.01, ***P⩽0.001, ****P⩽0.0001 (e) Robust but transient pharmacodynamic response of Axin2 to ETC-159: Axin2 mRNA and ETC-159 drug concentration were measured in MMTV-Wnt1 tumors at the indicated times following 1, 3 or 10 mg/kg dose. Data are presented as mean±s.d. n= 3/group.
Figure 3
Figure 3
ETC-159 inhibits Wnt autocrine signaling and growth of teratocarcinomas. (a) Specific inhibition of autocrine Wnt signaling in human teratocarcinomas cells: PA-1 cells transfected with Super 8xTOPFLASH reporter were treated with indicated concentrations of the compounds for 24 h before luciferase assay. Data represents mean±s.d. relative to the DMSO control. (b) ETC-159 decreases downstream activation of LRP6 and Dvl2: western blot analysis of PA-1 teratocarcinoma cells treated with 100 nM ETC-159 for 24 h. (c) Inhibition of anchorage-independent growth with ETC-159 and ETC-131: PA-1 cells were plated in soft agar and treated with indicated concentrations of ETC-159 and ETC-131. Total number of colonies was scored after 2–3 weeks. Each data point represents an average count of two wells. (d) ETC-159 prevents the growth of PA-1 teratocarcinomas: 3 weeks after inoculation with PA-1 cells, mice were divided into two treatment groups matched for tumor volume (average tumor volume ~150 mm3/group) and treated daily unblinded with vehicle or 30 mg/kg ETC-159. Data represents group mean±s.e.m. n=10 tumors/group. (e) ETC-159 inhibits the growth of NCCIT teratocarcinomas: As in d. n=9 tumors/group. (f) ETC-159 decreases Axin2 mRNA in PA-1 and NCCIT tumors: Tumors were harvested 4 h after the last dose and Axin2 expression was measured using qRT-PCR. Data represents mean±s.d. n=8 tumors/group. Data were analyzed using unpaired t-test, **P⩽0.01, ****P⩽0.0001.
Figure 4
Figure 4
ETC-159 prevents growth of colorectal tumors with RSPO fusions: (a) ETC-159 inhibits Wnt signaling induced by the RSPO fusion proteins: HEK293 cells transfected with the indicated RSPO expression plasmids and STF reporter were treated with DMSO or 100 nM ETC-159 for 16 h before analysis. The graph shows mean±s.d. and is representative of three independent experiments. Data were analyzed using unpaired t-test and corrected for multiple comparisons. ***P⩽0.001, ****P⩽0.0001. (b and c) Anti-tumor efficacy of ETC-159 in CRC patient-derived xenografts: female BALB/c nude mice with established subcutaneous tumors from two independent patient-derived xenografts CR-1 (b) and CR-2 (c) were treated daily with vehicle or 75 mg/kg ETC-159. The graph shows group means±s.e.m., n=12 tumors/group. Groups were matched for tumor size before unblinded treatment. The sequence chromatograms of the PTPRK-RSPO fusions in the tumors are shown below the respective graphs (d) ETC-159 promotes differentiation of colorectal tumors: tumors harvested at the end of the study were stained with hematoxylin and eosin (upper panel) or Alcian blue (lower panel). Representative sections from tumors of vehicle and ETC-159-treated group are shown. Scale bar=200 μm.
Figure 5
Figure 5
Global remodeling of gene expression in ETC-159 treated colon cancers with RSPO translocations. (a) Differential expression of genes in CRCs: The volcano plot shows the fold change in gene expression (x axis) vs the Benjamini–Hochberg adjusted P-value between vehicle and ETC-159 treated CR-1 tumors. n=4/group. Dotted line, Benjamini–Hochberg adjusted P-value=0.0001. (b) Downregulation of Wnt/β-catenin target genes in ETC-159 treated CRCs: the bar graph shows the fold changes in expression of Wnt/β-catenin target genes in ETC-159 treated CR-1 tumors. (c) Functional enrichment analysis of the genes downregulated and upregulated after ETC-159 treatment. False Discovery Rate <5%. (d) Analysis of over-representation of transcription factor-binding sites in the genes downregulated after ETC-159 treatment (top panel) and upregulated after ETC-159 treatment (bottom panel). False Discovery Rate <5%. (e) Functional enrichment analysis of the genes upregulated after ETC-159 treatment. False Discovery Rate <5%.
Figure 6
Figure 6
Treatment with ETC-159 prevents growth of RNF43 mutant pancreatic tumors and induces differentiation. (a) Three weeks after inoculation with HPAF-II cells, the mice were divided into four treatment groups matched for tumor volume (average tumor volume of ~150 mm3) and treated daily unblinded with vehicle or indicated doses of ETC-159. The graph shows group mean±s.e.m., n=9/group. (b) AXIN2 levels are decreased in ETC-159 treated HPAF-II and AsPC-1 tumors: HPAF-II and AsPC-1 tumors were harvested 6 h after the last dose and AXIN2 expression was measured by qRT-PCR. ****P⩽0.0001, n=6/group (HPAF-II) and n =16/group (AsPC-1). (c) ETC-159 treatment prevents the growth of AsPC-1 xenografts: 3 weeks after inoculation of AsPC-1 cells, mice were randomized into two matched groups and treated twice daily unblinded with 15 mg/kg/dose ETC-159 or vehicle. Data is plotted as group mean±s.d., n=16/group. (d) Increased expression of mucins with ETC-159 treatment: Mucin gene expression was assessed using qRT-PCR. Data were analyzed using unpaired t-test, ***P⩽0.001, ****P⩽0.0001, n=6/group (HPAF-II) and n=16/group (AsPC-1). (e) Representative images of Alcian blue stained tumor sections from vehicle and ETC-159 treated HPAF-II tumors. Scale bar=500 μm. (f) ETC-159 treatment prevents the growth of AsPC-1 xenografts after stopping treatment: following establishment of matched size AsPC-1 tumors, the mice were treated twice daily unblinded with 15 mg/kg/dose ETC-159 or vehicle for 21 days before the treatment was stopped. Tumor volumes were measured for up to 6 weeks after stopping the treatment. The graph shows group means±s.d., n=20/group.
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