Malic Enzyme 1 (ME1) is pro-oncogenic in ApcMin/+ mice

Cytosolic Malic Enzyme (ME1) provides reduced NADP for anabolism and maintenance of redox status. To examine the role of ME1 in tumor genesis of the gastrointestinal tract, we crossed mice having augmented intestinal epithelial expression of ME1 (ME1-Tg mice) with ApcMin/+ mice to obtain male ApcMin/+/ME1-Tg mice. ME1 protein levels were significantly greater within gut epithelium and adenomas of male ApcMin/+/ME1-Tg than ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice had larger and greater numbers of adenomas in the small intestine (jejunum and ileum) than male ApcMin/+ mice. Male ApcMin/+/ME1-Tg mice exhibited greater small intestine crypt depth and villus length in non-adenoma regions, correspondent with increased KLF9 protein abundance in crypts and lamina propria. Small intestines of male ApcMin/+/ME1-Tg mice also had enhanced levels of Sp5 mRNA, suggesting Wnt/β-catenin pathway activation. A small molecule inhibitor of ME1 suppressed growth of human CRC cells in vitro, but had little effect on normal rat intestinal epithelial cells. Targeting of ME1 may add to the armentarium of therapies for cancers of the gastrointestinal tract.

and adenomas in ilea of male mice. Quantification was via Aperio Imagescope. Boxes demarcate the inter-the KRAS gene had enhanced expression of ME1 16 . Mutations of both TP53 and KRAS are common in CRC 17 . Indeed, siRNA-mediated knockdown of ME1 leads to growth inhibition and senescence of CRC cell lines in vitro 9,15 . Moreover, ME1 abundance in some cancer cells was reported to be a prognostic marker for efficacy of radiation therapy 18 . Nevertheless, most of what is known about the actions of ME1 in cancer cells is derived from in vitro studies and xenograft transplants to mice.
To mechanistically define the contributions of ME1 to intestinal cancer genesis within a more physiological context, we generated an ME1 transgenic mouse (ME1-Tg) which over-expresses ME1 predominantly in intestinal epithelial cells under the control of the murine villin gene promoter-enhancer 19 . We reported that ME1-Tg mice had greater intestinal 5-bromodeoxyuridine labeling index and exhibited deeper intestinal and colonic crypts 19 . In contrast, a functionally null ME1 mouse (MOD-1 mouse line) displayed shallower colonic crypts and reduced intestinal expression of pro-proliferative Ccnd1 and Mtor genes compared to WT mice 20 . Intestinal expression of genes encoding proteins responsible for lipid and cholesterol biosynthesis were elevated in the ME1-Tg mice 19 , indicating a shift towards increased lipogenesis. While these studies suggested a stimulatory role for ME1 in proliferation of gut epithelium, ME1-Tg mice did not spontaneously develop intestinal adenomas at increased frequencies.
The Apc Min/+ mouse is a well-utilized model of Familial Adenomatous Polyposis (FAP), an inherited form of colorectal/intestinal cancer 21 . Here, we have used this mouse model to test the hypothesis that ME1 overexpression would lead to increased tumor burden. We characterized the male progeny of the novel intercross of heterozygous male Apc Min/+ mice with female ME1-Tg mice, namely, Apc Min/+ mice with intestine-specific augmentation of ME1 (designated Apc Min/+ /ME1-Tg) for tumor parameters and for expression of candidate tumor-associated genes. Further, we utilized small molecule inhibitors of ME1 and the canonical Wnt signaling pathway, respectively to elucidate single and combinatorial effects on two human CRC cell lines. Results document a stimulatory role for ME1 in intestinal tumor genesis.

Effects of enhanced intestinal epithelial ME1 expression.
