Oct1/Pou2f1 is selectively required for gut regeneration and regulates gut malignancy

The transcription factor Oct1/Pou2f1 promotes poised gene expression states, mitotic stability, glycolytic metabolism and other characteristics of stem cell potency. To determine the effect of Oct1 loss on stem cell maintenance and malignancy, we deleted Oct1 in two different mouse gut stem cell compartments. Oct1 deletion preserved homeostasis in vivo and the ability to generate cultured organoids in vitro, but blocked the ability to regenerate after treatment with dextran sodium sulfate, and the ability to maintain organoids after passage. In a chemical model of colon cancer, loss of Oct1 in the colon severely restricted tumorigenicity. In contrast, loss of one or both Oct1 alleles progressively increased tumor burden in a colon cancer model driven by loss of heterozygosity of the tumor suppressor gene Apc. The different outcomes are consistent with prior findings that Oct1 promotes mitotic stability, and consistent with different gene expression signatures associated with the two models. These results reveal that Oct1 is selectively required for gut regeneration, and has potent effects in colon malignancy, with outcome (pro-oncogenic or tumor suppressive) dictated by tumor etiology. Author summary Colorectal cancer is the second leading cause of cancer death in the United States. Approximately 35% of diagnosed patients eventually succumb to disease. The high incidence and mortality due to colon cancer demand a better understanding of factors controlling the physiology and pathophysiology of the gastrointestinal tract. Previously, we and others showed that the widely expressed transcription factor is expressed at higher protein levels in stem cells, including intestinal stem cells. In this study we use a conditional mouse Oct1 (Pou2f1) allele deleted in two different intestinal stem cell compartments. The results indicate that Oct1 loss is dispensable for maintenance of the mouse gut, but required for regeneration. We also tested Oct1 loss in the context of two different mouse colon cancer models. We find that Oct1 loss has opposing effects in the two models, and further that the two models are associated with different gene expression signatures. The differentially expressed genes are enriched for previously identified Oct1 targets, suggesting that differential gene control by Oct1 is one mechanism underlying different outcomes.

Oct1 is a widely expressed POU domain transcription factor related to the embryonic stem cell master transcription factor, Oct4 [1,14]. Oct1 promotes glycolytic metabolism and mitotic stability [15][16][17]. It also promotes poised gene expression states, i.e. the ability of transcriptionally silent target genes to remain inducible following the correct developmental cues [18,19]. Oct1 loss is associated with increased oxidative metabolism, elevated reactive oxygen species, hypersensitivity to oxidative and genotoxic stress, and a modest increase in abnormal mitoses [1,15,16,20,21]. Oct1 loss does not compromise cell viability, affect immortalization by serial passage or reduce growth rates in standard culture. Oct1 is however required for oncogenic transformation in soft agar [15]. In a wellcharacterized Tp53 null mouse model, loss of even one Oct1 allele suppresses thymic lymphoma [15]. In a variety of malignancies, Oct1 motifs are enriched in coordinately activated genes [3,[22][23][24].
These results indicate that Oct1 plays important roles in stress responses and tumorigenicity.
Here, we use a conditional Oct1 allele together with two gut stem cell Cre-drivers to determine the role of this protein in the maintenance and regeneration of normal gut cells, and in gastrointestinal malignancy. The Cre-drivers are tamoxifen inducible to provide both tissue-specific and temporal control over Oct1 deletion. We find that Oct1 is dispensable for colon homeostasis, but required for normal regeneration. In the small intestine, Oct1 deletion from Lgr5 + cells in vivo allows for the establishment of cultured gut organoids from isolated crypts, but not their maintenance following passage. In a chemical model of colon tumorigenesis, Oct1 loss greatly reduces tumor incidence and size. The tumors that do emerge in this model have escaped Oct1 deletion, indicating a requirement for Oct1. In contrast, a model of colon cancer driven by loss of heterozygosity (LOH) shows a progressive increase in tumor number, though not of grade, as one or both Oct1 alleles are lost. The increase in tumor initiation indicates a tumor-suppressive role of Oct1 in this context, and is consistent with prior findings that Oct1 promotes mitotic stability. Comparison of gene expression signatures in the two models identifies a set of genes correlating with the model of origin, indicating that the two models have distinct molecular features. This set enriched in direct Oct1 targets.

