Functional interplay between YY1 and CARM1 promotes oral carcinogenesis

Abstract

Coactivator associated arginine methyltransferase 1 (CARM1) has been functionally implicated in maintenance of pluripotency, cellular differentiation and tumorigenesis; where it plays regulatory roles by virtue of its ability to coactivate transcription as well as to modulate protein function as an arginine methyltransferase. Previous studies establish an oncogenic function of CARM1 in the context of colorectal and breast cancer, which correlate to its overexpressed condition. However, the mechanism behind its deregulated expression in the context of cancer has not been addressed before. In the present study we uncover an oncogenic function of CARM1 in the context of oral cancer, where it was found to be overexpressed. We also identify YY1 to be a positive regulator of CARM1 gene promoter, where silencing of YY1 in oral cancer cell line could lead to reduction in expression of CARM1. In this context, YY1 showed concomitant overexpression in oral cancer patient samples compared to adjacent normal tissue. Cell line based experiments as well as xenograft study revealed pro-neoplastic functions of YY1 in oral cancer. Transcriptomics analysis as well as qRT-PCR validation clearly indicated pro-proliferative, pro-angiogenic and pro-metastatic role of YY1 in oral cancer. We also show that YY1 is a substrate of CARM1 mediated arginine methylation, where the latter could coactivate YY1 mediated reporter gene activation in vivo. Taken together, CARM1 and YY1 were found to regulate each other in a positive feedback loop to facilitate oral cancer progression.

Keywords: CARM1; YY1; arginine methylation; oncogene; oral cancer.

Figure 1. CARM1 and YY1 are overexpressed in oral cancer patient tumor tissue.

(A) Representative immunohistochemistry images of paired oral cancer patient samples stained with CARM1 antibody. The numbers on images represent patient ID (e.g., 63, 65, 215 and 626). Image scale: 500 μm. (B) H-scoring for CARM1 staining in oral cancer patient tumor tissue compared to adjacent normal tissue (n = 26; Student’s t-test: ^****p < 0.0001). (C) Snapshot of Genome browser from ENCODE to show enrichment of different transcription factors on CARM1 promoter. Highlighted are E2F4, CTCF, YY1 and c-Myc. (D) Luciferase assay with transfection of increasing amounts of pcDNA3-HA-YY1 (e.g., 100ng, 200ng, 400ng, 600ng) in HEK293T cells in a dose dependent manner (n = 2). (E) Representative immunohistochemistry images of paired oral cancer patient samples stained with YY1 antibody. The numbers on images represent patient ID (e.g., 65, 268, 66911 and 74805). Image scale: 500 μm. (F) H-scoring for YY1 staining in oral cancer patient tumor tissue compared to adjacent normal tissue (n = 27 and ^***p < 0.001). (G) Correlation analysis of H-scores of CARM1 and YY1 immunohistochemistry in oral tumor tissues (n = 23, p = 0.01, r = 0.52, r² = 0.27).

Figure 2. YY1 regulates CARM1 expression.

(A) qRT-PCR to assess RNA expression of YY1 in AW8507_Tet-ON-shYY1 cells with Doxycycline treatment (n = 3, ^***p < 0.001). (B) Immunoblotting to analyze protein expression of YY1 and CARM1 in AW8507_Tet-ON-shYY1 cells with Doxycycline treatment (FC: Fold change). Data is representative of three independent experiments. (C) qRT-PCR to assess RNA expression of CARM1 in AW8507_Tet-ON-shYY1 cells with inducible silencing of YY1 (n = 3, ^*p < 0.05). (D) The sequence of the probe taken from CARM1 promoter for EMSA. Putative YY1 binding sites have been highlighted. (E) EMSA with recombinant full length His-YY1 and radiolabelled oligonucleotide taken from CARM1 promoter. (F) ChIP to assess recruitment of YY1 on CARM1 promoter in AW8507_Tet-ON-shYY1 cells (n = 3, ^**p < 0.01, Dox: Doxycycline).

Figure 3. Elucidation of oncogenic role of YY1 in oral cancer cell line.

