Viral mimicry acts as a tumor suppressor in colitis

Larsen, Frederikke; Jeong, Will; Schep, Daniel; Good, Hayley J.; Shin, Alice E.; Zhang, Liyue; Derouet, Mathieu F.; Wang, Timothy C.; Castellani, Christina A.; Shooshtari, Parisa; Asfaha, Samuel

doi:10.1038/s41467-026-68850-1

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Published: 04 February 2026

Viral mimicry acts as a tumor suppressor in colitis

Nature Communications volume 17, Article number: 1313 (2026) Cite this article

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Abstract

Transposable elements (TEs) comprising nearly 50% of the genome are generally silenced by epigenetic mechanisms. Epigenetic anti-cancer drugs can lead to their re-expression, however, the role of TEs in tumorigenesis is unknown. Here, we demonstrate that TEs and their activation of a viral mimicry response plays an important tumor suppressive role in inflammation. We discovered that both patients and mice with colitis express TEs that lead to a viral mimicry response. Interestingly, this response inhibits stemness of cancer-initiating cells. Further activation of viral mimicry by DNA hypomethylation inhibits tumorigenesis. Conversely, knockout of the anti-viral signaling protein MAVS promotes tumorigenesis and reverses the anti-tumor effect of DNA hypomethylation, confirming a tumor suppressive role of viral mimicry. Consistent with this finding, patients with colitis-associated dysplasia show decreased expression of TEs and interferon-related genes. These findings suggest that activation of viral mimicry inhibits stemness and plays a key tumor suppressive role in inflammation.

Introduction

The vast majority of the human genome consists of noncoding intergenic DNA and intronic regions of DNA. This noncoding DNA includes repetitive sequences that consist of transposable elements (TEs), microsatellites, and DNA duplications^1,2. Interestingly, TEs, including long-interspersed elements (LINEs) and small-interspersed elements (SINEs) that make up much of the repetitive DNA, are able to duplicate and move around in the genome, whereas endogenous retroviruses (ERVs) are mostly immobile³. In homeostasis, however, expression of these TEs is repressed by epigenetic mechanisms such as DNA methylation^4,5,6 and histone modification^{7,8,9,10,11,12}. In contrast, aberrant expression of TEs has been observed in disease states such as cancer^13,14,15 and autoimmune diseases^{16,17,18,19,20}, including the neuroinflammatory disease Aicardi–Goutières syndrome^16,21 and systemic lupus erythematosus^17,18,22.

Recent studies have demonstrated that anti-cancer drugs that affect DNA methylation, such as 5-AZA-2′-deoxycytidine (5-AZA), lead to re-expression of TEs that form immunostimulatory nucleic acids such as double-stranded RNAs (dsRNAs)^23,24,25,26. These dsRNAs in turn activate innate immune sensors melanoma differentiation-protein 5 (MDA5) and retinoic acid-inducible gene I (RIG-I) that signal through mitochondrial antiviral-signaling protein (MAVS) to induce an antiviral type I or III interferon response and cell death^{23,24,27,28,29}. This anti-viral-like immune response is known as a “viral mimicry” response. Indeed, DNA demethylating anti-cancer drugs are postulated to kill cancer cells, at least in part, through activation of “viral mimicry” and interferon signaling^{23,24,28,30,31,32,33}.

These observations have led to speculation that re-expression of TEs during injury serves as a natural defense mechanism to prevent tumorigenesis³⁴. It is not known, however, whether blocking the viral mimicry response makes cells prone to tumorigenesis or can reverse the anti-cancer effects of DNA demethylating drugs. Thus, we hypothesized that re-expression of TEs during injury (i.e., colitis) leads to a viral mimicry response that suppresses tumor initiation, and therefore, knockout of the viral mimicry response will promote tumorigenesis.

Consistent with this hypothesis, we here provide evidence that TEs become re-expressed during inflammatory injury of the colon in both mice and humans. Moreover, we demonstrate that further upregulation of TEs by DNA hypomethylation, using 5-AZA or knockout of the DNA methyltransferase enzyme DNMT1, inhibits colonic tumorigenesis by activation of a viral mimicry response. Importantly, we show that blocking the viral mimicry response by knocking out the innate immune sensor MAVS promotes tumorigenesis, reversing the effects of 5-AZA or DNMT1 KO. Remarkably, the viral mimicry response inhibits the stemness of cancer-initiating cells in a cell-autonomous manner independent of an immune cell response. These findings suggest that viral mimicry serves as an important tumor suppressor during cancer initiation.

Results

Viral mimicry is induced in active colitis and downregulated in patients with IBD-associated dysplasia

To first examine whether colonic injury leads to re-expression of transposable elements, we examined expression of transposable elements and interferon response genes in patients with inflammatory bowel disease (IBD). We examined gene expression in the RNA-sequencing dataset GSE83687³⁵ of surgical specimens from 61 control patients without IBD versus 77 inflamed tissues from patients with active IBD. We found that transposable elements of multiple subclasses including Alu elements (i.e., SINEs) known to engage MDA5^36,37,38 and interferon response genes such as IFNAR1/2, IRFs, ISG15, and MX1 (see Table S1 for a full list of genes) were significantly upregulated in patients with active IBD versus controls (Fig. 1a, b, Supplementary Fig. 1a). Indeed, the majority of TEs were upregulated with inflammation (p = 3.21 × 10⁻²¹) (Supplementary Fig. 1b), and we found a positive correlation between IFN and TE expression (Supplementary Fig. 1c), consistent with the activation of a viral mimicry response. Moreover, the evolutionary age of Alus has previously been associated with their predicted dsRNA foldability³⁷. Thus, we analyzed the Alus based on evolutionary age, but did not detect a difference in the expression levels of Alus between patients with IBD versus healthy controls when divided according to their evolutionary ages (Supplementary Fig. 1d). These data suggest that it may be specific TEs, rather than a broader class of TEs, that drive stimulation of downstream interferon signaling.

