BRAFV600E Mice Research Articles

Abstract A020: Exploring the effects of resveratrol on the gut microbiome in high fat diet-fed BRAFV600E mutant mice

Abstract The polyphenol resveratrol, widely known for its cancer protective activities has been shown to protect against the pro-tumorigenic effects induced by high-fat diets (HFD) in mouse models of colorectal cancer (CRC). Although, the mechanisms of actions of this compound in humans still remain elusive, recent studies suggest its effects may be mediated partly through its interaction with the gut microbiota. Here we sought to determine the changes induced by HFD on the intestinal microbiome and the impact of resveratrol supplementation on these alterations in a mouse model of BRAFV600E mutant CRC. BRAFV600E; BRAFWT (wild-type) mice were randomised into 8 groups and fed either a standard diet or HFD supplemented with low or high-dose of resveratrol (0.7mg/kg/day and 14mg/kg/day, respectively) for 6-weeks. The mouse faecal microbial profile was then assessed by targeting the V3-V4 area of the bacterial 16S rRNA gene. Consumption of HFD was associated with significant alternations in the gut microbiome composition in both BRAFV600E and BRAFWT based on beta-diversity analysis (PADONIS<0.05). HFD increased the Firmicutes and Clostridia abundance in BRAFWT compared to animals on a standard diet (P<0.01, P<0.008 respectively), while in BRAFV600E mice, alterations were observed in lower taxonomic levels including the enrichment of Faecalibaculum (P<0.02) and growth inhibition of Lactobacillus johnsonhii (P<0.05). High-dose resveratrol resulted in significant differences in the microbial composition compared to HFD-treated group in the BRAFV600E mice (PADONIS<0.05) but not in BRAFWT . Resveratrol administration did not counteract the HF-induced taxonomic changes but high-dose resveratrol changed Muribaculum, and UBA181 genera abundances in BRAFV600E mice (P<0.001 and P<0.05 respectively) compared to HF-treated mice. Low dose resveratrol had no significant effects on bacterial abundance in both BRAFV600E and BRAFWT mice. It was also observed that BRAFV600E mutation influenced the gut microbiome independently of diet based on beta and alpha-diversity (PADONIS<0.05, PChao1<0.05 respectively). Resveratrol administration modified the gut microbiota composition of BRAFV600E mice but did not completely reverse the HFD-induced changes indicating that alternative mechanisms may contribute to the protective effects of resveratrol in this mouse model. Citation Format: Despoina Theofanous, Hong Cai, Karen Brown. Exploring the effects of resveratrol on the gut microbiome in high fat diet-fed BRAFV600E mutant mice [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A020.

Pre-existing Thyroiditis Ameliorates Papillary Thyroid Cancer: Insights From a New Mouse Model.

Papillary thyroid cancer (PTC) often co-occurs with Hashimoto's thyroiditis, an association that has long been reported in clinical studies yet remains controversial. Some studies, in fact, have suggested a protective effect of thyroiditis while others have not. We generated a mouse model where PTC and thyroiditis develop in a predictable manner, combining the oncogenic drive of the BRAFv600E mutation (inducible by tamoxifen) to the thyroiditis susceptibility of the NOD.H2h4 strain (inducible by iodine). A total of 113 NOD.H2h4_TPO-CRE-ER_BRAFV600E mice (50 followed throughout lifetime and 63 sacrificed at 16 weeks post tamoxifen) were used to determine whether the PTC phenotype differs when thyroiditis precedes or coincides with the onset of PTC. Mice with pre-existing thyroiditis lived longer (median survival of 28.2 weeks post tamoxifen) than those with concomitant (25.6 weeks) or no (24.5 weeks) thyroiditis (P < 0.01 by Laplace regression). PTC developed less frequently (33%) in the pre-existing thyroiditis group than the concomitant (100%) or no (100%) thyroiditis groups (P < 0.001 by chi-squared) and showed less aggressive histopathological features. The intratumoral mononuclear cell infiltration was more prominent in mice with pre-existing thyroiditis (P = 0.002 vs the other groups) and sustained by a significant expansion of effector memory CD8 + T cells and CD19 + B cells. These findings shed light on the controversial PTC-thyroiditis association and emphasize the contribution of intratumoral T and B lymphocytes to the evolution of PTC.

β-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). β-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/β-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of β-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of β-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFβ pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual β-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that β-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/β-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

MAPK mutations and cigarette smoke promote the pathogenesis of pulmonary Langerhans cell histiocytosis.

