Mouse Models of Overexpression Reveal Distinct Oncogenic Roles for Different Type I Protein Arginine Methyltransferases

Abstract

Protein arginine methyltransferases (PRMT) are generally not mutated in diseased states, but they are overexpressed in a number of cancers, including breast cancer. To address the possible roles of PRMT overexpression in mammary gland tumorigenesis, we generated Cre-activated PRMT1, CARM1, and PRMT6 overexpression mouse models. These three enzymes are the primary type I PRMTs and are responsible for the majority of the asymmetric arginine methylation deposited in the cells. Using either a keratin 5-Cre recombinase (K5-Cre) cross or an MMTV-NIC mouse, we investigated the impact of PRMT overexpression alone or in the context of a HER2-driven model of breast cancer, respectively. The overexpression of all three PRMTs induced hyper-branching of the mammary glands and increased Ki-67 staining. When combined with the MMTV-NIC model, these in vivo experiments provided the first genetic evidence implicating elevated levels of these three PRMTs in mammary gland tumorigenesis, albeit with variable degrees of tumor promotion and latency. In addition, these mouse models provided valuable tools for exploring the biological roles and molecular mechanisms of PRMT overexpression in the mammary gland. For example, transcriptome analysis of purified mammary epithelial cells isolated from bigenic NIC-PRMT1 ^Tg and NIC-PRMT6 ^Tg mice revealed a deregulated PI3K-AKT pathway. In the future, these PRMT ^Tg lines can be leveraged to investigate the roles of arginine methylation in other tissues and tumor model systems using different tissue-specific Cre crosses, and they can also be used for testing the in vivo efficacy of small molecule inhibitors that target these PRMT. SIGNIFICANCE: These findings establish Cre-activated mouse models of three different arginine methyltransferases, PRMT1, CARM1, and PRMT6, which are overexpressed in human cancers, providing a valuable tool for the study of PRMT function in tumorigenesis.See related commentary by Watson and Bitler, p. 3.

Fig.1. Generation and validation of conditional transgenic overexpression of PRMTs

(A) Schematic diagram of the vector design strategy for conditional PRMT overexpression. The full open reading frames (ORFs) of PRMT1 and CARM1 were cloned downstream of 3×Flag and upstream of IRES-GFP-PolyA cassette. The design for PRMT6 was similar, but this vector did not harbor an IRES-GFP cassette. A floxed STOP signal element was placed downstream of a ubiquitous, synthetic chicken β-actin promoter (CAGGS). When crossed with CRE-expressing mouse lines, the STOP cassette will be removed, and the CAG promoter will drive the high expression of Flag-tagged PRMTs fusion proteins. (B) Relative mRNA expression of PRMT1, CARM1 and PRMT6 by qRT-PCR, using RNA isolated from the skin and purified mammary epithelial cells (MECs) from PRMT^Tg and K5-PRMT^Tg littermates. Data was calculated from three biological replicates. (C) Western blot analysis of PRMT1, CARM1 and PRMT6 expression in the purified mammary epithelial cells (MECs) isolated from PRMT^Tg and K5-PRMT^Tg. The asterisks represent non-specific protein bands. Triangles indicate expected protein bands. Note that CARM1 displays a smear and not a sharp band, which is likely due to extensive post-translational modifications, like glycosylation and O-GlcNAcylation, as reported previously (49,50)).

Fig.2. Phenotypic and histological analyses of K5-Prmt^Tg mice

(A) Whole mount images of the mammary glands by Carmine alum staining from the nulliparous females as indicated at different ages. Note the hyper-branching of the mammary ducts observed in the virgin bigenic K5-PRMT^Tg female mice, starting from different time points (left panel). H&E staining of the mammary duct epithelium with different genotypes as labeled (right panel). (B) H&E staining showing the MIN lesions in the non-palpable, nulliparous females of all three bigenic K5-PRMT^Tg models as indicated (left panel). Immunostaining of the epithelium by Ki-67 showing the proliferation status of the MIN lesions (right panel). Scale bar = 100 μm.

Fig.3. Analyses of the tumors induced by HER2 and PRMT overexpression

(A) Female PRMT^Tg mice were crossed with male MMTV-NIC (NEU-IRES-CRE) mice, in which the MMTV promoter drives the simultaneous expression of the activated NEU (NDL2–5) oncogene as well as the Cre enzyme in the same epithelial cells. Nulliparous pure NIC and bigenic NIC-PRMT^Tg females were selected for subsequent studies. (B) Kaplan-Meier curves of tumor-free survival in the cohorts of virgin NIC and bigenic NIC-PRMT^Tg females. T_1/2 indicates the age when 50% of mice developed tumors by palpation. The P-values were calculated by Log-rank test. (C) Histological analyses by H&E staining and immunostaining using the Flag antibody on non-palpable mammary epithelium. The average total number of MINs were counted from 5 mice for each genotype. *, p<0.05; (Student’s t-test). Scale bar = 60μm. (D) Histological analyses by H&E staining and immunostaining of Flag antibody in paraffin-embedded sections of mammary tumors collected from the end-point mice. Simultaneous overexpression of individual PRMTs in the HER2-induced mammary tumors led to the phenotypical HER2 characteristics showing the solid nodular growth with intermediate cells. Scale bar = 120μm.

Fig.4. Transcriptome analyses identifies deregulation of the PI3K-AKT pathway

(A) Scatter plots showing differentially expressed genes (DEGs) in pre-neoplastic mammary epithelium revealed by RNA-seq analyses between nulliparous NIC and bigenic NIC-PRMT^Tg females. Up, up-regulated DEGs. Dn, down-regulated DEGs. Cutoff: FC>2, FDR<0.05. Black circles mark overexpressed PRMT1, CARM1 and PRMT6, respectively. (B) Hierarchical clustering heatmap of the DEGs in the mammary epithelium among different biological replicates of NIC-PRMT^Tg females. Red and green colors represent the log2 ratios of the expression values in the NIC-PRMT^Tg epithelium relative to those in NIC epithelium. Euclidean distance and ward clustering methods were used to construct the dendrograms against genes and samples. Note that the NIC-CARM1^Tg samples segregated from the NIC-PRMT1^Tg and NIC-PRMT6^Tg samples, which clustered with each other. (C) KEGG pathway analysis showing the overrepresented pathways among the upregulated DEGs in the mammary epithelium of NIC-PRMT1^Tg and NIC-PRMT6^Tg females (left panel). The Venn diagram illustrates the overlapping genes among the top 3 hit pathways (right panel). (D) A heatmap displaying a list of representative upregulated genes involved in the PI3K-AKT pathway (left panel). The bar graphs representing RT-qPCR validation of six representative genes in the PI3K-AKT pathway, demonstrating their up-regulated expression levels in the mammary epithelium of NIC-PRMT1^Tg and NIC-PRMT6^Tg females, but not NIC-CARM1^Tg females, as compared to NIC females (right panel). *, p<0.05; **, p<0.01 (Student’s t-test).

Fig.5. Protein analyses identifies activation of the AKT pathway in NIC-PRMT1/6^Tg tumors

(A) Immunoblotting analyses of mammary tumor samples derived from different NIC and biogenic NIC-PRMTs virgin females. A panel of proteins involved in the PI3K-AKT pathway was tested on these lysates, as labelled. ACTIN and MAT2A serve as a loading control. (B) IHC staining on mammary tumors from the indicated genotypes using the pAKT^S473-specific antibody. Nuclei were counterstained by hematoxylin. Positive signals were visualized as brown. Scale bar = 50 μm.