Activation of Wnt signaling is among the earliest events in colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding β-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations affecting APC, a major β-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct β-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that MYC suppresses DKK3 transcription through one of miR-17-92 cluster miRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in Apc-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration/invasion in vitro and metastasis in a syngeneic orthotopic mouse colon cancer model.

Implications:

Our results suggest that the MYC→miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feed-forward oncogenic loop.

Colorectal cancer (or CRC) is the second most common cancer diagnosed and third leading cause of cancer-related deaths in the United States. Despite serving as the initial testing ground for cancer genetics and more recently—genomics, colorectal cancer remains a puzzling and deadly disease. There are projected to be 147,950 individuals newly diagnosed with colorectal cancer with an estimated 53,200 colorectal cancer deaths in 2020 (1). One reason for the lack of major breakthroughs is that focusing on individual signaling pathways is not enough to understand pathogenesis and progression of this disease. The MYC oncogene is a case in point. It is involved in a dizzying number of functional interactions, few of which have been fully understood or sufficiently validated.

In colorectal cancer, MYC is usually overexpressed due to activating mutations in the Wnt pathway (2, 3). Binding of a WNT ligand to its receptor Frizzled (FZD) and co-receptor low-density lipoprotein receptor related protein 5/6 (LRP5/6) initiates a sequence of signaling events, which reprograms protein–protein interactions and ultimately prevents adenomatous polyposis coli (APC) tumor suppressor-mediated degradation of β-catenin (4, 5). Stabilized β-catenin translocates into the nucleus (6), where it forms a complex with the TCF4 transcription factor (7, 8) and drives expression of MYC, along with numerous other target genes involved in stem cell self-renewal and proliferation (3). Not surprisingly, inactivating mutations in APC and activating mutations in CTNNB1 are mutually exclusive as they work toward the same goal of rendering Wnt signaling pathway constitutively active (9, 10). The question remains whether APC- or CTNNB1-mutant colorectal cancers accumulate complementary genetic or regulatory events that are needed to hyperactivate Wnt signaling.

In this study, we set out to identify such hyperactivating events by establishing epistatic relationships between various recurrent mutations in the Wnt pathway in colorectal cancer. We discovered that mutations in many additional components of the Wnt signaling co-occur with CTNNB1 mutations, consistent with our Wnt hyperactivation hypothesis. These mutations are exemplified by the four members of the DKK superfamily, which encode secreted glycoproteins capable of modulating the activity of LRP5/6 and KREMEN co-receptors in cells without genetic perturbation of the Wnt pathway (11–13). We then focused on the least frequently mutated member in the Wnt signaling pathway: DKK3, whose role in Wnt signaling (13, 14) and colorectal cancer is not well defined. We discovered that although it is seldom mutated, it is negatively regulated by MYC by a miRNA-dependent mechanism and that WNT, MYC, miR-92a and DKK3 form a previously unrecognized positive feed-forward loop wherein MYC and Wnt activate each other.

Cell culture

The human colorectal cancer cell lines HCT116, SK-CO-1, mouse Ras and RasMyc, colonocytes mouse L cells with or without Wnt3a were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen), 2 mmol/L L-glutamine, and penicillin/streptomycin at 37°C and 5% CO2. After thawing, cells were authenticated by short tandem repeat analysis, tested for Mycoplasma using the EZ-PCR Mycoplasma Detection Kit (Biological Industries), and used for up to 12 passages. Mouse Apc null, trp53 null, Smad4-null, Kras-mutant intestinal organoids were maintained in DMEM-F12 supplemented with 10% FBS, 2 mmol/L L-glutamine, penicillin/streptomycin, N2 supplement, B27 supplement, and N-acetyl-cysteine.

Organoid generation

The colonic crypts from a KrasG12D-LSL mouse (The Jackson Laboratory, stock no: 008179) were extracted and used to establish colonoid cultures. The KrasG12D mutation was then activated by transient transfection of Salk-Cre with pPGK-Puro (RRID:Addgene_11349) plasmids, followed by puromycin selection for 3 days. Next, Apc, trp53, and Smad4 mutations were introduced by CRISPR-Cas9 editing. Specifically, single-guide RNAs targeting Apc, trp53, and Smad4 were cloned into PX330 plasmid (RRID: Addgene_42230) and transiently transfected into the KrasG12D tumoroids. One week after the transient transfection, the tumoroids with Apc, trp53, and Smad4 mutations were selected by removing R-spondin, adding Nutlin-3 and removing Noggin from the culture media, respectively. A total of 10 subclones were picked from the engineered bulk tumoroids, and PCR and Sanger sequencing were used to verify the mutations in each subclone. A subclone with the recombined LSL-KrasG12D allele, and verified Apc, trp53, and Smad4 mutations was used for downstream experiments.

RNA extraction and quantitative real-time PCR

Total RNA, including miRNA, was isolated from tissues or cell lines using TRIzol reagent (Invitrogen) according to manufacturer's instructions. For DKK3 and MYC mRNA detection and miRNA expression analysis, reverse transcription was performed using the ABI cDNA reverse transcription kit (Applied Biosystems) with miR-92a–specific primers (Applied Biosystems). Quantitative PCR was performed using FAST SYBR green mix (Roche) on the ABI 7500 real-time PCR System (Applied Biosystems). U6 snRNA or hypoxanthine phosphoribosyltransferase (HPRT) was used as internal control. The primer sequences are available upon request. The relative expression levels were calculated by the equation 2−ΔΔCT.