Pulmonary Langerhans cell histiocytosis (PLCH) is a rare smoking-related lung disease characterized by dendritic cell (DC) accumulation, bronchiolocentric nodule formation, and cystic lung remodeling. Approximately 50% of patients with PLCH harbor somatic BRAF-V600E mutations in cells of the myeloid/monocyte lineage. However, the rarity of the disease and lack of animal models have impeded the study of PLCH pathogenesis. Here, we establish a cigarette smoke-exposed (CS-exposed) BRAF-V600E-mutant mouse model that recapitulates many hallmark characteristics of PLCH. We show that CD11c-targeted expression of BRAF-V600E increases DC responsiveness to stimuli, including the chemokine CCL20, and that mutant cell accumulation in the lungs of CS-exposed mice is due to both increased cellular viability and enhanced recruitment. Moreover, we report that the chemokine CCL7 is secreted from DCs and human peripheral blood monocytes in a BRAF-V600E-dependent manner, suggesting a possible mechanism for recruitment of cells known to dominate PLCH lesions. Inflammatory lesions and airspace dilation in BRAF-V600E mice in response to CS are attenuated by transitioning animals to filtered air and treatment with a BRAF-V600E inhibitor, PLX4720. Collectively, this model provides mechanistic insights into the role of myelomonocytic cells and the BRAF-V600E mutation and CS exposure in PLCH pathogenesis and provides a platform to develop biomarkers and therapeutic targets.

DNMT3B Oncogenic Activity in Human Intestinal Cancer Is Not Linked to CIMP or BRAFV600E Mutation.

SummaryApproximately 10% of human colorectal cancer (CRC) are associated with activated BRAFV600E mutation, typically in absence of APC mutation and often associated with a CpG island methylator (CIMP) phenotype. To protect from cancer, normal intestinal epithelial cells respond to oncogenic BRAFV600E by activation of intrinsic p53 and p16-dependent tumor suppressor mechanisms, such as cellular senescence. Conversely, CIMP is thought to contribute to bypass of these tumor suppressor mechanisms, e.g. via epigenetic silencing of tumor suppressor genes, such as p16. It has been repeatedly proposed that DNMT3B is responsible for BRAFV600E-induced CIMP in human CRC. Here we set out to test this by in silico, in vitro, and in vivo approaches. We conclude that although both BRAFV600E and DNMT3B harbor oncogenic potential in vitro and in vivo and show some evidence of cooperation in tumor promotion, they do not frequently cooperate to promote CIMP and human intestinal cancer.

Abstract 1357: The small GTPase Arf6 potentiates melanoma metastasis by activating Akt

Abstract Arf6 is a member of the Ras-superfamily of small GTPases and controls membrane trafficking and cytoskeletal remodeling, functioning mainly in endocytosis pathways at the cell periphery. Arf6 is activated by various extracellular signals and oncogenic events and has been shown to promote cell migration and pro-invasive phenotype in human cancer cells. Small molecule inhibition of ARF6 reduces spontaneous metastasis in xenograft models of human cutaneous melanoma, suggesting that ARF6 is necessary for disease progression. Using a genetically-engineered mouse model of BRAF-mutant melanoma, we determined whether activation of Arf6 is sufficient to induce spontaneous metastasis in vivo. For melanocyte-specific primary tumor induction, Cre recombinase was delivered via local injection of RCAS virus into DCT-TVA;Cdkn2alox/lox;BRAFV600E mice. In the experimental mice, a constitutively active mutant form of Arf6 (Q67L) was virally delivered with Cre. In this study, we observed a significant increase in spontaneous metastatic disease burden in Arf6 Q67L mice. Likewise, tail vein injection of melanoma cell lines derived from Arf6 Q67L tumors consistently show a diffuse pattern of pulmonary metastasis compared to controls that show rare, microscopic metastasis. Recently, it has been demonstrated that activation of Akt, but not Pten loss, leads to an aggressive phenotype in melanoma that includes the acquisition of brain metastases. Immunohistochemical analysis of phospho-Akt revealed that Arf6 Q67L is sufficient to induce Akt activation in tumors. In addition, ARF6 is necessary for AKT activation in human melanoma lines. We did not observe brain metastasis in DCT-TVA;Cdkn2alox/lox;BRAFV600E + Arf6 Q67L mice. When we added Ptenlox/lox allele to this genetic background, however, we observed microscopic brain metastases at a low frequency, suggesting that the combination of Pten loss and Arf6 activation reaches a threshold level of Akt activation that is sufficient to cause brain metastasis. Taken together our data indicate that activation of Arf6 is sufficient to potentiate melanoma metastasis, consistent with the proinvasive cellular phenotype attributed to Arf6. In addition, our data suggests a novel signaling mechanism by which Arf6, a small GTPase involved in trafficking, is somehow involved in Akt activation and that this step may be important for the acquisition of metastatic capacity. Citation Format: Lehi Acosta, Aaron Rogers, Jae H. Yoo, Shannon J. Odelberg, Dean Y. Li, Sheri Holmen, Allie H. Grossmann. The small GTPase Arf6 potentiates melanoma metastasis by activating Akt [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1357. doi:10.1158/1538-7445.AM2017-1357

