SMAD4 is critical in suppression of BRAF-V600E serrated tumorigenesis

Abstract

BRAF-driven colorectal cancer is among the poorest prognosis subtypes of colon cancer. Previous studies suggest that BRAF-mutant serrated cancers frequently exhibit Microsatellite Instability (MSI) and elevated levels of WNT signaling. The loss of tumor-suppressor Smad4 in oncogenic BRAF-V600E mouse models promotes rapid serrated tumor development and progression, and SMAD4 mutations co-occur in human patient tumors with BRAF-V600E mutations. This study assesses the role of SMAD4 in early-stage serrated tumorigenesis. SMAD4 loss promotes microsatellite stable (MSS) serrated tumors in an oncogenic BRAF-V600E context, providing a model for MSS serrated cancers. Inactivation of Msh2 in these mice accelerated tumor formation, and whole-exome sequencing of both MSS and MSI serrated tumors derived from these mouse models revealed that all serrated tumors developed oncogenic WNT mutations, predominantly in the WNT-effector gene Ctnnb1 (β-catenin). Mouse models mimicking the oncogenic β-catenin mutation show that the combination of three oncogenic mutations (Ctnnb1, Braf, and Smad4) are critical to drive rapid serrated dysplasia formation. Re-analysis of human tumor data reveals BRAF-V600E mutations co-occur with oncogenic mutations in both WNT and SMAD4/TGFβ pathways. These findings identify SMAD4 as a critical factor in early-stage serrated cancers and helps broaden the knowledge of this rare but aggressive subset of colorectal cancer.

Figure 1:. Loss of SMAD4 Promotes Microsatellite Stable (MSS) Tumors.

(A) 30% of human tumors with BRAF^V600E mutation have one or more oncogenic alterations in TGFβ pathways, with predominant mutation being truncation (black) or missense (green) in SMAD4. (B) MSI analysis of Wildtype, Msh2^KO, and Smad4^KO BRAF^V600E/+ Villin-Cre mice organoids. Microsatellite loci (A33, GA29, Bat37, Bat67) were assessed as either unchanged (gray box) or experienced a genetic shift (pink box) in tumor organoids when compared to matched genomic tail DNA. MSI status dictated by number of genetic shifts (MSS = grey, MSI-L = pink, MSI-Hi = Red). (C) Analysis of human patients with BRAF^V600E colorectal cancer (from AACR GENIE) shows that tumors with oncogenic alteration in SMAD4 have low mutation burden and are therefore most likely MSS. Red/blue boxes: BRAF^V600E cases with/without oncogenic alteration in SMAD4. (D) Similar analysis shows that BRAF^V600E tumors known to have an oncogenic mutation in other TGFβ pathway genes have almost an order of magnitude higher mutation burden than the remaining BRAF^V600E tumors and are therefore most likely MSI. Red/blue boxes: BRAF^V600E cases known/not known to have an oncogenic mutation in TGFBR2/SMAD2/SMAD3/BMPR1A (p-val < 10⁻¹⁵, 2-sided Wilcoxon rank-sum test).

Figure 2:. MSI Accelerates Serrated Tumorigenesis in Smad4^KO BRAF^V600E/+ Environment.

(A) Mice were treated with tamoxifen for 4 consecutive days and then aged for 2–3 months post-injection. (B) Whole mount of Msh2^KO Smad4^KO BRAF^V600E/+ and Smad4^KO BRAF^V600E/+ showing visible macroscopic tumors in duodenum and jejunum. (C) Counts of macroscopic tumors were based off 7 biological replicates of Msh2^KO Smad4^KO BRAF^V600E/+, Smad4^KO BRAF^V600E/+ and 3 biological replicates of BRAF^V600E/+ as a control within 3 months post tamoxifen treatment. The number of macroscopic tumors found within the Msh2^KO Smad4^KO BRAF^V600E/+ mice are higher than in the Smad4^KO BRAF^V600E/+ mice (p-val = 0.1055, Student’s T-Test). (D) Size of tumors in Msh2^KO Smad4^KO BRAF^V600E/+ and Smad4^KO BRAF^V600E/+. (E) H&E of Smad4^KO BRAF^V600E/+ and Msh2^KO Smad4^KO BRAF^V600E/+. Images are representative of 3 biological replicates (Scale bar = 0.5mm). Low-grade and high-grade dysplasias were both noted and together classified as dysplasia, as compared with invasive cancer and normal tissue. (F) Counts of microscopic tumors were based off 3 biological replicates of Msh2^KO Smad4^KO BRAF^V600E/+ and Smad4^KO BRAF^V600E/+ mice. The number of microscopic tumors found within the Msh2^KO Smad4^KO BRAF^V600E/+ mice are higher than in the Smad4^KO BRAF^V600E/+ mice. (* = p-val = 0.005, Student’s T-Test).