**Fig. 1: Viral mimicry is induced in active IBD and downregulated in patients with IBD-associated dysplasia.**

To further examine whether the viral mimicry response was altered in the setting of colitis-associated dysplasia, we analyzed a separate publicly available dataset that included RNA expression data of colonic biopsies from 41 healthy controls, 22 patients with IBD without dysplasia, and 7 patients with IBD-associated dysplasia (non-dysplastic adjacent region) (GSE230524³⁹). Similar to the first dataset, we found expression of interferon response genes was upregulated in patients with IBD when compared to healthy controls. In patients with IBD-associated dysplasia, however, the expression of these genes was downregulated when compared to those patients without dysplasia (Fig. 1c–e). Interestingly, when comparing IBD patients with or without dysplasia, the top 10 upregulated GO term pathways in those without dysplasia were those related to interferon signaling and responses to virus (Fig. 1f). These data suggest that in the disease progression from IBD to IBD-associated dysplasia, the viral mimicry response pathways are downregulated. Indeed, transposable element expression was downregulated in patients with IBD-associated dysplasia versus patients with IBD without dysplasia (Fig. 1g). This pattern also held true for the comparison between healthy controls and patients with IBD-associated dysplasia where we similarly observed downregulation of TEs (Supplementary Fig. 1e). Furthermore, patients with sporadic colorectal cancer from the GSE230524 dataset (n = 8) had decreased expression of TEs when compared to healthy controls, consistent with TE expression being reduced during tumorigenesis even in the absence of inflammatory injury (Supplementary Fig. 1f). Together, these data suggest that upon injury or inflammation, cells upregulate TEs and interferon signaling. However, with progression from inflammation to inflammation-associated dysplasia, the viral mimicry response is downregulated.

Activation of viral mimicry by DNA hypomethylation inhibits colitis-associated cancer

We next sought to validate our human observations in a mouse model of colitis. In colonic epithelial cells isolated from mice treated with dextran sodium sulfate (DSS), we found the expression of several classes of transposable elements such as LINE1, SINEs, and Mu-LV was significantly increased in colitis (Fig. 2a). Interferon response genes were similarly increased in epithelial cells from mice with colitis versus controls (Fig. 2b), consistent with the observations made in patients with IBD. To further examine the relationship between colitis and the viral mimicry response, we treated mice with different doses (0.5, 1.5, and 2.5%) of DSS (Supplementary Fig. 2a). Mice administered 2.5% DSS displayed greater body weight loss (Supplementary Fig. 2b) than those given 0.5% or 1.5% DSS, and mice administered either 1.5% or 2.5% DSS displayed decreased colon length, histological damage, and increased MPO activity when compared to controls or mice treated with 0.5% DSS (Supplementary Fig. 2c–e). Notably, mice treated with 2.5% DSS displayed the highest level of TE expression and expression of interferons or interferon response genes (Supplementary Fig. 2f, g). Taken together, these data suggest that induction of a viral mimicry response correlates with colitis severity.

To next test whether further activation of a viral mimicry response could affect inflammation-associated tumorigenesis, we examined the effects of the DNA demethylating drug 5-AZA-2′deoxycytidine (5-AZA) on colonic tumorigenesis. 5-AZA has previously been shown to induce a viral mimicry response^{23,24,26,28,40}. To induce colitis-associated cancer, we administered the carcinogen azoxymethane (10 mg/kg i.p. × 1 dose) followed by 2.5% dextran sodium sulfate (DSS) in the drinking water of C57BL6/J (wildtype) mice (Fig. 2c). Mice were then treated with six doses of 5-AZA (5 mg/kg i.p. weekly) or vehicle, with the first dose given 2 days prior to DSS and the remaining five doses being given weekly 1 week post DSS (Fig. 2c). Although all mice developed tumors, mice treated with 5-AZA had significantly fewer colonic tumors than vehicle treated mice (Fig. 2d, e), with no difference in tumor size being detected (Fig. 2f). Global 5-mC levels, a measure of overall methylation, were significantly reduced in colonic epithelial cells from mice treated with two doses of 5-AZA versus vehicle (Fig. 2g). No major differences in tumor histology were observed between vehicle and 5-AZA treated mice based on H&E staining of tumors (Fig. 2h). To validate our results in another model of colitis-associated cancer, we used our previously described Dclk1^CreERT2;Apc^f/f;Rosa26^mTmG mice⁴¹, and administered tamoxifen (6 mg p.o. q2d × 3 doses) followed by 5-AZA and 2.5% DSS as described above (Fig. 2i). Analogous to the AOM/DSS model, we found 5-AZA-treated mice had significantly fewer colonic tumors than vehicle-treated mice (Fig. 2j,l), with significantly fewer mice having tumors than vehicle-treated mice (Fig. 2k). Interestingly, in this model we also observed significantly smaller tumors in mice treated with 5-AZA versus vehicle treated mice (Fig. 2m). Importantly, we detected increased expression of transposable elements including LINEs, SINEs, and ERVs, as well as increased interferons and JAK/STAT signaling in both models of colitis-associated cancer (Fig. 2n, o, Supplementary Fig. 3a–g), consistent with DNA hypomethylation inducing a viral mimicry response. To confirm that the expression of interferons is induced by expression of TEs, we transfected HCT116 and HT29 colorectal cancer cell lines with plasmids overexpressing LINE1 or SINE B2. Expression of either LINE1 or SINE B2 induced expression of interferons and interferon response genes in both cell lines (Supplementary Fig. 4a–d), confirming that TEs induce interferon signaling. These data show that colitis injury induces a viral mimicry response and that further activation of this response by 5-AZA induced DNA hypomethylation inhibits colitis-associated tumorigenesis.

