HP1γ Promotes Lung Adenocarcinoma by Downregulating the Transcription-Repressive Regulators NCOR2 and ZBTB7A

Abstract

Lung adenocarcinoma is a major form of lung cancer, which is the leading cause of cancer death. Histone methylation reader proteins mediate the effect of histone methylation, a hallmark of epigenetic and transcriptional regulation of gene expression. However, their roles in lung adenocarcinoma are poorly understood. Here, our bioinformatic screening and analysis in search of a lung adenocarcinoma-promoting histone methylation reader protein show that heterochromatin protein 1γ (HP1γ; also called CBX3) is among the most frequently overexpressed and amplified histone reader proteins in human lung adenocarcinoma, and that high HP1γ mRNA levels are associated with poor prognosis in patients with lung adenocarcinoma. In vivo depletion of HP1γ reduced K-Ras^G12D-driven lung adenocarcinoma and lengthened survival of mice bearing K-Ras^G12D-induced lung adenocarcinoma. HP1γ and its binding activity to methylated histone H3 lysine 9 were required for the proliferation, colony formation, and migration of lung adenocarcinoma cells. HP1γ directly repressed expression of the transcription-repressive regulators NCOR2 and ZBTB7A. Knockdown of NCOR2 or ZBTB7A significantly restored defects in proliferation, colony formation, and migration in HP1γ-depleted lung adenocarcinoma cells. Low NCOR2 or ZBTB7A mRNA levels were associated with poor prognosis in patients with lung adenocarcinoma and correlated with high HP1γ mRNA levels in lung adenocarcinoma samples. NCOR2 and ZBTB7A downregulated expression of tumor-promoting factors such as ELK1 and AXL, respectively. These findings highlight the importance of HP1γ and its reader activity in lung adenocarcinoma tumorigenesis and reveal a unique lung adenocarcinoma-promoting mechanism in which HP1γ downregulates NCOR2 and ZBTB7A to enhance expression of protumorigenic genes.Significance: Direct epigenetic repression of the transcription-repressive regulators NCOR2 and ZBTB7A by the histone reader protein HP1γ leads to activation of protumorigenic genes in lung adenocarcinoma. Cancer Res; 78(14); 3834-48. ©2018 AACR.

Figure 1:. HP1γ levels are upregulated in lung adenocarcinoma and high HP1γ levels correlate with shorter survival in LUAD patients.

(A) An in silico screening strategy for the search of putative oncogenic histone methylation reader proteins. LUAD, Lung adenocarcinoma. (B) Bar graph showing alterations in the 58 histone methylation reader proteins in LUAD samples in TCGA database. The cutoff value for alterations was 8%. (C) Fourteen histone methylation reader proteins with >1.5-fold upregulation in LUAD tumors (n = 357) compared with adjacent normal tissue samples (n = 54) in TCGA dataset. P-value for the 14 reader proteins, <0.05. T, tumor; N, normal. (D) Kaplan-Meier survival rate analysis on the basis of HP1γ mRNA levels (probe set, 200037_s_at) using NCI LUAD dataset. Tumor samples (n = 443) were divided into HP1γ-high or HP1γ-low mRNA groups by using a cutoff value of the mean plus 2 standard deviations. (E) Representative images of IHC staining of HP1γ in normal lung and lung tumors. Scale bars, 50 μm. (F) Survival analysis of lung cancer patients on the basis of HP1γ protein levels (see also Supplementary Table S3 and S4). HP1γ protein levels in lung cancer samples in TMAs were determined by IHC staining (n = 73).

Figure 2:. HP1γ is required for in vivo growth of K-Ras^G12D-driven LUAD tumors.

(A) A scheme for monitoring the effect of in vivo knockdown of HP1γ on K-Ras^G12D-driven lung tumorigenesis. The lungs of 8-week-old mice were infected by an intratracheal intubation of shmHP1γ−1-Cre lentiviruses, which express both Cre recombinase and shRNA targeting mouse HP1γ. Lentiviruses with an empty vector expressing only Cre were used as a control. (B) Micro-CT-scan images of mouse lungs infected with shmHP1γ−1-Cre (n = 3) and control (n = 3) viruses at 3 months and 9 months post-infection. (C‒H) The effect of in vivo HP1γ knockdown on K-Ras^G12D-driven lung tumorigenesis. Lung tumors in mice infected with control viruses were compared with those in mice infected with shmHP1γ−1-Cre viruses. Representative images of massive lung tumors in the control group and of tiny lung tumors in the shmHP1γ−1-Cre group are shown; tumors are encircled by dotted green lines (C). Representative images of H&E-stained lung lobes effaced by lung tumors in the control and shmHP1γ−1-Cre groups are shown (D). The percentages of tumor area per lobe in control (n = 10) and shmHP1γ−1-Cre (n = 10) groups were quantified (E). Tumor grades, based on tumor size, in control (n = 4) and shmHP1γ−1-Cre (n = 4) groups were scored (F). IHC staining data for HP1γ and the cell proliferation marker Ki-67 in lung tumors from control and shmHP1γ−1-Cre groups are shown (G). Ki-67-positive cells in eight random fields of three different tumors of control and shmHP1γ−1-Cre groups were quantified (H). (I) Kaplan-Meier survival analysis of the control group of mice (n=17) and the shmHP1γ−1-Cre group of mice (n=14). Scale bars, 7 mm (D) and 100 μm (G).