Abstract 661: Establishment of transgenic mice with liver-specific BRAF V600E mutation

Abstract Diethylnitrosamine (DEN) is often used to induce hepatocarcinogenesis in rodents to investigate the pathogenesis of hepatocellular carcinoma (HCC). BrafV637E mutation, corresponding to the human BRAFV600E mutation, is highly frequent in DEN-induced hepatic tumors depending on mouse strains. To investigate the role of BrafV637E mutation in DEN-induced hepatocarcinogenesis, we established the transgenic mice (Alb-Cre/BRAFV600E), in which human BRAFV600E mutation is specifically expressed in the liver, and compared the properties of DEN-induced hepatic tumors and the livers of the transgenic mice. The hepatic tumors were induced by neonatal treatment with DEN, isolated at various time points, and stored as frozen or paraffin-embedded materials. For production of the transgenic mice, we obtained homozygous male BRAFV600E mice, in which a LoxP-flanked cassette containing the human BRAF exons 15-18 cDNA, mouse polyadenilation sequences and the neo gene was inserted into the mouse Braf intron 14 upstream of the human BRAF exon 15 cDNA that encodes the BRAFV600E mutation, and crossed them with female Alb-Cre mice by in vitro fertilization. RT-PCR and cDNA sequencing revealed that the liver of Alb-Cre/BRAFV600E mice expressed mRNA derived both from normal mouse and mutated human BRAF exon 15. The transgenic mice showed 20% decrease in body weight compared to normal mice, but 5 fold-increase in the liver/body weight. Histological examination revealed that the liver was entirely consisted of basophilic hepatocytes resembling to those in DEN-induced foci of cellular alteration at 8 weeks of birth, and bile duct cells were further increased at 10-12 weeks. The Ki-67 labeling index of hepatocytes in the Alb-Cre/BRAFV600E mice was 3 times higher than normal mice. Immunoblot analysis revealed that the liver of Alb-Cre/BRAFV600E mice expressed the BRAFV600E protein and showed hyperphosphlylation of ERK1 and AktS473 like the DEN-induced hepatic tumors. Furthermore, immunohistochemical staining detected increased C5/C5a expression in the hepatocytes of Alb-Cre/BRAFV600E mice, and by immunoblot analysis C5a, a protease-cleaved form of C5, was specifically detected in Alb-Cre/BRAFV600E liver like DEN-induced tumors. These results indicate that the liver specific expression of mutationally-activated BRAF led to the hepatocytic phenotype mimicking the DEN-induced hepatic tumors. Citation Format: Hiroki Tanaka, Masahiro Yamamoto, Kosuke Yamazaki, Keiko Shimizu, Katsuhiro Ogawa. Establishment of transgenic mice with liver-specific BRAF V600E mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 661.

Abstract A19: Expression of BRAFV600E in melanocytes induces Schwannian differentiation in vivo