To generate Apc Min/+ mice with enhanced intestinal expression of ME1, heterozygous male Apc Min/+ mice were intercrossed with female ME1-Tg mice. Sixteen-week-old male mouse progeny were used to quantify Me1 RNA abundance and adenoma burden in the small and large intestines. We observed a significant (~2.0-fold; P = 0.010) increase in expression of the endogenous (mouse) Me1 gene in the jejunums of Apc Min/+ /ME1-Tg mice when compared to WT mice (Fig. 1A). Similarly, total Me1 mRNA levels (i.e., endogenous plus transgenic Me1 RNAs) in mouse jejunum were significantly greater for ME1-Tg (P < 0.001) and Apc Min/+ /ME1-Tg (P < 0.001) mice when compared to WT and Apc Min/+ mice, respectively (Fig. 1B). As expected, no transgene-derived RNA was observed in the non-transgenic Apc Min/+ mouse intestine (Fig. 1C). We then evaluated relative abundance of ME1 protein in ileum by immunohistochemistry (IHC). An increase in ME1 protein (IHC staining) was observed within normal-appearing villi of the transitional mucosa (P = 0.002) as well as in adenomas (P = 0.026) of Apc Min/+ /ME1-Tg when compared to Apc Min/+ mice; by contrast, the crypts of both mouse lines did not differ ( Fig. 1D-J). The intestine smooth muscle layers (outer longitudinal and inner circular) stained intensely for ME1 ( Fig. 1D-G); although, as expected, this staining was unaffected by Me1 transgene. Apc Min/+ /ME1-Tg mice exhibited greater amounts of ME1 protein in adenomas when compared to those of Apc Min/+ . Interestingly, the borders of adenomas exhibited significantly greater ME1 staining than the corresponding inner regions irrespective of genotype ( Supplementary Fig. 1). However, the adenoma borders of Apc Min/+ /ME1-Tg mice displayed significantly greater (P = 0.042) ME1 staining than those of Apc Min/+ mice ( Supplementary Fig. 1).
Goblet cells are the most abundant secretory cell type in the villus epithelium, and their numbers serve as a readout of lineage determination in intestines. We performed Alcian blue histochemistry to evaluate goblet cell numbers as a function of ME1 status. There was no difference in number of goblet cells in villi of Apc Min/+ / ME1-Tg and Apc Min/+ mice ( Supplementary Fig. 2). Villi immediately adjacent to adenomas had significantly more goblet cells (P < 0.01) than those that were more distant from adenomas ( Supplementary Fig. 2). However, the number of goblet cells in adenoma-associated villi of Apc Min/+ /ME1-Tg vs. Apc Min/+ mice did not differ ( Supplementary Fig. 2).

Increased intestinal adenoma burden in male Apc
Min/+ /ME1-Tg mice. We next evaluated the effect of the Me1 transgene on intestinal adenoma burden. Male Apc Min/+ /ME1-Tg mice exhibited a significant increase (~1.5-fold; P = 0.009) in numbers of small intestine adenomas when compared to Apc Min/+ mice ( Fig. 2A); this increase occurred in the Ileum (P = 0.032) and jejunum (P = 0.020) (Fig. 2B). The number of colon adenomas did not differ (P = 0.076) between Apc Min/+ /ME1-Tg and Apc Min/+ mice (Fig. 2B). Apc Min/+ /ME1-Tg mice displayed significantly greater numbers of adenomas that were less than 1 mm in diameter in the duodenum (P = 0.011), jejunum (P = 0.014), and ileum (P = 0.040) when compared to male Apc Min/+ mice ( Fig. 2C-E). Interestingly, the number of adenomas between 3 mm and 4 mm in diameter was significantly greater (P = 0.034) in the colons of male Apc Min/+ /ME1-Tg mice compared to Apc Min/+ mice (Fig. 2F). quartile range of 25-75% with mean (thick line) and median (thin line); (n = 6/group). (J) Student's t-tests were used to examine for differences in IHC staining intensity of ME1 protein between groups, and the Mann-Whitney Rank Sum Test was used for comparing non-normally distributed data. Significant differences were identified by P < 0.05. A tendency for a difference also is indicated (0.1 > P > 0.05). Boxes indicate the inter-quartile range (25-75%) with mean (thick line) and median (thin line); whiskers: 10 th and 90 th percentiles; dots: outliers. Student's t-tests were used to examine for differences between groups and the Mann-Whitney Rank Sum Test was used for comparing non-normally distributed data. Significant differences were identified by P < 0.05. Tendencies for differences also are indicated (0.1 > P > 0.05).