DNA hypomethylation of cancer-initiating cells activates viral mimicry to reduce stemness

To test if activation of the viral mimicry response specifically in cancer-initiating cells affects tumorigenesis, we genetically knocked out the DNA maintenance methyltransferase enzyme DNMT1 to induce a viral mimicry response as previously described^40,42,43. To do this, we crossed Dclk1^CreERT2;Apc^f/f; Rosa26^TdTomato mice to Dnmt1^f/f mice. This allowed us to conditionally express a truncated form of APC and simultaneously delete DNMT1 in DCLK1+ cells upon tamoxifen administration. Mice were administered three doses of tamoxifen (6 mg p.o.) followed by 2.5% DSS in the drinking water 1 week later (Fig. 3a). Fourteen weeks post DSS, mice were sacrificed for analysis. Using this model, we examined the role of the viral mimicry response specifically within the cancer-initiating cells of colitis-associated cancer. Similar to 5-AZA treated mice, significantly fewer Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice developed colonic tumors when compared to Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato mice (Fig. 3c). Indeed, mice with DNMT1 loss in DCLK1+ cells also had significantly fewer and smaller tumors (Fig. 3b, d, e) when compared to Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato mice that were Dnmt1^+/+ or Dnmt1^+/f. Importantly, DNMT1 loss did not affect DCLK1+ cell number as determined by the number of TdTomato+ cells quantified in vivo (Supplementary Fig. 5a–f) or in intestinal organoids from Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice (Supplementary Fig. 6a–d). Loss of DNMT1 in tumors from Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice was confirmed by RT-qPCR for Dnmt1 expression. Consistent with effective knockout of DNMT1, tumors from Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice had significantly lower Dnmt1 expression when compared to tumors from Dnmt1^+/+ and Dnmt1^+/f mice (Fig. 3f). Moreover, we confirmed that the tumors from the Dnmt1^f/f mice contained only the recombined allele of Dnmt1, whereas tumors from Dnmt1^+/+ mice displayed the WT allele (Fig. 3g). Notably, as previously reported^44,45, DNA damage as detected by γH2AX staining was seen in 5-AZA treated mice. However, this was not the case in mice with DNMT1 loss, suggesting that the presence of DNA damage did not necessarily associate with the observed effects on tumorigenesis (Supplementary Fig. 7a–d). Importantly, global 5-mC levels showed loss of DNA methylation in tumors from Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice compared to tumors from Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato mice (Fig. 3h). To confirm activation of a viral mimicry response, tumors were isolated from Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato and Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice, and RT-qPCR expression of TEs and interferons examined. Indeed, DNMT1 KO induced TE and interferon expression (Fig. 3i–j), confirming activation of a viral mimicry response. These data demonstrate that loss of DNMT1 in cancer-initiating DCLK1+ cells activates a viral mimicry response and inhibits colonic tumor formation.

Next, we investigated whether activation of viral mimicry inhibits tumorigenesis by blocking stemness of cancer-initiating cells. To test this, we administered three doses of tamoxifen (6 mg p.o.) to Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato and Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice followed by a low dose of DSS (0.5%) (Fig. 3k) that initiates stemness (i.e., lineage tracing from DCLK1+ cells) without promoting dysplasia⁴⁶. We found significantly fewer TdTomato+ lineage-traced crypts in Dnmt1^f/f mice versus Dnmt1^+/+ mice (Fig. 3l–m), suggesting that loss of DNMT1 in DCLK1+ cells reduced stemness of DCLK1+ cells. To further validate these findings in vitro, we generated colonic organoids from Dclk1^CreERT2;Rosa26^TdTomato;Apc^f/f and Dclk1^CreERT2;Rosa26^TdTomato;Apc^f/f;Dnmt1^f/f mice and treated the organoids with 4-hydroxytamoxifen to label DCLK1+ cells. These organoids were then dissociated into single cells, and the number of TdTomato+ traced organoids quantified after 7 days in culture as previously described⁴⁷ (Supplementary Fig. 8a). The percentage of DCLK1+ cells that were able to give rise to lineage-traced organoids was significantly reduced upon DNMT1 loss when compared to DNMT1 wildtype cells (Supplementary Fig. 8b, c).

To test whether pharmacologic DNA hypomethylation by 5-AZA can similarly inhibit DCLK1+ cell stemness, we administered 0.5% DSS to Dclk1^CreERT2;Rosa26^mTmG;Apc^f/f mice in addition to either 5-AZA or vehicle (Fig. 3n). We observed significantly fewer GFP+ lineage-traced crypts in mice treated with 5-AZA versus vehicle (Fig. 3o, p). Moreover, in mice treated with 5-AZA, we detected a small number of single DCLK1+ cells that expressed GFP but remained quiescent and did not lineage trace crypts or tumors (Supplementary Fig. 8d, e). This is in contrast to vehicle-treated mice, where only fully lineage-traced tumors were seen. Taken together, these data suggest that DNA hypomethylation reduces the stemness of APC-mutant DCLK1+ cells.

5-AZA induces DNA hypomethylation of transposable elements in colonic epithelial cells

Given that 5-AZA inhibited tumor formation, we next sought to confirm that 5-AZA led to transposable element expression by DNA hypomethylation of these elements during cancer initiation. WT mice were treated with 5-AZA or vehicle in the presence or absence of 2.5% DSS induced colitis. Two weeks following DSS, colonic tissues were collected (Fig. 4a). Notably, 5-AZA-treated mice had significantly less weight loss during the acute period of DSS, but had no difference in colon length (Supplementary Fig. 9a, b) or histology (Supplementary Fig. 9c) when compared to vehicle-treated mice (day 19 post DSS initiation). To examine DNA methylation, we isolated DNA from epithelial cells dissociated from the colon of mice in each of the four groups and determined methylation status using the Infinium Mouse Methylation BeadChip Array from Illumina, covering more than 285,000 CpGs. As predicted, we found that 5-AZA significantly reduced global DNA methylation levels as indicated by lower mean β-values (Fig. 4b) in colonic epithelial cells treated with 5-AZA. When comparing control and 5-AZA treated mice we found that 54.5% of all CpGs analyzed were significantly differentially methylated (FDR < 0.01, effect size > 0.1) (Fig. 4c) with 154,488 CpGs being hypomethylated and one CpG being hypermethylated (CpG: uk10321) (Fig. 4d). When comparing DSS and 5-AZA + DSS treated mice we observed 43.7% of all CpGs analyzed were significantly differentially methylated (Fig. 4c) with hypomethylation of more than 120,000 CpGs, and only one CpG (CpG: cg47977693) being hypermethylated (FDR < 0.01, effect size > 0.1) (Fig. 4e). Similarly, we detected regional DNA hypomethylation with 5-AZA treatment both in the presence or absence of colitis (Fig. 4f). Importantly, we detected DNA hypomethylation of repeat elements as a genomic region (Fig. 4f). We next checked whether 5-AZA treatment leads to DNA hypomethylation of different classes of TEs within colonic epithelial cells. We found that all elements, including SINEs, LINEs, satellite repeats, and endogenous retroviruses (ERVs), were hypomethylated following 5-AZA treatment versus vehicle and DSS-treated mice (Fig. 4g).