Western blot analysis

Total cell lysates were prepared from cultured cells or organoids using RIPA buffer with protease and phosphatase inhibitors (Pierce Halt Inhibitor Cocktail, Thermo Fisher Scientific). After protein transference to PVDF (Immobilin-p, Millipore), the antibodies for p-LRP6, LRP6, AXIN2, CCND1, MYC, GSK3β, p-GSK3β (Ser9), phospho-β-catenin, β-catenin (Cell Signaling Technology), and DKK3 (Thermo Fisher Scientific) were used. Subsequently, recommended dilutions of horseradish peroxidase–conjugated secondary antibodies (GE Healthcare) were applied. Enhanced chemiluminescence (Millipore) was used to detect bands that were then captured by Chemiluminescence imager (GE Healthcare). Each sample was normalized to GAPDH or β-actin (Cell Signaling Technology).

Plasmids and transfection

For infection of colorectal cancer cell lines with pMX-IRES constructs, retroviral particles were generated by transfection of HEK 293GP cells with Lipofectamine 3000 (Invitrogen). After transfection, the cell supernatants were collected and used to infect colorectal cancer cells, and the stably transfected cells were selected using 12.5 μg/mL of blasticidin (Gemini Bio-Products) for over a week. SMARTpool siRNA for DKK3, antagomir-92a, miR-92a mimic, and corresponding control oligonucleotides (Dharmacon) were transfected into colorectal cancer cells by using Lipofectamine 3000 according to manufacturer's protocols. For stable infection of organoids, PMX-IRES GFP constructs were used. The retroviral particles were prepared as described above and concentrated using Retro-X concentrator (Takara Biosciences). Organoid fragments were prepared following the protocol from Ref. 15 and combined with retroviral suspension along with polybrene in a 24-well plate, sealed, and spinoculated at 1,800 rpm at 37°C for 1 hour 45 minutes. Following spinoculation, the plate was incubated for 6 hours at 37°C. This was followed by seeding of infected organoids and 5 days after infection, GFP-positive cells were sorted and resuspended in matrigel.

Luciferase reporter assay

Cells were transfected with either TOP FLASH (containing TCF binding sites; Addgene#12456) or FOP FLASH (with mutated TCF binding sites; Addgene#12457) luciferase expression vectors and with Renilla luciferase vector (control) using Lipofectamine 3000. 24 hours post-transfection, cells were incubated with Wnt3a CM. 20 hours after incubation, luciferase activity in total cell lysates was measuring using Dual-Luciferase Reporter Assay Kit (Promega) and normalized for transfection efficiency via dividing by the Renilla luciferase activity. The TOP/FOP ratio was used as a quantitative measure of β-catenin–mediated transcriptional activity as described by us recently (16). To determine whether DKK3 is a direct target of miR-92a, wild-type or mutant 3′-UTR (untranslated region) of DKK3 was cloned into the psicheck-2 vector (Promega). HCT116 cells were transfected with wild-type or mutant 3′-UTR-luc using Lipofectamine 3000. After 48 hours, cells were harvested and assayed with Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocols. The sequences of primers used for generating wild type or mutant 3′-UTR are available upon request.

Biotin-labeled miRNA pulldown assays

Biotinylated miR-92a (Dharmacon) pulldown assay with target mRNAs was performed as described earlier (17). Briefly, 1 × 106 HCT116 cells were seeded in 10-cm plates. 24 hours later, control miR or 3′ biotin-labeled miR-92a was transfected at a final concentration of 50 nmol/L. After 24 hours, whole-cell lysates were harvested. Streptavidin-Dyna beads (Dyna beads M-280 Streptavidin, Invitrogen, 50 μL each sample) were coated with 10 μL yeast tRNA (stock 10 mg/mL, Ambion) and incubated with rotation at 4°C for 2 hours. Then beads were washed with 500 μL lysis buffer and resuspended in 50 μL lysis buffer. Sample lysates were mixed with precoated beads (50 μL per sample) and incubated overnight at 4°C on a rotator. Beads were pelleted down the next day to remove unbound materials at 4°C for 1 minute, 5K rpm and washed five times with 500 μL ice-cold lysis buffer. To isolate RNA, 500 μL of TRIzol (Invitrogen) was added to both input and pulldown samples. Tubes were mixed well and kept in −20°C for 2 hours. RNA was then precipitated using standard chloroform-isopropanol method and subjected to qRT-PCR.

Migration and invasion assays

The Corning BioCoat Cell Culture Inserts and Matrigel inserts were used for migration and invasion assays, respectively. Briefly, the inserts were rehydrated with plain DMEM for 2 hours before use. A total of 5–7 × 104 cells (treated with 10 μg/mL of Mitomycin C) were trypsinized and resuspended in serum-free medium and then seeded onto 24-well transwell chambers with 8–micron pore membrane in 500 μL serum-free medium with condition medium from Wnt3A-producing L cells or control L cells. The lower chamber contained medium supplemented with 10% FBS. After incubation for 22 hours, the non-migrated/invaded cells on the upper side of membrane were removed with a cotton swab and the migrated/invaded cells were stained with crystal violet and photomicrographed.