Figure 3:. MSI Increases Mutational Burden, but there is Strong Selection for Oncogenic WNT Activation.

(A) Mice were treated with tamoxifen for 4 consecutive days and aged for 2–3 months post-injection. Msh2^KO Smad4^KO BRAF^V600E/+ (n=9) and Smad4^KO BRAF^V600E/+ (n=3) tumor organoids were submitted for whole exome sequencing along with matched genomic tail DNA. (B) Mutect2 analysis reveals Msh2^KO Smad4^KO BRAF^V600E/+ tumors have increased total variants (* = p-val < 0.0001, Student’s T-Test). (C) Stratification of variants show higher frequencies of both Single Nucleotide Variants (SNVs) and Insertion/Deletion events (Indels) in Msh2^KO Smad4^KO BRAF^V600E/+ tumors (* = p-val = 0.0218, Student’s T-Test). (D) Msh2^KO Smad4^KO BRAF^V600E/+ tumors also have more mutations within coding regions of genes (* = p-val < 0.0001, Student’s T-Test). (E) Analysis of documented oncogenic mutations in either Msh2^KO Smad4^KO BRAF ^V600E/+ or Smad4^KO BRAF ^V600E/+ (orange arrows) tumors revealed that the most common oncogenic mutation in Msh2^KO Smad4^KO BRAF ^V600E/+ and the only oncogenic mutation in Smad4^KO BRAF ^V600E/+ was CTNNB1. Blue bar: oncogenic mutations according to OncoKB, black bar: truncating mutations in other cancer genes. (F) Oncogenic Ctnnb1 mutations identified in mouse tumors (top) coincide with CTNNB1 “hotspot” mutations in human patient cases (bottom). (G) Oncogenic Ctnnb1 mutations were isolated to Exon 3, with the most frequent point of mutation at “hotspot” T41.

Figure 4:. Smad4^KO BRAF^V600E/+ Tumors Exhibit Elevated WNT Signaling.

Smad4^KO BRAF^V600E/+ and Msh2^KO Smad4^KO BRAF^V600E/+ mice were collected 3 months post tamoxifen treatment. (A) Ki67 of adjacent normal and tumors show increased proliferation that extend beyond crypt compartments in tumors. Images are representative of three biological replicates. (B-C) CD44 and β-catenin of adjacent normal and tumors reveal higher WNT signaling in tumors. Images are representative of 3 biological replicates. (Scale bar = 0.5mm). Low-grade and high-grade dysplasias were both noted and together classified as dysplasia, as compared with invasive cancer and normal tissue. (D) qPCR analysis of WNT target genes in wildtype (n=3), Smad4^KO BRAF^V600E/+ tumor (n=6), and Msh2^KO Smad4^KO BRAF^V600E/+ tumor (n=8) organoids. Results were normalized to gene expression of wildtype organoids (a = wildtype vs. Smad4^KO BRAF^V600E/+ tumor, p-val < 0.05, b = wildtype vs. Msh2^KO Smad4^KO BRAF^V600E/+ tumor, p-val < 0.05, c = Smad4^KO BRAF^V600E/+ tumor vs. Msh2^KO Smad4^KO BRAF^V600E/+ tumor, p-val < 0.05, two-way ANOVA). (E) Viability of wildtype (n=2), Smad4^KO BRAF^V600E/+ tumor (n=2), and Msh2^KO Smad4^KO BRAF^V600E/+ tumor (n=2) organoids after 7 days cultured in media +/- R-Spondin (RSPO). Organoids were passaged and cultured for 2 days in complete media prior to removal of RSPO (* = p-val = 0.0107, two-way ANOVA). (F) Immunoblot of wildtype (n=2), Smad4^KO BRAF^V600E/+ tumor (n=3), and Msh2^KO Smad4^KO BRAF^V600E/+ tumor (n=2).

Figure 5:. Loss of Smad4 is Key Step in Serrated Hyperplasia-to-Dysplasia Transition.