When looking at specific CpGs within each class of transposable elements, we found 18 CpGs in satellite repeats, 9 CpGs in LTRs, 43 CpGs in LINEs, 51 CpGs in ERV1s, 310 CpGs in ERV2s, 30 CpGs in ERV3s, and 16 CpGs in retroelements that were significantly hypomethylated between either control and 5-AZA-treated mice, or DSS and 5-AZA + DSS treated mice (Fig. 4h, Supplementary Fig. 10a, b). When comparing the CpGs that were hypomethylated between control and 5-AZA, and between DSS and 5-AZA + DSS treated mice, we found 127 CpGs (26.4%) common to both comparisons. In contrast, 223 CpGs (46.4%) were specifically hypomethylated between control and 5-AZA-treated mice, while 131 CpGs (27.2%) were specifically hypomethylated only between DSS and 5-AZA + DSS-treated mice (Supplementary Fig. 10c). This suggests that 5-AZA alters the expression of many of the same transposable elements regardless of the inflammation present. To exclude the possibility that interferon response genes, including Mavs, were upregulated due to hypomethylation of the promoter region of these genes, we looked at methylation of CpGs located in CpG islands, shelves, or shores of these genes. We found that these were not different between groups (Supplementary Fig. 10d, e), suggesting that activation of interferon response genes was indeed a result of TE upregulation. These data suggest that 5-AZA induces a viral mimicry response by hypomethylating DNA of TEs in colonic epithelial cells.

Knockout of the viral mimicry response promotes tumorigenesis and reverses the effect of DNA hypomethylation

Next, to examine whether the main antitumor effect of DNA hypomethylation is mediated via activation of a viral mimicry response, we compared colonic tumorigenesis in WT or Mavs-tm1Zjc (MAVS-KO) mice treated with 5-AZA or vehicle in the AOM/DSS colitis-associated cancer model (Fig. 5a). Given that MAVS-KO mice are more susceptible to DSS colitis^48,49, a lower dose of DSS (1.5 %) was used to induced colitis in these mice. Interestingly, MAVS-KO mice had reduced survival irrespective of treatment when compared to WT mice (Fig. 5c). Furthermore, the percentage of mice with tumors was significantly higher in MAVS-KO mice versus 5-AZA-treated WT mice (Fig. 5d). MAVS-KO mice also had increased tumors when compared to WT mice, regardless of treatment (5-AZA versus vehicle) (Fig. 5b, e). We confirmed that MAVS-KO mice did not have any Mavs gene expression as detected by RT-qPCR (Fig. 5f). As predicted, expression of transposable elements was significantly increased upon 5-AZA treatment in all groups (Fig. 5g), whereas increased gene expression of interferons was only seen in 5-AZA-treated WT mice and not MAVS-KO mice (Fig. 5h).

**Fig. 5: Knockout of viral mimicry promotes tumorigenesis and reverses the effect of DNA hypomethylation.**

We next validated these observations in a second model of colitis-associated cancer. Here, we crossed our Dclk1^CreERT2;Rosa26^TdTomato;Apc^f/f;Dnmt1^f/f mice to MAVS-KO mice and induced tumor formation (Fig. 5i). We found that the percentage of mice with tumors was significantly lower with DNMT1 loss compared to controls (Fig. 5j). Knockout of MAVS in the setting of DNMT1 loss resulted in 100% of mice developing tumors, and reversal of the anti-tumor effects of DNMT1 loss (Fig. 5j). Similar to 5-AZA treatment, we detected significantly fewer tumors with DNMT1 loss compared to controls, whereas knockout of MAVS reverted tumor number back to control levels (Fig. 5k). These data clearly suggest that the main antitumor effect of DNMT1 loss is activation of the viral mimicry pathway. No difference in tumor size was seen between groups (Fig. 5l). Importantly, tumors collected from controls, Dnmt1^f/f mice and Dnmt1^f/f;Mavs-tm1Zjc mice revealed that tumors with DNMT1 loss had lower Dnmt1 expression compared to controls (Fig. 5m). Tumors with MAVS KO had undetectable expression of Mavs compared to both control and Dnmt1^f/f tumors (Fig. 5m). Together, these data show that loss of the viral sensing protein MAVS reverses the anti-tumor effect of DNA hypomethylation, consistent with activation of a viral mimicry response being essential to the anti-tumor effect of DNA hypomethylation.

Viral mimicry suppresses stemness of cancer-initiating cells

To next investigate whether the effects of viral mimicry on stemness are cell autonomous versus dependent on extracellular signaling, we used an in vitro epithelial-derived organoid culture model. Colonic organoids were established from Dclk1^CreERT2;Rosa26^TdTomato mice wildtype for the Apc gene or crossed to Apc^f/f, Apc^f/f;Dnmt1^f/f, Apc^f/f;Mavs-tm1Zjc, or Apc^f/f;Dnmt1^f/f;Mavs-tm1Zjc mice. Following 4-hydroxytamoxifen treatment for 48 h, organoids were dissociated into single cells, and TdTomato+ cells counted. Seven days post dissociation, fully traced TdTomato+ organoids were counted (Fig. 6a). Importantly, while DCLK1+ cells wildtype for the Apc allele did not form organoids, 5–10% of DCLK1+ cells that were Apc^f/f showed organoid-forming ability (Fig. 6b). As shown previously (Supplementary Fig. 8b, c), DNMT1 loss significantly reduced the number of TdTomato+ cells able to give rise to organoids (Fig. 6b, c). Remarkably, knockout of MAVS promoted stemness of DCLK1+ cells as measured by increased lineage tracing capacity of TdTomato+ cells as well as increased organoid size even in the setting of DNMT1 loss (Fig. 6b–d). Similarly, 5-AZA significantly reduced the lineage tracing capacity of DCLK1+ Tomato labeled cells, whereas MAVS-KO reversed this effect and increased organoid size (Fig. 6e–h). These findings suggest that the viral mimicry response inhibits the stemness of APC-mutant DCLK1+ cells in a cell-autonomous manner (Fig. 7).