(A) MTT assay with AW8507_Tet-ON-shYY1 cells with inducible silencing of YY1 expression (n = 3, ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001, D1-D5:Day1-Day5). (B) In vitro wound healing assay with AW8507_Tet-ON-shYY1 cells with inducible silencing of YY1 expression. Image scale: 50 μm. Data is representative of three independent experiments. (C) Clonogenic assay with AW8507_Tet-ON-shYY1 cells with inducible silencing of YY1 expression. (D) Quantitation of colony number (n = 3, ^**p < 0.01).

Figure 4. Elucidation of oncogenic role of CARM1 in oral cancer cell line.

(A) qRT-PCR to assess RNA expression of CARM1 in AW8507_Tet-ON-shCARM1 cells with Doxycycline treatment (n = 3, ^***p < 0.001). (B) Immunoblotting to assess protein expression of CARM1 in AW8507_Tet-ON-shCARM1 cells with Doxycycline treatment. Data is representative of three independent experiments. (C) Immunoblotting to assess levels of H3R17me2a upon inducible silencing of CARM1 expression in AW8507_Tet-ON-shCARM1 cells. Data is representative of three independent experiments. (D) MTT assay with AW8507_Tet-ON-shCARM1 cells with inducible silencing of CARM1 expression (D1-D5:Day1-Day5). (E) In vitro wound healing assay with AW8507_Tet-ON-shCARM1 cells with inducible knock-down of CARM1 expression. Image scale: 100 μm. Data is representative of three independent experiments. (F) Clonogenic assay with AW8507_Tet-ON-shCARM1 cells in presence of Doxyxycline. (G) Quantitation of colony number (n = 3, ^*p < 0.05).

Figure 5. YY1 regulates multiple oncogenic pathways in oral cancer cells.

(A) Array correlation heat map to show differential gene expression with a fold change value of 1.5 above the median expression value with hierarchical clustering from microarray performed in AW8507_Tet-ON-shYY1 cells. (B) A molecular network with integration of multiple pathways which were modulated upon inducible silencing of YY1. Green indicates downregulation, Red indicates upregulation. (C) qRT-PCR experiment to assess the expression of various genes related to cancer upon inducible silencing of YY1 in AW8507_Tet-ON-shYY1 cells (n = 3). Genes tested were PGF, VEGFC, VIM, TWIST1, MMP7 and BCL3.

Figure 6. Xenograft study in nude mice with inducible silencing of YY1 and CARM1 in oral cancer cells.

(A) Xenograft study with AW8507_Tet-ON-shYY1 cells (n = 5, ^**p < 0.01). (B) Images of dissected tumors of AW8507_Tet-ON-shYY1 cells after 7 weeks of growth. (C) Xenograft study with AW8507_Tet-ON-shCARM1 cells (n = 5, p = 0.0675). (D) Images of dissected tumors of AW8507_Tet-ON-shCARM1 cells after 7 weeks of growth.

Figure 7. CARM1 methylates YY1.

(A) In vitro methylation assay with recombinant full length His-YY1 and FLAG-CARM1 in presence of tritiated SAM. (B) Immuno-pull down assay to assess in vivo interaction between YY1 and CARM1 (IP: Immunopulldown and IB: Immunoblotting). (C) Reporter luciferase assay to assess coactivation potential of CARM1 in the context of YY1 driven gene expression (n = 2, ^*p < 0.05). (D) In vitro methylation assay with recombinant His-YY1-DBD and FLAG-CARM1 in presence of tritiated SAM. (E) In vitro methylation assay with His-YY1-DBD and FLAG-CARM1 in presence of tritiated SAM and PRMT inhibitor V (25, 50 and 100 μM). (F) EMSA to determine effect of arginine methylation on DNA binding ability of YY1-DBD (15 minutes methylation assay + 15 minutes DNA binding assay).

Figure 8. A schematic to explain probable mechanisms of cooperative action between YY1 and CARM1 either to suppress or promote carcinogenesis by upregulating either YY1 responsive tumor suppressor genes (such as BRCA1, p53 and p73) or YY1 responsive oncogenes (such as c-Myc, c-Fos and Erb B2) respectively in a context dependent manner.