(A) Histology of wildtype and BRAF^V600E/+ Ctnnb1^Exon3/+ Villin-Cre mice treated with tamoxifen for 4 consecutive days to induce recombination in the intestinal epithelium. Mice were collected 6 days post tamoxifen treatment based on weight loss. H&E and histology of Ki67 and SMAD4 of wildtype normal epithelium and BRAF^V600E/+ Ctnnb1^Exon3/+ Villin-Cre mice. Images are representative of 4 biological replicates. (Scale bar = 0.5mm). (B) BRAF^V600E/+ Ctnnb1^Exon3/+ Lgr5-Cre mice treated with tamoxifen to induce recombination in the stem cells of the intestinal epithelium. Mice were collected 2 months post tamoxifen treatment based on weight loss. Whole swiss roll of a SMAD4 stained BRAF^V600E/+ Ctnnb1^Exon3/+ Lgr5-Cre mouse. Images are representative of 3 biological replicates. (C) SMAD4 immunohistochemistry of BRAF^V600E/+ Ctnnb1^Exon3/+ Lgr5-Cre mice reveal both SMAD4-positive and SMAD4-negative dysplasias. Images are representative of 3 biological replicates. (Scale bars=0.5mm). (D) Distribution of SMAD-positive and SMAD4-negative dysplasias found in BRAF^V600E/+ Ctnnb1^Exon3/+ Lgr5-Cre mice (n=3). (E) BRAF^V600E/+ Ctnnb1^Exon3/+ Lgr5-Cre dysplasias were dissected from adjacent tissue and extracted for RNA. (F) Smad4 expression is reduced in dissected tumors (n=4) when compared to paired adjacent tissue (* = p-val = 0.0477 Student’s T-Test).

Figure 6:. Activation of WNT in Smad4^KO BRAF^V600E/+ Mouse Model Accelerates Serrated Tumorigenesis.

(A) Mice were treated with tamoxifen for 4 consecutive days and collected 7–10 days post-injection. (B) Histology of wildtype, BRAF^V600E/+ Ctnnb1^Exon3/+, and Smad4^KO BRAF^V600E/+ Ctnnb1^Exon3/+ Villin-Cre mice. H&E and Ki67 revealed dysplasias in only the triple mutant model. Images are representative of 4 biological replicates. (Scale bar=0.5mm). (C) Counts of serrated dysplasias in Villin-Cre mice were based on 4 biological replicates of wildtype, BRAF^V600E/+ Ctnnb1^Exon3/+ and Smad4^KO BRAF^V600E/+ Ctnnb1^Exon3/+ within 3 days post tamoxifen treatment (* = p-val = 0.0004 two-way ANOVA). (D) Counts of serrated dysplasias in wildtype (n = 5), BRAF^V600E/+ Ctnnb1^Exon3/+ (n = 3), and Smad4^KO BRAF^V600E/+ Ctnnb1^Exon3/+ (n = 5) Cdx2-Cre mice within 10 days post tamoxifen treatment (a = p-val < 0.05 vs. control; b = p-val < 0.05 vs. BRAF^V600E/+ β-catenin^Exon3/+, two-way ANOVA).

Figure 7:. Activation of WNT in a Smad4^KO BRAF^V600E/+ Mouse Model Accelerates Serrated Tumorigenesis and Progression.

(A) Mice were injected with tamoxifen for 1 day and aged for 21 days post-injection. (B) Histology of wildtype, BRAF^V600E/+ Ctnnb1^Exon3/+, and Smad4^KO BRAF^V600E/+ Ctnnb1^Exon3/+ Lgr5-Cre mice treated with tamoxifen to induce recombination in the stem cells of the intestinal epithelium. H&E and Ki67 of the duodenum and dysplasias were identified. Images are representative of 3 biological replicates. (Scale bar=0.5mm). (C) Counts of serrated dysplasias in Lgr5-Cre mice within 21 days post tamoxifen treatment. (* = p-val = 0.0175, Student’s T-Test). (D) Counts of invasive tumors in Lgr5-Cre mice were based on 5 biological replicates of wildtype, and BRAF^V600E/+ Ctnnb1^Exon3/+ (n=4), and Smad4^KO BRAF^V600E/+ Ctnnb1^Exon3/+ mice (p-val = 0.0945, one-way ANOVA). (E) Human patient data reveals at least 56% of tumors with BRAF^V600E mutation and oncogenic TGFβ pathway alterations have one or more oncogenic alterations in the WNT pathway. (F) At least 37% of tumors with BRAF^V600E mutation and oncogenic WNT pathway alterations also had one or more oncogenic alterations in the TGFβ pathway.

Figure 8:. SMAD4/TGFβ Has Early-Stage Role in BRAF-V600E Serrated Tumor Progression.

(A) Summary of genotypes used in this study. Degree of dysplasia is ranked based upon average dysplasia counts per mouse relative to genotypes within Cre-specific drivers. Dysplasia formation and degree of dysplasia development reported is earliest timepoint recorded based upon this study. (* = dysplasias have been documented, but found at later timepoints)[9]. (B) Serrated cancers are predominantly driven by BRAF-V600E mutation. Loss of SMAD4 or suppression of TGFβ pathway in combination with oncogenic WNT elevation promotes hyperplasia-to-dysplasia transition. SMAD4 loss promotes the development of MSS serrated tumors, and SMAD4 loss can occur before or after oncogenic elevation of WNT.