SCIenTIfIC RepoRtS | (2018) 8:14268 | DOI:10.1038/s41598-018-32532-w β-catenin IHC of adenomas. An increase in nuclear β-catenin content is a hallmark of intestinal tumorigenesis. We therefore evaluated, by IHC, the number of nuclear β-catenin-positive cells in ilea from male Apc Min/+ /ME1-Tg and Apc Min/+ mice. No significant differences in numbers of nuclear β-catenin-positive cells were observed for crypts, villi or adenomas as a function of genotype ( Supplementary Fig. 3). However, all (H) Quantification of ileal villus length in male mice (n = 5/group). (I) Ratio of villus length to crypt depth in the ilea of male mice (n = 5/group). Boxes indicate the inter-quartile range of 25-75% with mean (thick line) and median (thin line); whiskers extend to the 10 th and 90 th percentiles. Student's t-tests were used to examine for differences between genotypes (significant difference, P < 0.05). adenomas stained very strongly for β-catenin (nuclear and cytoplasmic/membranes) (Fig. 3A,B), which facilitated the measurements of adenoma areas (in cross-section) by use of Aperio software. Both adenoma number and area were significantly greater (P = 0.032 and P = 0.004, respectively) for ilea of male Apc Min/+ /ME1-Tg mice compared to male Apc Min/+ mice (Fig. 3C,D). In addition, ileal crypts and villi of male Apc Min/+ /ME1-Tg mice were significantly deeper (P = 0.039) and longer (P = 0.003), respectively when compared to those of male Apc Min/+ mice; however, villus to crypt ratio was comparable between genotypes ( Fig. 3E-I). We examined whether the increases in crypt depth and villus length were due to increased numbers of cells within the corresponding epithelium. The number of cells lining the crypts did not significantly differ as a function of genotype; however, we observed a significant increase (~1.2-fold; P = 0.044) in the number of cells comprising the villus epithelium in the Apc Min/+ /ME1-Tg relative to Apc Min/+ mice ( Supplementary Fig. 3).

Gene expression, proliferation and apoptosis in male Apc
Min/+ /ME1-Tg mouse intestines. We performed targeted gene expression analysis to identify a pathway-oriented basis for the increased intestinal adenoma burden of Apc Min/+ /ME1-Tg mice. Among the oncogenes and tumor suppressor genes that were examined, only Sp5 was significantly (P = 0.013) altered (i.e., upregulated by more than two-fold) in the Apc Min/+ / ME1-Tg jejunum (Fig. 4A). Sp5 is a known induced target gene of the Wnt pathway [22][23][24] . Expression analysis of apoptosis-associated genes showed a two-fold up-regulation (P = 0.05) of anti-apoptotic Bcl2 in Apc Min/+ / ME1-Tg mice (Fig. 4B). No differences in expression of several epithelial to mesenchymal (EMT)-associated genes were noted between the two groups ( Supplementary Fig. 4). The jejunal expression of other Sp/Klf family member genes that were previously implicated in intestinal growth and homeostasis, did not differ between the mouse lines ( Supplementary Fig. 4). Moreover, the numbers of BrdU-positive and nuclear Ki67-positive cells were comparable between genotypes for the crypt and villus epithelium and adenomas (

Increased KLF9 protein abundance in crypts and villus lamina propria of male Apc Min/+ /ME1-Tg mice.
We previously reported that null mutation of the transcription factor Krϋppel-like factor 9 (Klf9) negatively affected small intestine crypt stem-progenitor cell proliferation and villus cell migration in mice 25 . As a consequence, the villi of Klf9 knockout mice were shorter than their wild-type counterparts 25 . In view of the positive effect of ME1 transgene on villus length, we evaluated, by IHC, the presence of nuclear-localized KLF9 in the ilea of Apc Min/+ /ME1-Tg and Apc Min/+ mice. Nuclear KLF9 protein levels were significantly greater in the crypts (P = 0.0008) and the villus lamina propria (P = 0.000006) of Apc Min/+ /ME1-Tg compared to Apc Min/+ mice ( Fig. 5A-E). Nuclear KLF9 immunoreactivity was minimal in the villus epithelium, but was robust in the muscularis externa of both genotypes (Fig. 5A,B). Moreover, KLF9 immunostaining in adenomas was negligible in both mouse lines (Fig. 5C-E).