Oct1 is required for colon epithelium regeneration but not homeostasis
We crossed an Oct1 (Pou2f1) conditional allele [18] to Lrig1-CreER T2 [32], which upon tamoxifen treatment (single injection of 2 mg in corn oil by gavage, see methods) induces Cre activity in stem cells of the colon. 4 wks after treatment, the colon epithelium was superficially normal (Fig 1A), despite near-global deletion of Oct1 (Fig 1B). Oct1 deletion and normal colonic architecture could be maintained for 150 days (not shown). Histological examination of tissue revealed that Oct1 was deleted in >90% of crypts ( Fig 1C). In contrast, non-epithelial cells retained staining (Fig 1D, asterisk).
Rarely there were crypt structures that escape deletion (red arrow), whose frequency did not change over time (not shown). Crypts lacking Oct1 appeared normal compared to those that had escaped deletion (Fig 1D, right) and compared to Oct1 sufficient mice (Fig 1D, left). These results show that Oct1 loss has no superficial effect on the colon epithelium.
We treated animals with 2.5% DSS to damage the GI tract and mobilize gut stem cells to regenerate the epithelium (Fig 1E). Mice lacking Oct1 in their colon were somewhat more sensitive to DSS treatment ( Fig 1F, day 0-10), consistent with the finding that cells lacking Oct1 are hypersensitive to stress [20]. Upon switching back to water, Oct1 sufficient mice rapidly began to gain weight, while mice lacking Oct1 in the colon continued to lose weight ( Fig 1F, day 12-15) and did not regenerate their colon epithelia ( Fig 1G). The failure to regenerate was associated with increased colitis ( Fig 1H) and loss of barrier function with microbial infiltrate in the spleen ( Fig 1I) and liver (data not shown). Collectively, the data indicate that Oct1 loss from the colon results in failure to regenerate following DSS-mediated damage.

Oct1 is required for regeneration of the small intestinal epithelium following DSS-mediated damage, and required for the passage of intestinal organoids in vitro
To determine if Oct1 behaves similarly in the small intestine, we deleted Oct1 using Lgr5-EGFP-IRES-CreER T2 mice [33]. These mice express tamoxifen-inducible Cre and GFP under the control of the native Lgr5 gene locus, which encodes a R-Spondin receptor expressed in stem cells of the small intestine that stabilizes Frizzled receptors, allowing for increased Wnt signaling [34]. As with the colon, deletion of Oct1 in the small intestine preserved a normal-appearing small intestine (Fig 2A) despite efficient deletion from the epithelium (Fig 2B). Following a similar scheme as in the colon (Fig 2C), failure to thrive and increased weight loss was observed upon removal of DSS from drinking water ( Fig 2D). Histological examination revealed that the small intestine had regenerated crypt structures but failed to regenerate nutrient-and fluid-absorbing villi after DSS removal (Fig 2E). These results are similar to the colon in that Oct1 loss results in regeneration-specific defects, but differ in that only villi are affected, with little associated colitis.
In vitro cultured intestinal organoids are a powerful tool to study intestinal stem cells and their differentiated progeny. They are relatively easy to culture and manipulate [33,35]. We additionally profiled the effect of Oct1 loss in the small intestine using organoids from mice 4 weeks posttamoxifen treatment. Organoids were generated directly ex vivo from intestinal crypts. No difference in size, viability or morphological were noted (Fig 2F, primary culture). However, upon passage Oct1 fl/fl ;Lgr5-EGFP-IRES-CreER T2 , disaggregated crypts were unable to regenerate new villus and crypt structures to generate complete organoids, resulting in severe abnormalities ( Fig 2F, after passage). In contrast, control Lgr5-EGFP-IRES-CreER T2 organoids grew normally following passage.
Colon tumors were efficiently generated in control mice but were fewer in number and size in Oct1 deleted mice ( Fig 3B). Quantification from 6 experimental and 4 control mice is shown in Fig 3C. In the Oct1 fl/fl ;Lrig1-CreER T2 tumors that did occur, a high proportion of tumor tissue stained positively for Oct1 compared to gross uninvolved (GU) tissue the same section (~10%, Fig 3D). Quantification from multiple mice indicated that ~90% of tumor tissue retained Oct1 staining, while only ~10% of GU tissue did so ( Fig 3E). This result indicates a selection against Oct1 deletion in the low-grade tumors that do occur, consistent with Oct1 promotion of tumorigenicity in this model.