Orthotopic mouse model

All surgical procedures were performed using aseptic methods in compliance with Institutional Animal Care and Use Committee guideline for rodent survival surgery. C57BL/6J mice (The Jackson Laboratory stock no: 000664) were used in all experiments. After the mouse was fully anesthetized, the surgical site was shaved and locally disinfected. The mouse was placed under a dissecting microscope and using watchmaker forceps and fine dissection scissor an incision of about 2 cm was made in the ventral part of the mouse skin. Through the first incision, the abdominal musculature was picked up with watchmaker forceps and a second 1.5 cm incision was made to access the cecum. The cecum was pulled out using two sterile cotton tips and placed on top of the surgical site. Using an insulin syringe with a 30G needle, the organoids were injected under the serosa membrane. To prevent the cecum from drying, sterile saline was used to keep the site moist. After tumoroids harboring engineered oncogenic mutations in Apc, trp53, Kras and Smad4 with Vector or MYC overexpression were injected (4 × 106 cells), the cecum was introduced back in the body cavity and three simple interrupted stitches were placed in the abdominal wall to close the body cavity. For closing the skin, two stainless steel wound clips were applied. After 6 weeks, primary tumors and livers were resected and analyzed histologically to evaluate for metastatic lesions in the liver.

Bioinformatics analysis

Publicly available data for “Colorectal Adenocarcinoma (TCGA, PanCancer Atlas)” were downloaded from cBioPortal (coadread_tcga_ pan_can_atlas_2018.tar.gz) or accessed from dbGaP via Request 3861. From these data, we examined the mutual exclusivity of mutations and the normalized RNA sequencing expression. We correlated MYC and DKK3 expression using the lm methods as implemented in the R language to determine r2 and significance. Microarray (Array express database under accession number E-MEXP-757) intensities were normalized and analyzed using the Bioconductor package limma (RRID:SCR_010943). The volcano plots only display the probes with the highest absolute fold change. Results were processed and visualized using the R packages dplyr and ggplot2 (RRID:SCR_014601).

Statistical analyses

All statistical analyses were carried out using Graph Pad Prizm (version 7) by unpaired Student t test for two group comparisons or one-way ANOVA correcting for multiple comparisons, with similar variance between groups being compared. Error bars represent means ± SEM, and statistical significance was defined as P < 0.05.

Multiple Wnt pathway mutations co-occur in APC/CTNNB1-mutant colorectal cancer

In 2012, The Cancer Genome Atlas project (TCGA) reported genome-scale analysis of 276 samples, including exome sequence, DNA copy number, promoter methylation and messenger RNA and miRNA expression (18). In addition to APC and CTNNB1, that paper identified several key members of the Wnt pathway being mutated, including Frizzled (FZD10), LRP co-receptors, DKK1–4, AXIN2, FBXW7 and TCF7L2. In 2017, this dataset was supplemented with additional specimens resulting in the larger 594-sample colorectal cancer subset of TCGA PanCancer Atlas (19). We excluded from our analysis APC mutations given their very high prevalence (75%). The Oncoprint implemented on cBio portal provided updated frequency numbers (Fig. 1A), and the mutual exclusivity analysis surprisingly revealed that all statistically significant relationship were in fact co-occurrences (Fig. 1B). For example, CTNNB1 mutations co-occurred with those in LRP6, suggesting that even in β-catenin–mutant colorectal cancer receptor-mediated events still play an important role. In turn, LRP6 mutations co-occurred with those in three members of the DKK family (1, 2, and 4), suggesting the importance of deregulating both receptors and their cognate ligands. Of note, DKK3 did not appear in this list, casting uncertainty on its role in Wnt signaling and colorectal cancer pathogenesis in general. However, by analyzing its expression across various solid cancer types profiled in the Broad Cell Line Encyclopedia, we observed that colorectal cancer has the lowest DKK3 mRNA expression levels (Supplementary Fig. S1A), suggesting that this least frequently mutated DKK family member is subject to additional mechanisms of dysregulation, such as gene repression.

Figure 1.

MYC regulates DKK3 expression in colorectal cancer cells. A, Oncoprint analysis showing frequency of mutations in Wnt pathway members. B, Mutual exclusivity analysis showing significant co-occurrence of mutations in Wnt pathway members. C, Inverse correlation between MYC and DKK3 expression in human colorectal cancers. r = −0.14. D, Volcano plot showing expression of select TGFβ and Wnt pathway components in KRAS and RAS+MYC cells. E, Immunoblotting on total lysates from the same cells performed using an anti-DKK3 antibody. α-Tubulin was used as a loading control. F, qRT-PCR analysis performed on parental and MYC-overexpressing HCT116 cells. Bar graph represents log2-transformed ratios of MYC and DKK3 expression normalized to HPRT. G, Immunoblotting showing protein levels of MYC and DKK3 in the same cells. β-Actin was used as a loading control. Also shown are levels of DKK3 in the conditioned medium. H, Immunoblotting showing protein levels of MYC and DKK3 in HCT116 cells treated with IBET-151 (1 μmol/L) for 0–8 hours. I, Immunoblotting showing protein levels of MYC and DKK3 in HCT116 cells treated with Chiron-99021 (3 μmol/L) for 0–6 hours.

Figure 1.

MYC regulates DKK3 expression in colorectal cancer cells. A, Oncoprint analysis showing frequency of mutations in Wnt pathway members. B, Mutual exclusivity analysis showing significant co-occurrence of mutations in Wnt pathway members. C, Inverse correlation between MYC and DKK3 expression in human colorectal cancers. r = −0.14. D, Volcano plot showing expression of select TGFβ and Wnt pathway components in KRAS and RAS+MYC cells. E, Immunoblotting on total lysates from the same cells performed using an anti-DKK3 antibody. α-Tubulin was used as a loading control. F, qRT-PCR analysis performed on parental and MYC-overexpressing HCT116 cells. Bar graph represents log2-transformed ratios of MYC and DKK3 expression normalized to HPRT. G, Immunoblotting showing protein levels of MYC and DKK3 in the same cells. β-Actin was used as a loading control. Also shown are levels of DKK3 in the conditioned medium. H, Immunoblotting showing protein levels of MYC and DKK3 in HCT116 cells treated with IBET-151 (1 μmol/L) for 0–8 hours. I, Immunoblotting showing protein levels of MYC and DKK3 in HCT116 cells treated with Chiron-99021 (3 μmol/L) for 0–6 hours.