Inhibition of ME1 activity suppresses growth of human CRC cells in vitro.
We next evaluated the involvement of ME1 in the Wnt/β-catenin pathway using intestinal cell lines. In initial studies, we inhibited ME1 enzyme activity in the non-cancerous IEC6 intestinal epithelial cell line with a small molecule inhibitor of ME1 26 (designated ME1*). A small but significant decrease in colony formation was noted for these cells but only at the highest dose evaluated ( Supplementary Fig. 5). Since our earlier results for Sp5 (Fig. 4A) indicated a possible functional connection between ME1 and the canonical Wnt/β-catenin signaling pathway, we next treated HCT116 and HT29 CRC cells, singly and in combination, with the small molecule ME1 inhibitor and an inhibitor of the canonical Wnt pathway (JW74) 27 . Non-confluent cells were treated with 50 uM ME1*, 15 uM JW74, 50 uM ME1* plus 15 uM JW74, or vehicle (DMSO; control) for 72 h. Results showed a significant reduction in total cell numbers with ME1* or ME1* + JW74 for both HCT116 and HT29 cell lines (Fig. 6A,B). In HT29 cells, the combination of ME1* and JW74 had an additive inhibitory effect on cell counts compared to vehicle (P < 0.001) (Fig. 6B). JW74 alone did not alter cell numbers (Fig. 6A,B). Diameters of HCT116 and HT29 cells treated with ME1* or ME1* + JW74 were reduced when compared to vehicle; JW74 alone did not affect HCT116 cell diameter but showed a tendency for this in HT29 cells (P = 0.067) ( Supplementary Fig. 5). We also examined HCT116 and HT29 cell viability/metabolism by use of the MTS reagent. A significant reduction in cell viability/metabolic activity was observed with ME1* treatment, which was further diminished with co-addition of JW74 (Fig. 6C,D). Unexpectedly, JW74 alone had a small but significant (P < 0.001) stimulatory effect on HCT116 cells.
We performed colony formation assays to confirm the above effects. ME1* and ME1* + JW74 dramatically reduced (P < 0.001) the number of colonies formed by both cell lines, while JW74 alone had no effect (Fig. 6E-H). ME1* dose-dependently reduced the number of colonies formed by both HCT116 (overall P < 0.001) and HT29 (overall P < 0.001) cells ( Supplementary Fig. 5), albeit HCT116 cells were more sensitive than HT29 cells to ME1*. Since the number of colonies formed after treatment with ME1* or ME1* + JW74 were so few, we inferred that the ME1 inhibitor was causing cell death. To investigate this further, HCT116 cells were seeded at high density and treated with ME1*, JW74, ME1* + JW74, or vehicle (DMSO). ME1* caused significant loss of cells, while the combination of ME1* and JW74 showed an additive effect on cell numbers (Fig. 6I,K). IEC6 cells when treated similarly showed no effect with ME1* while JW74 alone and combination of JW74 and ME1* had comparable inhibitory effects (Fig. 6J,L). the inter-quartile range of 25-75% with mean (thick line) and median (thin line); whiskers extend to 10 th and 90 th percentiles. Student's t-tests were used to examine for differences between groups and the Mann-Whitney Rank Sum Test was used for non-normally distributed data. Significant differences were identified by P < 0.05. Tendencies for differences also are indicated (0.1 > P > 0.05).
SCIenTIfIC RepoRtS | (2018) 8:14268 | DOI:10.1038/s41598-018-32532-w ME1 expression in human colon adenocarcinomas. ME1 was immunolocalized to specific cells of human normal colon and colon tumor tissue using a commercial tissue array. Robust expression of ME1 was observed in the more differentiated epithelial regions of both human colorectal adenocarcinomas and normal colon ( Supplementary Fig. 6).