**Fig. 7: Activation of viral mimicry suppresses tumorigenesis.**

Discussion

Epigenetic-modifying drugs can induce re-expression of transposable elements that stimulate an interferon-mediated viral mimicry response and cancer cell death^{23,24,40,42,43,50,51,52,53,54,55,56,57,58,59}. Increased interferon in response to chemo- or radio-therapy can lead to cancer cell cytotoxicity^60,61,62, and inhibition of interferon signaling by interferon receptor knockout has been shown to promote tumorigenesis^49,63,64. The role of transposable elements and the viral mimicry response in inflammation and cancer initiation, however, has not previously been examined.

In this study, we provide evidence that inflammation, which predisposes to tumorigenesis, can lead to re-expression of normally silent transposable elements in both mice and humans. Further expression of these elements by DNA hypomethylation inhibits the stemness of cancer-initiating cells and suppresses tumorigenesis, suggesting that the viral immune-sensing response inhibits tumorigenesis. Importantly, we found this effect to be cell autonomous and independent of an adaptive immune response. Indeed, knockout of the viral-sensing immune protein MAVS promoted stemness of DCLK1+ cells and led to increased colonic tumors. This suggests that re-expression of transposable elements and induction of a viral mimicry response serve to suppress tumor formation. Consistent with this observation, we found that the viral mimicry response is downregulated in IBD-associated dysplasia, suggesting that overcoming or inhibiting the viral mimicry response may be required to form tumors.

Re-expression of transposable elements has also been suggested to stimulate innate immune signaling and enhance immune checkpoint blockade responsiveness in cold tumors^{25,51,65,66,67,68,69}. In this study, we found that normally quiescent APC-mutant DCLK1+ cells transition to a proliferative stem cell state upon knockout of MAVS. Importantly, this transformation occurred in the absence of non-epithelial components and independent of an adaptive immune response. Notably, some transposable elements, such as SINE B2 and LINE1, are more likely to induce a viral mimicry response than other elements^36,70, such as LTRs that act in antigen presentation and induce T-cell mediated killing⁷¹. In our analysis, we detected broad expression of SINEs, LINEs, and LTRs upon DNA hypomethylation. In the context of colitis, we similarly observed broad upregulation of transposable elements. However, additional studies are needed to determine how different transposable elements affect the viral mimicry response and cancer initiation in colitis.

There are currently numerous clinical trials examining the effect of epigenetic modifiers in cancer^72,73. The results of this study suggest that refining these therapies to target transposable elements directly or induce similar downstream responses in a targeted approach may improve cancer treatment and cancer prevention. Moreover, combining therapies such as DNA demethylating drugs with chemotherapeutics^74,75 or immunotherapies^76,77,78 may further require consideration of effects on transposable elements. Interestingly, the global DNA hypomethylation changes often seen early in colorectal cancer have recently been proposed to be tumor suppressive due to upregulation of TEs, and increased DNA methylation is associated with a higher risk of colon cancer⁷⁹. Indeed, patients with high expression of ERVs and interferon response genes have improved survival outcomes and better prognosis than those with low ERV expression and interferon response^{14,80,81,82,83}. This is in line with our observation that expression of transposable elements has a tumor suppressive role.

Several groups have also suggested that negative regulators of the viral mimicry response, such as ADAR1 or the DNA/RNA helicase DHX9, can similarly be targeted to induce cancer cell death^{36,84,85,86,87,88,89}. Knockout of ADAR1 not only synergizes with 5-AZA to kill cancer cells³⁶, but also improves response to immunotherapy^65,84. Whether combining 5-AZA with inhibitors of ADAR1 or DHX9 can further suppress cancer initiation, however, requires further investigation.

In this study, we found TEs to be tumor suppressive; however, we acknowledge that there is also some evidence that TEs may be tumor-promoting. For instance, high expression of HERV-H in colorectal cancer patients has been associated with worse overall survival⁹⁰ and expression of other TEs associated with expression of certain oncogenes⁹¹. LINE1 retrotransposition into the APC gene has also been proposed to potentially be an initiating event in CRC⁹². Thus, disease-specific and/or TE-specific roles may exist in tumorigenesis, and further studies will need to be done to determine whether specific classes of TEs are responsible for the tumor suppressive effects we observe during cancer initiation.

In summary, we demonstrate that transposable elements play an important tumor suppressive role during cancer initiation. Specifically, we show that inhibition of the viral sensing immune response promotes stemness and tumorigenesis. DNA hypomethylation prevents tumor formation by inducing the expression of transposable elements and a viral mimicry response. Our findings suggest that transposable elements play a tumor suppressive role and may be a target for cancer prevention strategies, particularly in patients at high risk of cancer.

Methods

Animals

Animal studies were approved by the Western University Animal Care Committee according to guidelines established by the Canadian Council on Animal Care. Mice were housed in the London Regional Cancer Program vivarium. Mice were exposed to a 12-h light-dark cycle and housed with controlled temperature (19–22 °C) and humidity (40–60%), with water and regular chow ad libitum. Equal numbers of male and female mice were used for each experiment. All mice were inbred on a C57BL/6 J background. For practical reasons, the strains were maintained as homozygous when possible. Mouse lines used include C57Bl6/J (Jackson Labs), Dclk1^CreERT2 (Westphalen and Asfaha⁴¹), Rosa26^mTmG (Jackson Labs), Rosa26^TdTomato (Jackson Labs), Dnmt1^f/f (Mutant Mouse Resource and Research Centers), Mavs-tm1Zjc (kindly gifted by Dr. Daniel De Carvalho from University of Toronto), and Apc^f/f (National Cancer Institute).