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MYC regulates DKK3 expression in colorectal cancer cells

To identify possible mechanisms of repression, we analyzed transcription factors-encoding mRNAs which anti-correlated with DKK3 mRNA in human samples from TCGA colorectal cancer dataset. Interestingly, one of the transcription factors that showed significant anticorrelation with DKK3 was MYC (Fig. 1C). To establish the causality, we utilized the previously generated MYC-dependent colorectal cancer model system from our lab (20). Specifically, we compared the transcriptomic profiles of MYC-overexpressing and parental KRAS-transformed mouse colonocytes. Volcano plot analysis of the MYC-upregulated genes yielded some of key Wnt pathway components (Lef1, Dvl2, Fzd2/9, etc.; Fig. 1D) indicating that overexpression of MYC alters Wnt signaling pathway. It also demonstrated lower expression levels of Dkk3 along with several TGFβ-responsive genes reported by us previously (21), suggesting that MYC negatively regulates DKK3. Immunoblotting experiments confirmed that MYC-overexpressing colonocytes have reduced expression of DKK3 at the protein level (Fig. 1E). To extend our findings to human colorectal cancer cells, we used the HCT116 derivative stably overexpressing MYC (22). We found that MYC-overexpressing cells have decreased expression of DKK3 mRNA (Fig. 1F) and DKK3 protein levels were decreased in lysates as well as in the conditioned media (Fig. 1G), indicative of negative regulation of DKK3 by MYC.

To confirm the effect of MYC on DKK3 independently of retrovirus-mediated overexpression, we used chemical inhibitors (I-BET 151 and CHIR99021) to repress or stabilize MYC expression in HCT116 cells, respectively. First, using the chemical inhibitor of the Brd4 transcription factor, I-BET 151 (23), we were able to inhibit MYC expression and observed a concomitant increase in DKK3 levels at corresponding time intervals (Fig. 1H). Using a reverse strategy, we stabilized MYC levels in HCT116 cells using CHIR99021 treatment, as described by us previously (24). CHIR99021 inhibits the phosphorylation of MYC at Threonine-58 by GSK-3-β, which leads to phospho-MYC being targeted for ubiquitination and subsequent degradation (25). MYC levels thus stabilized resulted in decreased DKK3 expression (Fig. 1I), confirming an inverse relationship between MYC and DKK3.

MYC regulates DKK3 expression via miR-92a

To further understand the regulation of DKK3 expression by MYC, we mined the publicly available dataset of MYC chromatin immunoprecipitation (ChIP) from the colon cancer cell line HCT116 (Gene Expression Omnibus dataset GSM2576763 from ref. 26). We observed that MYC does not bind to the promoter of DKK3 as evidenced by the absence of peaks within 3Kb of the transcription start site. This is in contrast to other genes reported to be directly repressed by MYC, such as NDRG1 (Fig. 2A; ref. 27). This negative result was indicative of an indirect mechanism of regulation, for example by a MYC-regulated miRNA (28).

Figure 2.

MYC regulates DKK3 expression via miR-92a in colorectal cancer cells. A, CHIP-seq analysis of NDRG1 and DKK3 promoters for MYC binding. B, Inverse correlation between miR-92a and DKK3 expression in human colorectal cancer. r = −0.26. C, Nucleotide sequence of wild-type and mutant 3′ UTR of DKK3. D, Luciferase activity of DKK3 wild-type and mutant 3′-UTR-base reporters in HCT116 cells. E, Schematic for biotinylated miR-92a pulldown with streptavidin beads. F, Enrichment for DKK3 mRNA in streptavidin pulldowns from cells transfected with biotinylated miR-92a mimic. HPRT transcript was used as a control. G, Quantitation of DKK3 mRNA expression normalized to HPRT (log2-transformed ratio) by qRT-PCR analysis in control and miR-92a–transfected cells. H, Immunoblotting showing level of DKK3 in the same cells. GAPDH was used as loading control. I, Quantitation of miR-92a effects on DKK3 levels by immunoblotting. Vector-transduced and MYC-overexpressing HCT116 cells were treated with control or miR-92a inhibitor. Total cell lysates were probed for MYC and DKK3 as indicated. β-Actin was used as loading control. J, Quantitation of miR-92a effects on DKK3 levels by immunoblotting. Parental HCT116 cells were treated with control or miR-92a inhibitor in the presence or absence of Chiron-99021 (3 μmol/L for 6 hours). Total cell lysates were probed for MYC and DKK3 as indicated. β-Actin was used as loading control.

Figure 2.