Discussion
This study reports a novel in vivo association between gastrointestinal ME1 expression and small intestine adenoma burden. We observed increases in: a) adenoma number and size distribution, b) ME1 abundance within adenomas, and c) crypt depth and villus height, in the non-adenoma (transitional) mucosa with Me1 overexpression in the background of Apc haplo-insufficiency. We also found a significant increase in Sp5 transcript levels and enhanced numbers of nuclear KLF9 positive-cells in the crypt epithelium and lamina propria of Apc Min/+ / ME1-Tg mice. These findings link an important cytosol-residing metabolic enzyme namely ME1 with alterations in expression (and by inference, downstream pathway effects) of two members of the SP/KLF family of transciptional regulators, which are themselves increasingly considered as context-dependent participants in oncogenesis and canonical Wnt pathway signaling. Moreover, our in vitro experiments utilizing two human CRC cell lines confirmed that ME1 is important for cancer cell growth (hyperplasia and hypertrophy). Importantly, we found that growth of cancer cells was highly sensitive to inhibition of ME1 enzyme activity; by contrast, growth of non-tumorigenic intestinal epithelial cells (IEC6) was relatively resistant to ME1 inhibition albeit sensitive to Wnt pathway inhibition. Finally, we showed that ME1 is abundantly expressed within epithelial-like regions of human colon adenocarcinoma akin to what was observed in the adenoma borders in Apc Min/+ /ME1-Tg mice. Collective  results implicate ME1 as a functional contributor to intestinal cancer development and suggest that targeting ME1 should be explored as a potential therapeutic strategy to improve patient outcome.
Analogous to our previous findings for ME1-Tg mice 19 , the Apc Min/+ /ME1-Tg mice exhibited greater ME1 RNA and protein levels in the small intestine, when compared to their littermate controls. The greater numbers of small adenomas and the larger adenoma sizes, respectively within the small intestine of Apc Min/+ /ME1-Tg mice, coincident with increased ME1 expression, are suggestive of ME1 promotion of the initial step(s) of adenoma formation and of ME1 participation in tumor progression. Moreover, the significant elevation in ME1 abundance within the adenoma borders is consistent with previously reported increases in lipid content in the epithelial-like adenoma borders of Apc Min/+ mice 28 .
The lack of differences in the expression of proliferation-associated genes, which were corroborated by results of BrdU and Ki67 staining, between the two mouse lines with distinct intestinal ME1 expression, suggest that the increased adenoma burden in vivo may not result from increased cell proliferation but rather to decreased apoptotic status. Consistent with this, we found an increase in anti-apoptotic Bcl2 gene expression in the jejunum and a decrease in TUNEL staining in the villi of Apc Min/+ /ME1-Tg mice. The lack of observed changes in the expression of several canonical EMT-associated genes may be related to EMT occuring at later stages of tumorigenesis than studied here or to the lack of functional relationship of ME1 and EMT. Further studies conducted at later stages of tumorigenesis should address this question.
Both crypt depth and villus length were enhanced in the transitional mucosa of Apc Min/+ /ME1-Tg mice. We found no increase in the number of cells resident in the crypt epithelium of Apc Min/+ /ME1-Tg mice, although the average number of cells per crypt was numerically greater than for Apc Min/+ mice; thus, a combination of increased cell number and cell size likely contributed to the increased crypt depth. Consistent with this, ME1 inhibition conferred smaller cell diameters in vitro. Moreover, in a previous study, we reported that mice functionally null for Me1 had shallower colon crypts when fed a high fat diet 20 . By contrast, ME1-Tg mice (on the WT Apc background) fed a high-fat diet exhibited increased jejunum crypt depth and more crypt stem-progenitor proliferation then wild-type littermate controls 19 . The ME1-Tg mice on the WT Apc background and fed a high fat diet, also had altered liver metabolism, reflecting gut-liver communication 19 ; similar effects may have contributed to the current findings. We conclude that ME1 may play a role, either directly or indirectly, in the maintenance of intestinal crypt stem-progenitor cell number and/or size.
We noted a virtual absence of goblet cells within the central regions of adenomas, whereas their borders contained a high frequency of these cells. While we found no quantitative effects of the Me1 transgene on this pattern, there was a significant increase in the number of goblet cells in the adenoma-associated villi when compared to normal villi. Perhaps the greater number of goblet cells within the transitional mucosa reflects a tumor-protective function through promotion of mucus production and of paracrine signaling elicited by tumor-promoting mucins 29 .