Dclk1^CreERT2;Apc^f/f mice were crossed to Dnmt1^f/f mice to generate inducible knockout mice. For induction of Cre-mediated recombination, mice of 6–10 weeks of age received three doses of 6 mg tamoxifen (MP Biomedicals) dissolved in corn oil every 2 days by oral gavage. For AOM administration, mice of 6–8 weeks of age received a single i.p. injection of 10 mg/kg AOM (Sigma-Aldrich). Mice were exposed to 0.5–2.5% dextran sodium sulfate (DSS) (Gojira Fine Biomedicals) in the drinking water for 5 days. During DSS and the 5 days following DSS, mice were scored daily in regards to weight loss, stool consistency, movement, and survival. Following the acute period of DSS, body weights were measured once a week until experimental or humane endpoint. 5-AZA-2′-deoxycytidine (Sigma-Aldrich) was dissolved in PBS and given at a dose of 5 mg/kg by i.p. injection.

Tissue preparation, immunofluorescence, and microscopy

Tissue was harvested from euthanized mice and fixed in 4% paraformaldehyde (PFA) for 6–8 h at 4°. The tissue was cryopreserved in 30% sucrose at 4°, embedded in optimal cutting temperature (OCT) compound (Fisher Healthcare), and frozen on dry ice. Sections were cut at 6 μm. For paraffin embedding, tissue was harvested, fixed in 10% formalin (Fisher Chemical) for 24 h, and processed, followed by embedding in paraffin. Sections were cut at 5 μm.

For hematoxylin and eosin staining, sections were deparaffinized and rehydrated, followed by staining with CAT hematoxylin (BioCare Medical) and Eosin-Y (Fisher Chemical). Slides were mounted with Permount Mounting Medium (Fisher Scientific) for viewing.

Fluorescence and brightfield images were acquired using EVOS FL Auto.

Plasmid preparation

Plasmid E.coli strains of pB2-neo-TET (Plasmid #51287) and pBS-L1PA1-CH-mneo (Plasmid #51288) were purchased from Addgene. Plasmid preparation was carried out with Presto Mini Plasmid Kit (Geneaid Biotech). Briefly, an individual bacterial clone was picked from an E.coli LB agar plate and cultured at 37 °C overnight with shaking in TB medium with ampicillin (100 μg/ml). Bacteria were then pelleted by centrifugation at 8000 × g for 1 min. The pellet was next resuspended and lysed. Neutralization buffer was added to the lysate and centrifuged at 16,000 x g for 5 min. The supernatant was then transferred to a binding column and centrifuged at 16,000 x g for 30 s. The column was washed, and the plasmid eluted.

Transfection of cell lines

HT29 (ATCC, HTB-38) and HCT116 (ATCC, CCL-247) colorectal cancer cells were seeded at 150,000 cells per well in a 6-well plate in DMEM media (Multicell) supplemented with 1× Glutamax (Gibco) and Pen Strep (Gibco). Transfection of cells was performed 24 h after seeding using jetPRIME transfection reagents (PolyPlus) according to the manufacturer’s instructions. Cells were collected 48 h after transfection for downstream analysis.

Small intestinal and colonic mouse organoid culturing

Organoid culturing was performed as previously described⁹³. Briefly, the scraped mouse small intestine (SI) or colon was harvested and incubated in 2.5 mM (SI) or 10 mM (colon) EDTA for 60 min. Following mechanical disruption of tissues, isolated crypts were plated in Matrigel (Corning), and cultured in Advanced DMEM/F12 (Gibco) supplemented with Primocin (InvivoGen), 10 mM HEPES (Gibco), 1x Glutamax (Gibco), 1x B27 (Gibco), 1 mM N-acetyl-L-cysteine (Sigma-Aldrich), and 1x N-2 (Gibco) as well as 50 ng/mL EGF (PeproTech), 100 ng/mL Noggin (PeproTech), and 1 μg/mL R-spondin1 (ENR). 50% Wnt3a-conditioned media (made in house) was used for colonic cultures. 2–3 days post-culture, 2 μM 4-hydroxytamoxifen was added to the culture media for 48 h for Cre-mediated recombination. For 5-AZA treatment, 1 μM 5-AZA was added to the media for 24 h.

For single-cell dissociation, TrypLE Express was added to each well following 4-hydroxytamoxifen treatment and incubated until organoids were dissociated into single cells. The cells were replated into new Matrigel, and the ROCK inhibitor Y-27632 (Selleck Chemicals LLC) added to the media to prevent cell death. The number of TdTomato+ cells was counted on day 1, and the number of TdTomato+ organoids was counted on day 7 post single cell dissociation. Notably, when we quantified traced organoids, these were additionally confirmed to be consistent with stem cell labeling as they could be passaged a minimum of five times, except APC-WT TdTomato+ cells and TdTomato+ cells treated with 5-AZA or with DNMT1 loss that could not be maintained post passaging.

Organoid size was measured using ImageJ⁹⁴.

RNA isolation and RT-qPCR

RNA was isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. Lithium chloride purification of the RNA was performed for all tissues, as DSS is known to inhibit RT-qPCR amplification of mRNA⁹⁵. The RNA was reverse transcribed using iScript RT Supermix (Biorad). RT-qPCR was conducted using SYBR Green (Applied Biosystems). Reactions were run on a QuantStudio5 thermocycler. Gene expression levels were quantified using the ddCT method and normalized to the CT value of GAPDH. Primers are listed in Table S2. For tumors from Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice, RT-qPCR was only performed on tumors confirmed to have recombination of Dnmt1.

Small intestinal and colonic epithelial cell isolation and FACS

In preparation for hybridization to the Illumina Mouse Methylation BeadChip array, colonic tissue was harvested and incubated in 10 mM EDTA in PBS for 1 h. After incubation, the tissue pieces were transferred to 10% Fetal Bovine Serum (FBS) (Multicell) in PBS and mechanically disrupted. The solution was transferred through a 100 μm strainer and centrifuged at 300 × g for 5 min to pellet colonic crypts.