MYC regulates DKK3 expression via miR-92a in colorectal cancer cells. A, CHIP-seq analysis of NDRG1 and DKK3 promoters for MYC binding. B, Inverse correlation between miR-92a and DKK3 expression in human colorectal cancer. r = −0.26. C, Nucleotide sequence of wild-type and mutant 3′ UTR of DKK3. D, Luciferase activity of DKK3 wild-type and mutant 3′-UTR-base reporters in HCT116 cells. E, Schematic for biotinylated miR-92a pulldown with streptavidin beads. F, Enrichment for DKK3 mRNA in streptavidin pulldowns from cells transfected with biotinylated miR-92a mimic. HPRT transcript was used as a control. G, Quantitation of DKK3 mRNA expression normalized to HPRT (log2-transformed ratio) by qRT-PCR analysis in control and miR-92a–transfected cells. H, Immunoblotting showing level of DKK3 in the same cells. GAPDH was used as loading control. I, Quantitation of miR-92a effects on DKK3 levels by immunoblotting. Vector-transduced and MYC-overexpressing HCT116 cells were treated with control or miR-92a inhibitor. Total cell lysates were probed for MYC and DKK3 as indicated. β-Actin was used as loading control. J, Quantitation of miR-92a effects on DKK3 levels by immunoblotting. Parental HCT116 cells were treated with control or miR-92a inhibitor in the presence or absence of Chiron-99021 (3 μmol/L for 6 hours). Total cell lysates were probed for MYC and DKK3 as indicated. β-Actin was used as loading control.

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Previous data by several labs, including ours, has shown that MYC upregulates the miR-17-92 cluster of miRNA (20, 29, 30). Per TargetScan (31), one of the predicted targets for miR-92a is DKK3 (Supplementary Fig. S1B). Accordingly, we observed highly significant anti-correlation between miR-92a and DKK3 mRNA in human samples from TCGA colorectal cancer dataset (Fig. 2B). To further confirm that DKK3 is a target of miR-92a, we generated WT-DKK3-3′UTR and MUT-DKK3-3′UTR luciferase reporter plasmids (Fig. 2C). Luciferase assays demonstrated that the reporter activity was significantly decreased in HCT116 cells transfected with the WT-DKK3-3′UTR construct compared with the parental UTR-less cassette. However, no significant difference in activity was observed in cells transfected with MUT-DKK3-3′UTR, where the predicted miR-92a binding sites had been mutated (Fig. 2D).

We next sought to determine whether miR-92a is directly bound to DKK3 mRNA using biotinylated miRNA/mRNA pulldown (Fig. 2E). Pulldown of miR-92a itself was confirmed by stem-loop RT-PCR (Supplementary Fig. S1C). As anticipated, levels of DKK3 mRNA were markedly elevated in biotin-labeled miR-92a pulldown fraction compared with control, while there was no difference in the levels of HPRT mRNA. (Fig. 2F). Consistent with this finding, DKK3 mRNA (Fig. 2G) and protein (Fig. 2H) levels were significantly decreased compared with those seen in control miR-transfected cells. To determine whether MYC regulates DKK3 via miR-92a, we treated MYC-overexpressing cells with a miR-92a antisense inhibitor. The level of miR-92a expression in response to the inhibitor were confirmed by stem-loop RT-PCR (Supplementary Fig. S1D). In the presence of this inhibitor, downregulation of DKK3 by MYC was completely abolished (Fig. 2I). As an independent approach, we treated the cells with CHIR99021, which resulted in elevated levels of MYC and reduced levels of DKK3. However, when the experiment was performed in the presence of the miR-92a inhibitor, the suppression of DKK3 by MYC was once again lost (Fig. 2J). These findings led us to conclude that miR-92a is the major, if not the sole mediator of DKK3 repression by MYC.

DKK3 represses Wnt signaling in colorectal cancer cells and Kras-mutated, trp53-, Apc-, and Smad4-null organoids

To evaluate the role of DKK3 in the context of WNT activating mutations in colorectal cancer, we analyzed the effect of DKK3 overexpression and knockdown on Wnt signaling using HCT116 cells which harbor an activating β-catenin mutation and SK-CO-1 cells which harbor an APC mutation. To induce Wnt signaling, media conditioned by murine Wnt3A-producing or control L cells were used (32). In HCT116 and SK-CO-1 cells upon Wnt3a treatment, LRP6 phosphorylation at Ser 1490, a hallmark of Wnt/β-catenin pathway activation, was increased, while the protein level of LRP6 remained unchanged. We also observed higher phosphorylation of GSK3β and increased level of Wnt target genes AXIN2 and CCND1. However, in the presence of overexpressed DKK3, all these effects were ablated (Fig. 3A, left and right). Conversely, knockdown of DKK3 in HCT116 cells increased basal as well as Wnt-induced phospho-LRP6 levels compared to control siRNA-transfected cells (Fig. 3A, middle). We further confirmed these observations using the β-catenin–responsive TOP FLASH reporter, whose activity was enhanced by Wnt3a, but significantly repressed by DKK3 overexpression in the same cell lines (Fig. 3B and D). Conversely, this reporter's activity was enhanced by DKK3 knockdown (Fig. 3C) indicating that DKK3 suppresses Wnt signaling at endogenous levels. To rule out off-target effects of siRNAs, we also used CRISPR-Cas9–mediated genome editing. DKK3-specific short guide RNAs and homology directed repair plasmids containing the puromycin resistance genes were transfected into HCT116 cells and puromycin-resistant clones were selected. Effective DKK3 knockdown in pooled clones was confirmed by Western blotting (Supplementary Fig. S1E). The effect of DKK3 downregulation on Wnt signaling was analyzed as above, and stronger Wnt-dependent increase in phospo-LRP6 was observed in DKK3-edited cultures (Supplementary Fig. S1F). Taken together, these results suggest that DKK3 inhibits Wnt signaling in colorectal cancer cells even in the presence of activating APC and CTNNB1 mutations.

Figure 3.