Our studies implicate two members of the Sp/Klf-family of transcription factors as potential mediators of ME1-induced GI tumorigenesis. SP5, a downstream target of the Wnt/β-catenin signaling pathway in colon and several other tissues, is reported to be up-regulated in CRC and to promote tumor cell growth [22][23][24] . Given that Sp5 transcript levels were elevated in the jejunums of Apc Min/+ /ME1-Tg mice yet we observed no differences in nuclear β-catenin localization in the two mouse lines, we infer that the Sp5 induction in response to ME1 occured via an alternate pathway or via interconnectivity with the Wnt/β-catenin signaling pathway but downstream of nuclear β-catenin. Alternatively or in addition, the increase in Sp5 mRNA abundance may reflect increased numbers of adenoma cells expressing Sp5 mRNA at high levels. However, since the effect of ME1 transgene on Sp5 is relatively specific and is not accompanied by differential expression of genes (e.g., c-Myc, cyclin D1) known to be overexpressed in intestinal adenomas, the latter scenario is not likely. Future studies should address this non-canonical link between SP5 and Wnt-β-catenin signaling.
KLF9 is another potential player in ME1-enhanced tumorigenesis. In a previous study, we reported that mice null for Klf9 had shorter intestinal villi than wild-type mice, due in part to reduced crypt cell proliferation and slower epithelial cell migration to the villus tip 25 . Thus, the increased villus length observed in the present study may be a result, in part, of the enhanced number of KLF9-positive cells in the crypts of Apc Min/+ /ME1-Tg mice. Interestingly, numbers of nuclear KLF9-positive cells were also elevated within the villus lamina propria of Apc Min/+ /ME1-Tg mice. At present, the identities of KLF9-positive cells in the villus lamina propria remain unknown, although this tissue compartment harbors lymphoid cells, macrophages and myofibroblasts, among other cell types. KLF9 is reported to be oncogenic in some contexts and to be tumor suppressive in others [30][31][32][33] . In a previous study, we found that Klf9 KO caused a significant reduction in adenoma number in the colon but had no effect on adenoma number in small intestines of Apc Min/+ mice 33 . Taken together with the findings reported here, our results suggest the tissue and context-dependent functions of KLF9. We speculate that the enhanced frequency of KLF9-positive cells in the small intestine crypts is growth-promoting for normal appearing and transitional villi, whereas KLF9 exerts tumor-suppressive actions in adenomas of the colon but not small intestine.
Remarkably, normal rat intestinal cells (IEC6) were more resistant than cancer cells to the ME1 inhibitor but were highly sensitive to Wnt pathway inhibition; the latter is in keeping with the well-known stimulatory role of with 50 uM ME1*, 15 uM JW74, 50 uM ME1* plus 15 uM JW74, or vehicle (DMSO). After 3 days, cells were stained with crystal violet. (K,L) Quantification of remaining cells from (I,J) expressed as % area of stained cells per well. Boxes show the inter-quartile range of 25-75% with mean (thick line) and median (thin line); whiskers: 10 th and 90 th percentiles. One way ANOVA was used to examine for differences between treatment groups. Different lowercase letters (a-d) designate groups that differ (P < 0.05); bars sharing the same letter are not significantly different.
SCIenTIfIC RepoRtS | (2018) 8:14268 | DOI:10.1038/s41598-018-32532-w the Wnt/β-catenin signaling pathway in intestinal stem-progenitor cell proliferation. The suppressive effect of ME1 inhibitor on cancer cell size in vitro is consistent with our previous work with the global Me1 hypomorphic null mouse in which we observed significant reductions in colon Mtor expression 20 . The mTOR pathway is dominant in determining cell size for many tissues/cells 34 ; hence, reductions in its expression may partly explain our in vitro data.
In conclusion, our results significantly extend previous findings from other laboratories implicating a role for ME1 in gastrointestinal cancers. The body of work implicating fatty acid synthesis in CRC initiation, progression and metastasis [12][13][14] , coupled with the documented role of ME1 in promoting lipogenesis in gut epithelium via the NADPH supply 9,19 , further identifies this pathway as a vulnerability to exploit for CRC treatment and therapy.