Oct1 restricts tumorigenicity in a model of colon cancer driven by loss of heterozygosity
Oct1 functions physiologically not to promote tumors, but rather to promote stem cell potency. The stem cell properties that Oct1 promotes are largely pro-oncogenic, but in one respect Oct1 can be tumor suppressive: like its paralog Oct4 [40], Oct1 promotes mitotic stability [16], a hallmark of stem cells [40][41][42]. To test the hypothesis that loss of Oct1 can accelerate tumor initiation in models of malignancy dependent on mitotic errors and LOH, we used conditional deletion of the Apc gene, which is mutated in a large proportion of human colon cancers [43]. Over time, Apc LOH results in adenocarcinomas in the distal colon that mimic human disease in many respects [32]. We crossed Apc fl mice [44] with Oct1 (Pou2f1) conditional mice, generating an allelic series of Oct1 +/+ , Oct1 +/fl , and Oct1 fl/fl Lrig1-CreER T2 mice with heterozygous Apc fl . Mice were followed for 100 days post-tamoxifen treatment before sacrifice. Progressive deletion of one or both Oct1 alleles progressively increased gross tumor number in this model (Fig 4A). Quantification of H&E sections from multiple animals confirmed that tumor number was increased (Fig 4B) but average area per tumor was not (Fig 4C).
Using Oct1 IHC we also found that Oct1 was again efficiently deleted from normal (gross uninvolved) crypts, with ~10% of crypts escaping deletion (Fig 4D, arrows). In this case and in contrast to AOM-DSS-mediated tumors, Oct1 was deleted in most tumor cells (Fig 4D). Oct1 was present in nonepithelial cells within the tumor, providing a measure of specificity (Fig 4D, asterisks). We also assessed b-catenin by IHC. Apc protein restricts b-catenin by ubiquitin-mediated degradation [45], and hence Apc LOH would be predicted to augment b-catenin specifically in tumors in this model. As expected, we found accumulated b-catenin, including nuclear b-catenin, in the tumor cells (Fig 4D).
To study tumor aggressiveness in this model, we conducted immunofluorescence (IF) using phospho-histone H3 antibodies. The number of mitotic events was low, and equivalent in the presence or absence of Oct1 (Fig 4E). Consistent with this result, pathological scoring of tumor sections indicated that despite the increased tumor incidence, there was no significant difference in tumor grade (Fig 4F).