For FACS, SI, and colonic tissues were harvested and washed in PBS. SI and colonic tissues were incubated in 2.5 mM and 3 mM EDTA, respectively, for 1 h. Tissues were then mechanically disrupted in 10% FBS in PBS and passed through a 100 μm strainer. Samples were then centrifuged at 300 × g for 5 min. The pellet was then resuspended and incubated in TrypLE Express (Gibco) for 30 min until dissociation into single cells. The cells were then filtered through a 40 μm strainer, centrifuged at 300 × g for 5 min, and resuspended in 2% FBS in PBS for sorting. TdTomato-expressing cells were sorted on a FACS SH800 (SONY) into 2% FBS in PBS. Viable single cells were gated based on the FSC-W versus FSC-A profile.

DNA isolation and genotyping

DNA isolation was performed using the Invitrogen DNA isolation kit. Briefly, tissues were digested in the Digestion Buffer provided by the kit, followed by the addition of the Lysis/Binding buffer. Lysate was added to a PureLink Spin Column, washed, and eluted. For the Infinium Mouse Methylation BeadChip Array, the DNA was extracted from isolated colonic epithelial cells. For confirmation of Dnmt1 recombination, DNA was isolated from tumors from Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato and Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice. Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and primers listed in Table S3 were used for confirmation of Dnmt1 recombination, whereas 2XTaq Frogga mix (FroggaBio) was used for all other genotyping.

Western blot

Whole colonic tissue from mice treated with two doses of 5 mg/kg 5-AZA or vehicle was homogenized in 200 μL RIPA buffer (containing 0.2 mM PMSF, 0.5 mM NaF, 0.1 mM Na3VO4, 1x protease/phosphatase inhibitor cocktail). Protein was quantified using a Bradford assay (BioRad). 20 μg protein lysates were loaded on an 8% SDS-PAGE gel and transferred to nitrocellulose membrane (BioRad) using a wet electroblotting system (BioRad). The membrane was incubated using the manufacturer protocols for p-STAT1 Ab (#9167, Cell Signaling Technologies, 1:1000), STAT1 Ab (#9172, Cell Signaling Technologies, 1:1000), β-Actin Ab (#4970, Cell Signaling Technologies, 1:1000), and Anti-rabbit IgG-HRP-linked Ab (#7074, Cell Signaling Technologies, 1:2000). The membrane was rinsed briefly in enhanced chemiluminescence substrate (BioRad) and imaged using the BioRad ChemiDoc MP Image System. Images were analyzed with ImageJ (version 1.53).

Myeloperoxidase (MPO) measurement

MPO measurements were performed on distal colonic tissues as previously described⁹⁶. Briefly, colonic tissues were homogenized in hexadecyltrimethylammonium bromide (HTAB) buffer according to tissue weight. Homogenate was added in triplicate to a 96-well plate. O-dianisidine solution was prepared by combining 16.7 mg o-dianisidine dihydrochloride, 90 mL dH₂O, 10 mL 50 mM potassium phosphate buffer, and 50 μL 1.2% H₂O₂. Next, 200 μL O-dianisidine solution was added to each well containing tissue homogenate. Absorbance was measured at 450 nm, and MPO calculated as units of MPO/mg tissue.

Global DNA methylation

Global DNA methylation was measured on DNA isolated from colonic epithelial cells or tumors from either Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato and Dclk1^CreERT2;Apc^f/f;Rosa26^TdTomato;Dnmt1^f/f mice using the MethylFlash Global DNA methylation (5-mC) ELISA Easy Kit (EpigenTek) according to the manufacturer’s instructions. Briefly, the binding solution and DNA were added to a 96-well plate. Wells were then washed, and 5-mC Detection Complex solution added to each well followed by Developer Solution. Stop Solution was then added, and the plate read at 450 nm. Percentage methylated DNA was calculated based on a standard curve.

Infinium mouse methylation beadchip array

DNA was isolated from colonic epithelial cells isolated from mice treated with vehicle (controls), DSS, 5-AZA, or 5-AZA + DSS (n = 3 in each group) and run on the Infinium Mouse Methylation BeadChip Array (GSE283386). Preprocessing of IDAT files was done using the R package SeSaMe (version 1.20)⁹⁷ and included calculation of detection p values (probes with a detection p value of <0.05 were retained), background subtraction, strain interference, and dye bias correction. Differentially methylated CpGs were identified using the DML function, which models β values using a linear model. Sample group was used as the predictor. Sex-specific CpGs in colonic tissues were removed from the analysis to account for unequal distribution of sexes between the four groups analyzed (Ctrl, DSS, 5-AZA, and 5-AZA + DSS). This was done by identifying differentially methylated CpGs in colonic tissues between male (n = 2) and female (n = 3) mice using samples available in the dataset GSE184410⁹⁸. Only colonic samples in this dataset were used for analysis. IDAT files were preprocessed as described above, and the DML function was used to identify differentially methylated CpGs in GSE184410 samples with sex as the predictor. CpGs were considered significant for a sex effect if the false discovery rate (FDR) was less than 0.05 and the effect size was greater than 0.1. 1223 CpGs were identified as being sex-specific and removed from the analysis. Following these quality control steps, differentially methylated CpGs were defined as those with FDR less than 0.01 and an effect size greater than 0.1, unless otherwise stated. Heatmap were generated using the pheatmap package (version 1.0.12) and volcano plots using ggplot2 (version 3.5.1).