Methods
Animals. All procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Arkansas for Medical Sciences in accordance with federal guidelines and regulations. Mice were housed under a 12 h light/12 h dark cycle and were fed a regular chow diet. Apc Min/+ mouse breeders (Strain Name: C57BL/6J-Apc Min /J; Apc Δ850 , stock number: 002020) were from the Jackson Laboratory (Bar Harbor, ME, USA). Mice with augmented intestinal epithelial expression of ME1 (rat ME1 cDNA under the control of the murine villin promoter-enhancer; ME1-Tg; C57BL/6 J background) were described previously 19 . To generate Apc Min/+ mice with gut-specific enhanced expression of ME1, heterozygous male Apc Min/+ mice were intercrossed with female ME1-Tg mice. Age-matched male mice were used to quantify tumor burden in the small and large intestines. At 16 weeks of age, mice (n = 17 Apc Min/+ male mice and n = 10 Apc Min/+ /ME1-Tg male mice) were euthanized for tissue collection. Their small intestines and colons were removed, flushed with phosphate-buffered saline (PBS) and opened longitudinally. The small intestine was divided into three equal parts, which were operationally designated as duodenum, jejunum, and ileum. The junction (~1 cm in length) between the jejunum and ileum was snap-frozen in liquid nitrogen and stored at −80 °C for later analysis. Jejunum-ileum junctions (referred to as jejunum in Results) were also obtained from age-matched non-transgenic wild-type and ME1-Tg male mice (both strains wild-type for Apc). After microscopic examination of tissues (below), each Ileum was coiled into a Swiss-roll and fixed in methanol Carnoy solution (60% methanol, 30% chloroform, 10% glacial acetic acid) for 24 h and then transferred to 70% ethanol, followed by embedding in paraffin. We chose ilea for embedding and subsequent IHC, since this region typically displays the most adenomas in Apc Min/+ mice 33 . To evaluate gastrointestinal proliferation, Apc Min/+ /ME1-Tg and Apc Min/+ mice were injected intraperitoneal with BrdU at a dose of 100 mg/kg of body weight (Sigma Aldrich, St. Louis, MO, USA) 2 h before euthanizing, as described previously 20 .
Microscopic examination of adenomas. Intestine segments and colons were examined for number and size of adenomas, in blinded fashion, with a dissecting microscope (Discovery 8, Carl Zeiss MicroImaging GmbH, Jena, Germany). All adenomas were counted, measured and categorized by size

Mouse gene
Forward primer (5′-3′) Reverse primer (3′-5′) Histology and immunohistochemistry. Five-micron sections of ilea (Swiss rolls) were used for immunohistochemistry (IHC). Paraffin-embedded sections were dewaxed and rehydrated through a graded alcohol series as described previously 19,20 . Antigen unmasking was conducted by boiling the sections in Coplin jars in a microwave using Citra Plus (Biogenex, San Ramon, CA, USA) for a duration of 2 min high power and then for 10 min at low power setting. After cooling for 30 min at room temperature, sections were treated with 3% Slides were imaged with a Aperio CS2 image capture device (Leica Biosystems Nussloch GmbH, Germany), or Nikon Eclipse E400 Microscope (Nikon Instruments, Melville, NY) fitted with an Olympus Dp73 digital camera. Quantification of positive antibody staining was performed using Aperio ImageScope algorithms or manually by counting the number of individual nuclear-stained cells. All positive staining was represented as a percentage of total staining (positive + negative) within a given field; with the exception of Ki67 and KLF9 for which the Aperio nuclear stain algorithm was used. Five representative areas per slide/mouse, each with 3 to 8 representative crypts or villi, were used for quantification. Apoptosis assay. Sections were stained using the ApopTag Peroxidase In Situ Apoptosis Detection kit following the manufacturer's instructions (Millipore, Burlington, MA, USA). Staining was evaluated using the Aperio ImageScope.

Measurement of crypts and villi.