DKK3 represses Wnt signaling in colorectal cancer cells and tumor organoids. A, Induction of Wnt signaling by Wnt3a-containing conditioned media. HCT116 and SK-CO-1 cells stably expressing DKK3 or vector alone (left and right) and HCT116 cells treated with control and anti-DKK3 siRNA (middle) were serum starved and treated with Wnt3a-CM or control L-CM. Total cell lysates was analyzed by immunoblotting using antibodies against pLRP6, pGSK-3β, AXIN2, CCND1, MYC, and DKK3 as indicated. Total LRP6, total pGSK-3β and GAPDH were used as loading control. B–D, Relative luciferase activities driven by Wnt-responsive TOP FLASH and control FOP FLASH reporters in cells from A. E, Schematic showing the generation of organoids from mouse colonic crypts followed by genome editing. F, Immunoblotting showing MYC and DKK3 levels in MYC-overexpressing and control organoids. G, Immunoblotting showing levels of active and inactive β-catenin, AXIN2, CCND1, MYC, and DKK3 in organoids overexpressing vector/DKK3 and treated with 200 ng of recombinant Wnt3a for 48 hours.

Figure 3.

DKK3 represses Wnt signaling in colorectal cancer cells and tumor organoids. A, Induction of Wnt signaling by Wnt3a-containing conditioned media. HCT116 and SK-CO-1 cells stably expressing DKK3 or vector alone (left and right) and HCT116 cells treated with control and anti-DKK3 siRNA (middle) were serum starved and treated with Wnt3a-CM or control L-CM. Total cell lysates was analyzed by immunoblotting using antibodies against pLRP6, pGSK-3β, AXIN2, CCND1, MYC, and DKK3 as indicated. Total LRP6, total pGSK-3β and GAPDH were used as loading control. B–D, Relative luciferase activities driven by Wnt-responsive TOP FLASH and control FOP FLASH reporters in cells from A. E, Schematic showing the generation of organoids from mouse colonic crypts followed by genome editing. F, Immunoblotting showing MYC and DKK3 levels in MYC-overexpressing and control organoids. G, Immunoblotting showing levels of active and inactive β-catenin, AXIN2, CCND1, MYC, and DKK3 in organoids overexpressing vector/DKK3 and treated with 200 ng of recombinant Wnt3a for 48 hours.

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For a more genetically defined ex vivo model of colon cancer, we used colonic crypts from mice bearing the KrasG12D-LSL mutation (33) to generate transformed organoids. Briefly, using CRISPR/Cas9 genome editing, we further modified them to carry trp53-, Apc-, and Smad4-null mutations commonly found in human colorectal cancer (Fig. 3E; Supplementary Fig. S2). We additionally engineered them to overexpress retrovirally encoded MYC, which resulted in reduced expression of Dkk3 (Fig. 3F).

Alternatively, to analyze the effect of DKK3 on Wnt signaling, organoids were stably transduced with control and DKK3-encoding retroviruses and treated with recombinant Wnt3a. We observed that compared to control cultures, DKK3-overexpressing organoids showed higher level of phosphorylated (inactive) β-catenin and reduced levels of non-phosphorylated (active) β-catenin. Consistent with this finding, overexpression of DKK3 blunted induction by Wnt3a of AXIN2 and CCND1, its canonical targets (Fig. 3G). These results further establish the role of DKK3 as a negative regulator of Wnt signaling in diverse genetic backgrounds.

MYC enhances Wnt signaling by enabling the miR-92a/DKK3 axis

Because overexpressed MYC downregulates DKK3, which acts as a Wnt signaling inhibitor, we hypothesized that in addition to being downstream of Wnt in the signal transduction pathway, MYC could reciprocally enhance Wnt signaling and in doing so boost its own expression. To evaluate this scenario, control and MYC-overexpressing HCT116 cells were treated with Wnt3a-CM or control CM. HCT116-MYC cells indeed showed elevated phospho-LRP6 levels (Fig. 4A, left) and higher levels of Wnt targets AXIN2, CCND1, and MYC. Notably, these effects were abolished when HCT116-MYC cells were additionally transfected with either the miR-92a inhibitor or the DKK3 expression cassette (Fig. 4A, middle and right), fully supporting our hypothesis about the role of the miR-92a/DKK3 axis downstream of MYC. Similar effects were observed using the TOP FLASH reporter assay performed in the same three types of cells (Fig. 4BD). On the basis of our findings, we proposed an amended model for MYC and Wnt interplay in colorectal cancer cells wherein MYC and Wnt signaling are linked in a feed-forward loop involving suppression of DKK3 expression by miR-92a (Fig. 4E).

Figure 4.

MYC enhances Wnt signaling by enabling the miR-92a/DKK3 axis. A, Induction of Wnt signaling by Wnt3a-containing conditioned media. HCT116 cells overexpressing vector or MYC (left) and HCT116-MYC transfected with either miR-92a inhibitor (middle) or DKK3 expression cassette (right) were serum starved and treated with Wnt3a-CM or control L-CM. Total cell lysates was analyzed by immunoblotting using antibodies against pLRP6, AXIN2, CCND1, MYC, and DKK3 as indicated. Total LRP6 and GAPDH were used as loading controls. B–D, Relative luciferase activities driven by Wnt-responsive TOP FLASH and control FOP FLASH reporters in cells from A. E, The overall model depicting the effect of the MYC→miR-92a-|DKK3 axis on Wnt signaling.

Figure 4.

MYC enhances Wnt signaling by enabling the miR-92a/DKK3 axis. A, Induction of Wnt signaling by Wnt3a-containing conditioned media. HCT116 cells overexpressing vector or MYC (left) and HCT116-MYC transfected with either miR-92a inhibitor (middle) or DKK3 expression cassette (right) were serum starved and treated with Wnt3a-CM or control L-CM. Total cell lysates was analyzed by immunoblotting using antibodies against pLRP6, AXIN2, CCND1, MYC, and DKK3 as indicated. Total LRP6 and GAPDH were used as loading controls. B–D, Relative luciferase activities driven by Wnt-responsive TOP FLASH and control FOP FLASH reporters in cells from A. E, The overall model depicting the effect of the MYC→miR-92a-|DKK3 axis on Wnt signaling.

