Degree of Tissue Differentiation Dictates Susceptibility to BRAF-Driven Colorectal Cancer

Abstract

Oncogenic mutations in BRAF are believed to initiate serrated colorectal cancers; however, the mechanisms of BRAF-driven colon cancer are unclear. We find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation. Correspondingly, tumor formation is inefficient in BRAF-driven mouse models of colon cancer. By reducing levels of differentiation via genetic manipulation of either of two distinct differentiation-promoting factors (Smad4 or Cdx2), stem cell activity is restored in BRAF^V600E intestines, and the oncogenic capacity of BRAF^V600E is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Additionally, our results predict that genetic and/or environmental factors that reduce tissue differentiation will increase susceptibility to serrated colon cancer. These findings offer an opportunity to identify susceptible individuals by assessing their tissue-differentiation status.

Keywords: BRAF; Cdx2; Smad4; intestinal homeostasis; serrated colorectal cancer.

Figure 1. BRAF^V600E activation alters intestinal homeostasis, promoting differentiation

(A) GSEA analysis reveals that MAPK target genes (Rad et al., 2013), were elevated in the BRAF^V600E/+ mutants, as expected (K-S test). (B) Assessment of morphological changes (H&E), proliferation (Ki67), differentiation (AP), stem cells (OLFM4), and senescent cells (p21) in BRAF^V600E/+ and BRAF^V600E/600E mutants. Note increasing levels of differentiation markers and decreasing levels of the stem cell marker with the increased number of activated BRAF alleles. Representative images from 3 biological replicates. Scale bars = 50 μm (C) intestinal stem cell gene expression levels are significantly reduced in BRAF^V600E/+ mutants whereas (D) intestinal differentiation gene signatures are significantly increased compared to controls.

Figure 2. Ablation of CDX2 reverses BRAF^V600E-driven changes in stem/differentiation homeostasis

(A) Immunohistochemistry of OLFM4 in uninjected control, BRAF^V600E/+, Cdx2^f/f; BRAF^V600E/+, BRAF^V600E, and Cdx2^f/f; BRAF^V600E mice, 5 days post-tamoxifen treatment. (B) Primary organoid cultures isolated from control, BRAF^V600E/+, and Cdx2^f/+; BRAF^V600E/+ mice. BRAF^V600E/+ mutant cells do not give rise to viable organoids, but reduction of CDX2 levels permits their growth. Representative of 3 biological replicates. Scale bars=50μm. (C) Lgr5-GFP expression. Scale bars=50μm. (D) Recombination at the BRAF^V600E locus, induced by tamoxifen treatment of the stem cell specific, conditional Cre (Lgr5-^CreERT2-IRES-eGFP), is not sustained unless Cdx2 is simultaneously inactivated in the stem cells. Samples from crypt epithelium were harvested at 2 days or 10 months after tamoxifen treatment. (E) AP staining reveals increased expression in BRAF^V600E/+ and BRAF^V600E, which is reduced when Cdx2 is lost. (F) GSEA shows intestinal stem cell genes are elevated while differentiation genes are reduced upon loss of CDX2, reversing gene expression changes induced by BRAF^V600E activation. (G) Examples of the types of differentiation-associated genes that are elevated upon BRAF activation, and suppressed upon CDX2-loss.

Figure 3. Loss of CDX2 accelerates BRAF-driven tumorigenesis

(A) H&E stains of age-matched control, BRAF^V600E/+, and Cdx2^f/+; BRAF^V600E/+ mice reveal characteristics of serrated cancer morphologies. Dysplasias and carcinomas had characteristics of human hyperplastic polyps (HPPs), SSAs, and serrated carcinomas (Also see Figure S3) (B) More serrated lesions were found in Cdx2^f/+; BRAF^V600E/+ compound mice than age-matched BRAF^V600E/+ counterparts. Counts were based upon complete swiss roll sections. Scale bars=50μm.

Figure 4. Loss of SMAD4 restores the stem-differentiation balance that is altered upon BRAF^V600E/+ activation

(A) AP staining reveals that Smad4 loss reverses the increase in differentiation induced by BRAF^V600E/+. (B) OLFM4 staining suggests that loss of SMAD4 in BRAF^V600E/+ tissue reverses stem cell loss. Representative of 3 biological replicates. Scale bars=50μm. (C) Lgr5-GFP expression is lost upon BRAF^V600E expression, but restored upon SMAD4-loss. (D) Organoid cultures isolated from control, BRAF^V600E/+, and Smad4^f/f; BRAF^V600E/+ mice reveal a rescue in viability in Smad4^f/f; BRAF^V600E/+ organoids. (E) GSEA shows intestinal differentiation genes are reduced, while stem cell gene expression is elevated upon loss of SMAD4, reversing gene expression changes induced by BRAF^V600E/+ activation.

Figure 5. Tumorigenesis in BRAF^V600E/+ mice is accelerated upon the loss Smad4

(A) H&E stain shows overall morphology of adjacent normal and tumor regions of the intestine of control, BRAF^V600E/+, and Smad4^f/f; BRAF^V600E/+ mice. Similar to Cdx2^f/+; BRAF^V600E/+ mice, dysplasias and carcinomas featured serrated morphologies (See also Figure S5). Counts of macroscopic (B) and microscopic (C) lesions observed in braf^v6ooe/+mice are lower than Smad4^f/f; BRAF^V600E/+ mutants. Scale bars=50um.

Figure 6. Elevated WNT signaling may restore oncogenic potential in Cdx2^f/f; BRAF^V600E/+and Smad4^f/f; BRAF^V600E/+ double mutant mice

(A) Transcriptome analysis of WNT signature genes (Van der Flier et al., 2007) indicates increased WNT target gene expression in the double mutant mice when compared to BRAF^V600E/+ mice (K-S test). (B) Common intestinal WNT target genes are highlighted. (C) Organoid cultures derived from BRAF^V600E/+ crypts survive when treated with either WNT3A or the WNT-pathway agonist CHIR-98014. (D) Immunohistochemistry of WNT targets CD44 and SOX9 show elevated levels in Cdx2^f/f; BRAF^V600E/+an6 Smad4^f/f;BRAF^V600E/+ mutants compared to BRAF^V600E/+ mutants. (E) Organoid cultures derived from compound mutant mice show sensitivity to WNT inhibitors. Table describes viability in days of mature organoids after treatment with XAV-939 or IWP-2. Representative of 2 biological replicates and 3 technical replicates each. Bars=50 μ.

Figure 7. Differentiation gene expression levels in normal tissues corresponds to serrated tumor susceptibility in humans

(A) Non-tumor, right colon tissue from serrated tumor patients (n=15) exhibit lower levels of differentiation-gene expression when compared to control patient tissues (n=10, K-S test). (B) K-means clustering of patients into tumor and non-tumor classes is improved when combining intestinal stem (Munoz et al., 2012) and differentiation cell (Chong et al., 2009) gene sets that are differentially regulated in mouse models (performing in the top 2.5% of 1000 random gene lists of equivalent size), or by either gene set individually (Figure S6). (C) Patients exhibit a range of differentiation gene expression, with serrated cancer patients exhibiting lower levels of intestinal differentiation genes (K-W Test, p<0.05). (D) Human tissue samples assessed for differentiation marker KRT20 stain intensity. (E) Adjacent normal tissue of serrated tumor patients exhibits a trend towards reducedstain intensity relative to non-tumor patients (Mann-Whitney p=0.13362). (F) KRT20transcript levels in normal tissue is lower in serrated tumor patients when compared tonon-tumor patient samples (ANOVA, p<0.05). Scale bars=50 μm.