Ileal sections were scanned using the ScanScope CS2 slide scanner and Aperio ImageScope software. Crypt depths were measured from the base of the crypts to the base of the villus, while villus lengths were measured from the base of the villus to its tip. The number of cells along the sides of crypts and villi were manually counted from representative images. Three to five representative areas per slide/ mouse, each with 3 to 5 representative crypts or villi, were used for quantification. Alcian Blue staining. Alcian Blue staining was performed using a kit (Alcian Blue (pH 2.5) stain kit H-3501, Vector Laboratories, Burlingame, CA, USA). The counter-stain was nuclear fast red. Goblet cells were manually counted from representative slides. Analysis of cell numbers and cell sizes. Cells were plated in 6-well tissue culture plates at a density of 2 × 10 4 cells in 2 ml of medium (DMEM + 10% heat-inactivated FBS) per well. After 24 h, medium was removed, and ME1 inhibitor (ME1*), canonical Wnt signaling pathway inhibitor (JW74), the combination of ME1* and JW74, or (0.5%) DMSO was added to plated cells in 2 ml of medium (containing 2% heat-inactivated FBS) and incubation continued for an additional 72 h. All treatments contained 0.5% DMSO. Wells were gently washed with PBS and then incubated with 1 ml of trypsin (Gibco ™ Trypsin-EDTA (0.25%), with Phenol Red) and the entire 1 ml sample analyzed in a Vi-CELL ™ XR viability counter (Beckman Coulter, Brea, CA, USA). Cell diameter was simultaneously recorded using this same instrument.
Colony-formation assays. HT29 and HCT116 cells were plated at a density of 1 × 10 3 cells in 1 ml/ well (however, 2 × 10 3 IEC6 cells were plated) in 24-well tissue culture plates (medium was DMEM + 10% heat-inactivated FBS) followed by incubation for 24 h. One ml containing the treatment (ME1*, JW74, the combination of ME1* and JW74, or 0.5% DMSO) were added in DMEM containing 10% heat-inactivated FBS (complete media), followed by incubation for six days (all treatment media contained 0.5% DMSO). Cells were washed with Dulbecco's phosphate-buffered saline and stained with 0.1% crystal violet in 10% formalin for 1 h. Plates were scanned using an Epson Perfection V600 Photo Scanner at 600 DPI, and colonies counted using OpenCFU 36 colony counting software. For OpenCFU, the threshold was set to 'regular' with a value of 3; minimum radius was set to 1, and maximum radius was set to auto. For IEC6 cells, colony areas were calculated using FIJI software 37 . For additional evaluation of the acute cytotoxic effects of inhibitors, cells were plated at high density (100,000 cells) per well in 12-well tissue culture plates (medium was DMEM containing 10% heat-inactivated FBS). After 24 h, treatments (ME1*, JW74, the combination of ME1* and JW74, or 0.5% DMSO) were added in 1 ml of DMEM containing 2% heat-inactivated FBS, and incubation was continued for 3 days (all treatment samples contained 0.5% DMSO). Cells were washed with Dulbecco's phosphate-buffered saline and stained for 1 h with 0.1% crystal violet in 10% formalin. The plates were air-dried and scanned using an Epson Perfection V600 Photo Scanner at 600 DPI. Area of remaining cell coverage in each well was quantified using FIJI software as a percentage of total area of well.

Statistics.
Power analysis indicated an ability to detect a difference of 10 adenomas per mouse (per experimental group) with n = 12 animals per genotype (SD = 8, P < 0.05, power = 0.8173, two sample t-test test). To detect a 50% difference in mRNA abundance as a function of ME1 transgene at the 0.05 level required a minimum of 6 animals per group (S.D. = 0.282; power = 0.8035; two sample t-test). Statistical analysis was performed using SigmaPlot V13.0 (Systat Software, San Jose, CA, USA). One-Way ANOVA and Student's t-tests were used to examine for differences between groups. The Normality Test (Shapiro-Wilk) was used to check if data were normally distributed before conducting Student's t-tests. The Mann-Whitney Rank Sum Test was used to compare data between two groups that were not normally distributed. Only two-tailed P values were used and are listed in the figures. Significant differences were identified by P < 0.05.

Data Availability
Additional data relating to the manuscript will be made available upon request.