Genes that are differentially expressed between chemical and Apc-LOH tumors are preferentially enriched for Oct1 targets
The finding that Oct1 loss resulted in opposing effects in the chemical-vs. Apc/LOH-driven colon cancer models suggested that the two models may differ at a molecular level, such that Oct1's dominant activity can switch from pro-oncogenic to tumor suppressive. To test this hypothesis, we sampled gene expression from control Oct1 wild-type FFPE and frozen tumor samples. We used a custom 60-gene panel enriched in gut-associated stem cell function together with 31 AOM-DSSinduced and 25 Apc fl ;Lrig1-CreER T2 samples, all of which were wild-type for Oct1. Unsupervised hierarchical clustering of gene expression resulted in interdigitation of the samples (Fig 5A, top). The interdigitation was robust using multiple settings and cutoffs (not shown), indicating that global tumorto-tumor gene expression variation is dominant over differences between the models. To identify model-associated molecular signatures, we grouped samples by the model of origin and identified subsets of profiled genes whose expression partitioned with the model. A group of 17 genes was identified (Fig 5A, bottom) whose expression tended to correlate with tumor model (Fig 5B, Table S1).
We compared this group of genes with Oct1 targets identified in T cells [18] and with Oct1/Oct4 targets identified in embryonic stem cells (ESCs, [46]. Oct4 levels are far higher than Oct1 in ESCs, resulting in most targets being occupied exclusively by Oct4, which has similar DNA binding specificity [46]. Of the 17 identified genes whose expression correlates with the tumor model, 8 were previously identified as direct Oct1 targets in T cells and 9 as Oct4 targets in ESCs (Fig 5C). Additionally Lef1, a gene on the tumor panel expressed more strongly in the Apc-LOH-driven model (Fig 5B), was identified as one of only 356 direct Oct1-exclusive target genes in ESCs. Across both ChIPseq datasets, all but four of the 17 genes overlapped with direct Oct1 targets (Table S1). To further test the robustness of this result, we performed a second analysis with a larger 770 gene panel together with 23 AOM-DSS-induced and 12 Apc fl ;Lrig1-CreER T2 Oct1 wild-type samples, in this case identifying 83 differentially expressed genes (Table S1). These genes were also enriched for Oct1 and Oct4 targets (Fig 5C). In this case, three of the genes overlapped with a set of Oct1-exclusive ESC targets: Ercc2, Lifr and Myb (Table S1). The enrichment for Oct1/Oct4 target genes in the differentially expressed gene set was statistically significant in all cases (Fig 5C). Collectively, these results indicate that Oct1 regulates genes that tend to be differentially expressed tumors from the two models, potentially contributing to the opposing effects of Oct1 in the two models.

Discussion
Here we show that the transcription factor Oct1 is dispensable for mouse gut epithelial homeostasis, but is essential for regeneration and the passage of intestinal organoids in vitro. Oct1 can be deleted in the colon with normal architecture for up to 150 days, consistent with the interpretation that Oct1 is dispensable for maintenance of the normal gut. In contrast to homeostatic conditions, Oct1 is required to regenerate the gut following DSS exposure. The findings are consistent with prior findings from others indicating that homeostasis and regeneration are molecularly and physiologically distinct [47][48][49][50][51]. DSS treatment of mice lacking Oct1 in small intestinal epithelium also had no effect on homeostasis, but resulted in continued weight loss after switching back to water. However the effect was weaker than in the colon and inflammation and loss of barrier function were not observed. The difference is likely due to the fact that crypts but not fluid-and nutrient-absorbing villi were efficiently generated. The difference in regeneration potential between the two organs and Cre drivers (Lrig1 vs.