Abstract Transgenic mice expressing BRAFV600E targeted to melanocytes via the tyrosinase promoter display hyperpigmentation of the skin, ears, tail, and paws, as well as profound graying of the hair, benign melanocytic hyperplasias and a low frequency of spontaneous melanomas on a mixed genetic background. In order to investigate the underlying mechanism of BRAFV600E-induced alterations in skin and hair, mice were backcrossed extensively onto a C57BL/6 background. Backcrossed BRAFV600E mice retained the phenotype of mixed background transgenic animals with respect to skin hyperpigmentation as well as premature hair graying. Analysis of skin samples from mice of different ages revealed an accumulation and mislocalization of hyperproliferative melanocytes, suggesting a developmental defect in melanocyte homing to hair follicles in response to activated BRAF. Moreover, melanocytic lesions in the skin developed into benign tumors resembling nerve sheath tumors of Schwannian origin, presumably due to the phenotypic switching of melanocytes into Schwann cells in response to chronic BRAFV600E stimulation. These benign tumors seldom progressed to melanoma in the absence of Akt pathway activation. A role for Akt activation in repressing the Schwannian differentiation program is suggested, as treatment of melanoma-derived cell lines with the Akt inhibitor MK-2206 results in elevated expression of Schwannian lineage markers at the transcript level. Therefore, in addition to induction of oncogene-induced senescence, eliciting lineage-switching from melanocytes into Schwann cells in response to oncogenic BRAF represents a potential tumor suppression mechanism to protect melanocytes from transformation, and repression of this phenotypic switch via activation of the Akt pathway may facilitate the ability of BRAFV600E to drive melanomagenesis. Citation Format: Jodie R. Pietruska, Chi Luo, Philip W. Hinds. Expression of BRAFV600E in melanocytes induces Schwannian differentiation in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A19.

Abstract 3140: Context specific effects of the BRAFV600E mutation on hematopoiesis identifies novel models of BRAF mutant hematopoietic disorders

Abstract BRAFV600E mutations have recently been identified in nearly 100% of patients with the chronic lymphoproliferative disorder hairy cell leukemia (HCL), as well as a small percentage of patients with the plasma cell malignancy multiple myeloma. Despite extensive knowledge regarding the functional effects of BRAFV600E expression in epithelial tissues, very little is understood about the role of the BRAFV600E mutation in hematopoietic transformation. We therefore utilized a conditional BRafV600E murine model crossed with Mx1-cre, Vav-cre, Cd19-cre, and Cγ1-cre transgenic mice to delineate the effects of mutant BRaf expression in pre-natal and post-natal hematopoietic stem and progenitor cells (HSPCs), B-lineage cells, and germinal center B cells respectively. We also investigated the origin of the BRAFV600E mutation in HCL patient bone marrow samples using prospective isolation of sorted HSPC populations followed by quantitative sequencing for the BRAFV600E mutation. Surprisingly, we identified the presence of the BRAFV600E mutation in long-term hematopoietic stem cells of HCL patients, and we also observed marked alterations in HSPC frequencies. Consistent with the human genetic data, expression of BRafV600E in HSPCs of mice resulted in a lethal transplantable hematopoietic disorder characterized by splenomegaly, anemia, thrombocytopenia, increased circulating soluble CD25, and increased clonogenic capacity of B-lineage cells- all classic features of human HCL. In contrast, restricting expression of BRafV600E to B-lineage cells did not result in disease even up to 1.5 years of age. We next assessed the effects of the BRafV600E mutation on HSPC self-renewal and lineage specification. We plated whole BM cells from Mx1-cre BRafV600E mice in methylcellulose containing myeloid/erythroid cytokines or lymphopoietic cytokes (IL-7). BRafV600E cells demonstrated impaired colony formation in myeloid/erythroid conditions. However, BRafV600E HSPCs exhibited limitless replating capacity when plated in the presence of IL-7, indicating that the BRAF mutation induces aberrant B lineage cell self-renewal. A clear competitive advantage was also seen with competitive transplantation of BRafV600E BM cells, identifying an increase in HSPC self-renewal associated with the BRAF mutation. Data from the murine models studied here and characterization of the BM compartment in HCL patients suggest that the cytopenias seen in HCL patients are due in part to HSPC-intrinsic effects of the BRAFV600E mutation on erythropoiesis, megakarypoiesis, and myelopoiesis. Moreover, these data suggest that the use of therapies targeting MAP-kinase signaling in HCL may lead to durable remissions not only through effects on mature leukemic cells, but also through targeted inhibition of signaling and survival in mutant HSPCs. Citation Format: Eunhee Kim, Stephen S. Chung, Jae H. Park, Young Rock Chung, Piro Lito, Julie Feldstein, Wenhuo Hu, Wendy Beguilin, Sebastien Monette, Cihangir Duy, Raajit Rampal, Leon Telis, Minal Patel, Min Kyung Kim, Ari M. Melnick, Neal Rosen, Martin S. Tallman, Christopher Y. Park, Omar Abdel-Wahab. Context specific effects of the BRAFV600E mutation on hematopoiesis identifies novel models of BRAF mutant hematopoietic disorders. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3140. doi:10.1158/1538-7445.AM2014-3140