Close modal

MYC induces migration and invasion of colorectal cancer cells by repressing DKK3 and activating Wnt signaling, resulting in enhanced metastasis

Because MYC is a known regulator of epithelial–mesenchymal transition (EMT) and the motile/invasive phenotype (34), we asked whether DKK3 downregulation plays a role in these processes. We first evaluated the expression of EMT markers in HCT116 cells stably overexpressing MYC. We found that HCT116-MYC cells have increased expression of mesenchymal markers such as N-cadherin and α-smooth muscle actin, with a concomitant decrease in the expression of epithelial markers such as E-cadherin. However, restoring DKK3 in these cells noticeably decreased the level of MYC and reversed its effects on EMT markers (Fig. 5A).

Figure 5.

MYC induces migration and invasion of colorectal cancer cells by repressing DKK3 and activating Wnt signaling, resulting in enhanced metastasis. A, Immunoblotting showing expression of EMT markers in HCT116 cells stably expressing vector, MYC, or MYC+DKK3. B, Migration patterns of the HCT116 cell line derivatives 24 hours after wounding. Bars represent the closure of the initial wounded area, in percentages. C, Migration and invasion potential of the HCT116 cell line derivatives as assessed using the transwell assay, with or without Matrigel, respectively. Numbers of migrated and invaded cells in the bottom chambers were plotted, with each bar representing mean ± SEM. All results are representative of three separate experiments each analyzed using Student t test. D, Migration patterns of the HCT116 cell line derivatives 24 hours after wounding with or without Wnt inhibitor ICG-001. Bars represent the closure of the initial wounded area, in percentages. E, Migration and invasion potential of the HCT116 cell line derivatives as assessed using the transwell assay, with or without Wnt inhibitor ICG-001. Quantitation of the results was performed as in C. F, 4× and 10× micrographs showing hematoxylin and eosin staining of metastatic lesions in liver sections (top) and primary tumor (bottom). The plot on the right shows quantitation of metastatic lesions per liver in mice implanted with vector (n = 3) and MYC-overexpressing (n = 5) organoids.

Figure 5.

MYC induces migration and invasion of colorectal cancer cells by repressing DKK3 and activating Wnt signaling, resulting in enhanced metastasis. A, Immunoblotting showing expression of EMT markers in HCT116 cells stably expressing vector, MYC, or MYC+DKK3. B, Migration patterns of the HCT116 cell line derivatives 24 hours after wounding. Bars represent the closure of the initial wounded area, in percentages. C, Migration and invasion potential of the HCT116 cell line derivatives as assessed using the transwell assay, with or without Matrigel, respectively. Numbers of migrated and invaded cells in the bottom chambers were plotted, with each bar representing mean ± SEM. All results are representative of three separate experiments each analyzed using Student t test. D, Migration patterns of the HCT116 cell line derivatives 24 hours after wounding with or without Wnt inhibitor ICG-001. Bars represent the closure of the initial wounded area, in percentages. E, Migration and invasion potential of the HCT116 cell line derivatives as assessed using the transwell assay, with or without Wnt inhibitor ICG-001. Quantitation of the results was performed as in C. F, 4× and 10× micrographs showing hematoxylin and eosin staining of metastatic lesions in liver sections (top) and primary tumor (bottom). The plot on the right shows quantitation of metastatic lesions per liver in mice implanted with vector (n = 3) and MYC-overexpressing (n = 5) organoids.

Close modal

Cells undergoing EMT acquire migratory and invasive phenotypes (35). Thus, in vitro wound healing (scratch) assays were performed to evaluate the role of MYC in colorectal cancer cell migration. We determined that HCT116-MYC cells closed the wound twice as efficiently as the control cells, and this effect was fully reversed upon DKK3 reexpression (Fig. 5B). Furthermore, MYC overexpression significantly enhanced migration and invasion of HCT116 cells compared with the vector-expressing controls in the regular and matrigel-coated Boyden chambers, respectively. Once again, these effects were reversed in HCT116 MYC+DKK3 cells (Fig. 5C). To determine whether MYC-induced migration and invasion are Wnt pathway-dependent, we treated HCT116-MYC cells with ICG-001, a well-validated Wnt inhibitor (36). In both wound healing and Boyden chamber assays, pro-EMT effects of MYC were reversed by ICG-001 (Fig. 5D and E). Finally, to establish the effects of MYC on metastasis in vivo, we utilized a syngeneic, orthotopic model of metastatic colorectal carcinoma. The tumoroids with vector or MYC overexpression were orthotopically transplanted into the cecal walls of syngeneic C57BL/6J mice. Six weeks post-transplant, primary tumors and livers were resected. IHC analysis showed that while primary tumors formed in all recipients, the number of liver metastatic lesions were significantly higher in the mice transplanted with MYC-overexpressing organoids as compared with vector transplanted mice (Fig. 5F). Collectively, these data suggest a role for MYC in the induction of EMT and the associated motile/invasive phenotypes strongly dependent on DKK3 downregulation and ensuing Wnt pathway activation.