Human RNA-sequencing data retrieval and processing

Two publicly available datasets containing RNA-sequencing was analyzed for this study. Shaw et al.³⁹ contained RNA-sequencing of colonic biopsies from 41 healthy controls, 22 patients with IBD without dysplasia, and 7 patients with IBD-associated dysplasia (biopsy of non-dysplastic region adjacent to dysplasia). Peters et al.³⁵ contained RNA-sequencing of surgical specimens from 61 control patients that had colon resection due to sporadic CRC and inflamed tissues from 77 patients with active IBD. Using the SRA toolkit (version 3.1.0), fastq files obtained and processed by Shaw et al.³⁹ and Peters et al.³⁵ were retrieved. FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to identify sequencing artifacts that were then removed using bbduk (http://jgi.doe.gov/data-and-tools/bb-tools/). The resulting reads were aligned to the hg38 human genome (GRCh38.p14, GENCODE) with HISAT2⁹⁹ (version 2.2.1), excluding multi-mapping reads and incorporating splice junctions as previously described³⁶. These splice junctions were specified through a custom GTF file merging GRCh38.p14 transcripts (GENCODE) and RepeatMasker repeat element loci from the UCSC genome browser. Samtools¹⁰⁰ (version 1.18) indexed the resulting alignments, which were later passed to htseq-count¹⁰¹ to quantify custom GTF gene expression (version 0.11.1). Final datasets from Shaw et al. and Peters et al. were compiled into separate matrices for further analysis in R (version 4.3.2).

Downstream gene expression analysis

DESeq2¹⁰² (version 1.42.0) was chosen for differential gene expression analysis across the specified groups, for which the default parameters were used. When comparing IBD patients to those with IBD and dysplasia in data from Shaw et al., genes identified as upregulated and significant (Benjamini-Hochberg adjusted p value < 0.05) were passed into enrichGO (clusterProfiler¹⁰³, version 4.10.0) for gene ontology (GO) enrichment analysis. The ten most significantly enriched biological processes (BP) GO terms were selected from these results and visualized with ggplot2 (version 3.4.4).

To identify patterns in interferon genes, the count matrix was adjusted for sequencing depth (using the DESeq2 counts function), normalized relative to the average expression in the noninflamed control samples, and then log2 transformed. The resulting data was visualized using ComplexHeatmap¹⁰⁴ (version 2.18.0). Additionally, the differential expression of repetitive elements, identified through RepeatMasker, was visualized using an EnhancedVolcano (https://github.com/kevinblighe/EnhancedVolcano, version 1.20.0) plot, emphasizing the most significantly altered elements.

Similarly, DESeq2 was used to compare control and IBD samples from the data in Peters et al. Repetitive elements with significant differences in expression were visualized using ComplexHeatmap (version 2.18.0). A two-sided binomial test, performed under the null hypothesis (alpha = 0.05), demonstrated that the number of repetitive elements upregulated in IBD samples (174) compared to control samples (36) was statistically significant (p = 3.21 × 10⁻²¹).

Statistical analysis

Statistical analyses were performed using two-tailed Student’s t-tests when comparing two groups or standard One-way ANOVA analysis with Tukey’s multiple comparison test when comparing 3 or more groups. For survival analysis, a Log-rank test was performed. Error bars denote the mean ± SEM. Unless otherwise specified, a P value of less than 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Infinium Mouse Methylation BeadChip Array data have been deposited in GEO with the accession number GSE283386 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE283386]. Publicly available RNA-sequencing datasets can be found in GEO with accession numbers GSE83687 and GSE230524. Source data are provided with this paper. The remaining data that support the findings of this study are available from the corresponding author (S.A.) upon request.

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Acknowledgements

This work was supported by operating grants from the Canadian Institutes of Health Research (CIHR) and Cancer Research Society awarded to S.A, the Canadian Association of Gastroenterology PhD Studentship awarded to F.L., and the National Institutes of Health U01-DK103155 awarded to Timothy C. Wang. The Mavs-tm1Zjc mice were a kind gift from Dr. Daniel De Carvalho from University of Toronto. Graphical depictions were created using Biorender.com.

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Authors and Affiliations

Department of Medicine, University of Western Ontario, London, Ontario, Canada
Frederikke Larsen, Will Jeong, Daniel Schep, Hayley J. Good, Alice E. Shin, Liyue Zhang, Mathieu F. Derouet & Samuel Asfaha
Department of Pathology and Laboratory Medicine, University of Western Ontario, London, Ontario, Canada
Frederikke Larsen, Hayley J. Good, Alice E. Shin, Christina A. Castellani & Parisa Shooshtari
Verspeeten Family Cancer Centre, London Health Sciences Centre, London, Ontario, Canada
Frederikke Larsen, Will Jeong, Daniel Schep, Hayley J. Good, Alice E. Shin, Liyue Zhang, Mathieu F. Derouet & Samuel Asfaha
Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
Liyue Zhang, Mathieu F. Derouet & Samuel Asfaha
Division of Digestive & Liver Diseases, Irving Medical Center, Columbia University, New York, USA
Timothy C. Wang
Department of Epidemiology and Biostatistics, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada
Christina A. Castellani
Children’s Health Research Institute, Lawson Research Institute, London, Ontario, Canada
Christina A. Castellani & Parisa Shooshtari
McKusick-Nathans Institute, Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Christina A. Castellani
Department of Computer Science, Western University, London, Ontario, Canada
Parisa Shooshtari
Ontario Institute of Cancer Research (OICR), Toronto, Ontario, Canada
Parisa Shooshtari
Department of Oncology, University of Western Ontario, London, Ontario, Canada
Samuel Asfaha

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Frederikke Larsen
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Will Jeong
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Daniel Schep
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Hayley J. Good
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Alice E. Shin
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Liyue Zhang
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Mathieu F. Derouet
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Timothy C. Wang
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Christina A. Castellani
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Parisa Shooshtari
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Samuel Asfaha
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Contributions

F.L. designed, performed, and analyzed experimental studies and wrote the manuscript. W.J. analyzed RNA-sequencing data. D.S. performed FACS analysis. H.J.G, A.E.S., L.Z, and M.F.D. contributed to data analysis. T.C.W. edited the manuscript. C.A.C supported the mouse methylation analysis and edited the manuscript. P.S. supported the RNA-sequencing analysis and edited the manuscript. S.A supervised the work and edited the manuscript.

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Correspondence to Samuel Asfaha.

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Larsen, F., Jeong, W., Schep, D. et al. Viral mimicry acts as a tumor suppressor in colitis. Nat Commun 17, 1313 (2026). https://doi.org/10.1038/s41467-026-68850-1

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Received: 22 January 2025
Accepted: 18 January 2026
Published: 04 February 2026
Version of record: 04 February 2026
DOI: https://doi.org/10.1038/s41467-026-68850-1