Lgr5) is unknown.
Intestinal organoids have been used before to study regeneration [52]. Consistent with the finding that Oct1 loss did not affect small intestinal homeostasis in vivo, organoids could be directly explanted from tamoxifen treated mice. Interestingly, we found that intact organoid structures could not be maintained by passage in vitro. Instead isolated crypts structures could close but not generate new villus and crypt domains. The underlying causes may be similar to the failure to regenerate in vivo, though more study is required to test this idea.
We also show that Oct1 loss has potent effects on tumorigenicity in two different mouse models of colon malignancy. In one model (AOM-DSS), Oct1 loss strongly protects mice from tumors. Elevated Oct1 mRNA expression is a negative prognostic factor in colon but not breast cancer ( Fig   4D). The normal appearance of the colon following Oct1 deletion, coupled with the protection afforded by Oct1 loss, suggests a possible "therapeutic window" in which targeting Oct1-associated pathways could be used to treat GI malignancies with minimal side effects. However, in a second model (Apc fl/+ ;Lrig1-Cre), Oct1's dominant activity is tumor suppressive, with more tumors generated though of equal grade. More study will be required to determine the conditions in which targeting Oct1 and its associated pathways in familial and non-familial gut malignancy could be beneficial.
In HeLa cells and MEFs, Oct1 loss slightly increases the rate of mitotic chromosome segregation abnormalities, resulting in increased lagging chromosomes and aneuploidy [16].
Consistent with these prior findings, Oct1 loss accelerated tumorigenesis and increased tumor number in a colon cancer model driven by LOH of the tumor suppressor gene Apc in Lrig1 + cells [32].
In additions to increased LOH, differences in the molecular pathways and vulnerabilities associated with Oct1 in the two tumor models could contribute to the difference. To test this idea, we profiled gene expression in AOM-DSS tumors and tumors in which Apc is deleted in Lrig1 + cells, identifying a set of differentially expressed genes. These genes were enriched in Oct1 targets. The results were robust, as they were reproduced with two different gene expression panels. Differential regulation of gene expression by Oct1 in the two models could therefore explain in part the opposing effects of Oct1 loss in the two models. A model for the effect of Oct1 loss in the two tumor models is shown in Fig 4E. In this model, Oct1 promotes AOM/DSS-induced tumors through actions on target genes controlling metabolism and stem cell identity. Because Oct1 promotes mitotic stability and because Oct1 target genes are differentially expressed in the more aggressive Apc-LOH model, Oct1 instead acts as a tumor suppressor. Cumulatively, the findings indicate that Oct1 is a potent regulator of colon malignancy, but that its functions are dictated by the colon tumor model used.

Laboratory mice
All mice used in this study were mixed C57BL/6:129/Sv background. The Oct1 conditional allele has been described previously [18]. The Lrig1-CreER T2 mouse allele [32] was a gift of Robert Coffey (Vanderbilt). The Lgr5-EGFP-IRES-CreER T2 allele [33] was purchased from Jackson labs. Apc loxp exon14 (Apc fl ) has been previously described [44] and was a gift from Ömer Yilmaz (MIT). Food and water were available ad libitum. Tamoxifen (200 µL 10 mg/mL in corn oil) was administered by oral gavage.
All mice were treated at 6-8 weeks of age. Regeneration and azoxymethane (AOM) chemical tumorigenesis experiments used a single tamoxifen treatment. The Apc tumor model received three treatments on consecutive days. Dextran sodium sulfate (DSS, MP Biomedicals) was provided in drinking water at a concentration of 2.0% for AOM-induced tumors, and 2.5% for regeneration. AOM (Sigma) was provided by IP injection (10mg/kg). AOM-DSS treatments followed the protocol in [39].
All in vivo experiments were reviewed, approved, and conducted in compliance with the University of Utah's Institutional Animal Care and Use Committee and the NIH Guide for Care and Use of Laboratory Animals guidelines.

Preparation of colon and small intestine epithelial cells and crypts
Total epithelial cells from the colon were isolated as previously described [53,54].

Immunohistochemistry
IHC was performed as in [7]. The slides were developed with DAB peroxidase substrate (Vector Laboratories, SK-4100) as per manufacturer instructions, and were counterstained with hematoxylin.

Intestinal organoids
Organoids were maintained as previously described [53,54] with modifications. Crypts from tamoxifentreated or untreated mice were plated in 8-well chambered slides in 40µl of Matrigel at a density of ~40 crypts per Matrigel droplet. Organoids were grown for 5-7 days until fully grown. Mature organoids were passaged every 5-7 days. Images were taken using an Olympus FV1000 confocal inverted microscope. Quantifications were performed using Image J software (National Institutes of Health).       A second analysis is also shown using genes enriched from a larger PanCancer Pathways probeset (Table S1) Table S1. Genes with expression correlating to tumor model (AOM-DSS vs Apc-LOH;Lrig1-CreER).