MYC is the central player in colorectal cancer, activated either by gene amplification or, by virtue of being a Wnt target gene, via APC deletions or β-catenin–activating mutations. However, the full repertoire of MYC-regulated pathways in colorectal cancer has not been established. Previous work from other investigators and our own laboratory have demonstrated that many of MYC effects on gene expression are realized through dysregulation of miRNAs (reviewed in ref. 28), in particular the miR-17-92 cluster miRNA (29, 30). Earlier we demonstrated that in colorectal cancer MYC-induced miR-17-92 directly targets thrombospondin-1 and other anti-angiogenic factors bringing about the angiogenic switch (20). In addition, miR-17-92 broadly dampens TGFβ signaling (e.g., via targeting of SMAD4; refs. 21, 37, 38), which is often a barrier to colorectal cancer neovascularization (22).

Here we demonstrate that another miR-17-92 direct target is DKK3. While this cluster encodes six distinct miRNAs (miR-17, -18a, -19a, -20a, -19b, and -92a), miR-92a by far is the most abundant (39), serves as a proposed biomarker for early detection of colorectal cancer (40, 41), and according to some reports is associated with worse prognosis in patients with colorectal cancer (42). Notably, ectopic overexpression of miR-92a has been shown to promote stem cell characteristics, proliferation, and migration of colorectal cancer cells by downregulating multiple components of the Wnt pathway including, but not limited to, DKK3 (43, 44). However, neither the oncogenic mechanism of miR-92a overexpression nor its direct binding to DKK3 mRNA have been established. Here we demonstrate direct binding of miR-92a to the DKK3 transcript, establishing DKK3 as a bona fide miR-92a target. We also show that miR-92a mediates repression of DKK3 by MYC via the seed homology sequence in DKK3 3′UTR.

The miR-92a-DKK3 connection was recently reported to contribute to osteosarcoma cell proliferation by a yet to be determined mechanism (45). Similarly, the functional relationship between DKK3 and another member of the MYC family N-MYC was previously reported to exist in B-cell acute lymphoblastic leukemia and neuroblastoma, but the underlying miRNA-based mechanism (46) and the connection to Wnt signaling (47) were left unexplored. In contrast, our work places the miR-92a-DKK3 axis in the middle of the feed-forward loop by which MYC levels and Wnt signaling sustain each other (Fig. 4E). Surprisingly, this interplay unfolds in cells where Wnt signaling is thought to be constitutively active because of acquired mutations in this pathway. Furthermore, as a consequence of hyperactive Wnt signaling, MYC is able to induce expression of EMT markers and promote migration and invasion of colorectal cancer cells by a DKK3 downregulation-dependent mechanism. This effect of MYC was also observed in a syngeneic, orthotopic model of metastatic colorectal carcinoma where mice implanted with MYC-overexpressing tumoroids showed enhanced liver metastasis.

The broad role of DKK3 in Wnt signaling has been controversial and highly context-dependent. For example, unlike DKK1, DKK3 is unable to inhibit Wnt signaling in Xenopus embryos to set up secondary axis induction (48). More recent work demonstrated that DKK3 promotes Wnt signaling in Muller glia MIOM1 and HEK293 cell lines (49), inhibits it in pheochromocytoma PC12 and osteocarcinoma Saos-2 cells (50, 51), and has no effect in LNCaP prostate cancer cells (52). From broader studies with multiple cell lines, the consensus is emerging that DKK3 generally antagonizes Wnt signaling and cell proliferation in lung (53), breast (54), and cervical (55) cancers. The main caveat is that these studies were performed in cells presumably lacking Wnt pathway mutations. We found that manipulating DKK3 levels profoundly affects Wnt signaling in murine and human colon cancer cells with preexisting CTNNB1 and APC mutations. This observation is conceptually similar to the finding that restoration of secreted frizzled-related proteins (SFRP) expression in colon cancer cells attenuates Wnt signaling despite the presence of APC/CTNNB1 mutations (56). Interestingly, SFRP1 and DKK1 are repressed by MYC in mammary epithelial cell lines (57) suggesting that inhibition of secreted Wnt antagonist might be a common theme in MYC-driven oncogenic programs. This could be a potent complement to the reported ability to MYC to activate transcription (58) and potentiate translation (59) of several key Wnt signaling components including LEF1. More broadly speaking, MYC effects on Wnt signaling highlight the importance of “secondary” hyperactivating events and help explain the surprising pattern of seemingly redundant but still co-occurring mutations in human colorectal cancer.

No disclosures were reported.

P. Sehgal: Conceptualization, formal analysis, investigation, writing–original draft, writing–review and editing. C. Lanauze: Writing–review and editing. X. Wang: Investigation, methodology. K.E. Hayer: Data curation, software, formal analysis. M. Torres-Diz: Data curation, software, formal analysis. N. Leu: Investigation, methodology. Y. Sela: Supervision, investigation, methodology. B.Z. Stanger: Conceptualization, supervision, methodology, writing–review and editing. C.J. Lengner: Conceptualization, supervision, methodology, writing–original draft, project administration, writing–review and editing. A. Thomas-Tikhonenko: Conceptualization, supervision, investigation, methodology, writing–original draft, project administration, writing–review and editing.

The authors thank members of their laboratories for many helpful discussions. Initial experiments on DKK3 downregulation in the ATT group were performed by Grace Tan. Wnt3a-overexpressing mouse L cells were a kind gift from Patrick Viatour (CHOP). All the schematics in the article have been created with BioRender.com

This work was supported by NIH grant R01 CA196299 (A. Thomas-Tikhonenko) and T32 CA009140 (C. Lanauze) and funds from the Colorectal Cancer Translational Center of Excellence of the Abramson Cancer Center at the University of Pennsylvania (P. Sehgal, C.J. Lengner, A. Thomas-Tikhonenko).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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