Figure 3:. HP1γ and its binding activity are essential for the proliferation, colony formation and migration of LUAD cells.

(A and B) Analysis of mRNA levels of ectopically expressed HP1γ and mtHP1γ and endogenous HP1γ by quantitative RT-PCR (A) and Western blot analysis of HP1γ, mtHP1γ, H3K9me3, and H3 levels (B). In HP1γ-depleted H1792 cells, HP1γ and mtHP1γ were ectopically expressed using a lentivirus system. (C) The effect of ectopic expression of HP1γ and mtHP1γ on the proliferation of HP1γ-depleted H1792 cells. (D) The effect of ectopic expression of HP1γ and mtHP1γ on the colony formation ability of HP1γ-depleted H1792 cells in a clonogenic cell survival assay. (E and F) The effect of ectopic expression of HP1γ and mtHP1γ on the migration (E) and anchorage-independent colony growth (F) of HP1γ-depleted H1792 cells. Black scale bars: 200 μm; white scale bars, 400 μm.

Figure 4:. HP1γ directly represses NCOR2 and ZBTB7A expression while upregulating expression of oncogenes, such as AXL, PVT1, and ELK1.

(A) Venn diagrams and heat maps for genes upregulated (>1.5-fold) or downregulated (<0.5-fold) by HP1γ knockdown. RNA was isolated from HP1γ-depleted H1792 cells (shHP1γ−16 and shHP1γ−17) and control (shLuc) H1792 cells. The mRNA levels were assessed by Affymetrix Human Genome U133 plus 2.0 Array in duplicate. (B) Ontology analysis of genes upregulated or downregulated by HP1γ knockdown. The functional annotation tool DAVID was used. (C) Analysis of the effect of HP1γ knockdown on EHF, NCOR2, ZBTB7A, AXL, PVT1, ARF1 and ELK1 mRNA levels using quantitative RT-PCR. (D) Western blot analysis of NCOR2, ZBTB7A and EHF protein levels in HP1γ-depleted (shHP1γ−16 and shHP1γ−17) H1792 cells. (E‒G) Chromatin levels of HP1γ at the NCOR2 (E), ZBTB7A (F), and EHF (G) genes. Schematic representations of individual genes are shown (top panels). Quantitative ChIP assay was performed using shLuc-infected cells (control) and HP1γ-depleted cells. Arrows indicate the primer sites for PCR amplification of ChIP-enriched DNAs. TSS, transcription start site.

Figure 5:. Knockdown of NCOR2 or ZBTB7A substantially rescues the proliferation, colony formation, and migration of HP1γ-depleted LUAD cells.

(A and B) Analysis of relative HP1γ, NCOR2, ZBTB7A, and EHF mRNA (A) and protein (B) levels in H1792 cells that were treated with the following five groups of shRNA-containing viruses: 1) shLuc, 2) shHP1γ−16, 3) shHP1γ−16 and shNCOR2–1, 4) shHP1γ−16 and shZBTB7A-1, and 5) shHP1γ−16 and shEHF-1. (C‒E) The effect of NCOR2 or ZBTB7A knockdown on the colony formation (C), proliferation (D), and migration (E) of HP1γ-depleted H1792 cells. shLuc-infected cells were used as controls. Scale bars, 200 μm.

Figure 6:. Low NCOR2 or ZBTB7A levels are linked to enhanced cell proliferation and migration, high HP1γ levels in LUAD tumors, and poor prognosis in LUAD patients.

(A) IHC levels of NCOR2 and ZBTB7A in K-Ras^G12D lung tumors and mHP1γ-depleted K-Ras^G12D tumors. (B and C) Scatter plots showing an inverse correlation between HP1γ mRNA levels and either NCOR2 (B) or ZBTB7A (C) mRNA levels in TCGA LUAD sample dataset. (D and E) The effect of NCOR2 knockdown on colony formation (D) and migration (E) of H1792 cells. (F and G) The effect of ZBTB7A knockdown on colony formation (F) and migration (G) of H1792 cells. (H) Box plots showing downregulation of NCOR2 (left panel) and ZBTB7A (right panel) mRNA levels in LUAD tumor samples (n = 357) compared with their adjacent normal samples (n = 54) in TCGA dataset. T, tumor; N, normal. (I and J) The Kaplan-Meier survival analysis showing the correlation of low NCOR2 (I) and ZBTB7A (J) mRNA levels with shorter survival in LUAD patients. The auto cutoff was used to divide samples into low and high groups in the KM Plotter database (http://kmplot.com/analysis). NCOR2 cutoff, 2870 in the range between 578 and 10323; ZBTB7A cutoff, 788 in the range between 259 and 20494; NCOR2 probe set, 207760_s_at; ZBTB7A probe set, 226554_at; black scale bars, 100 μm (A) and 200 μm (E and G).

Figure 7:. HP1γ indirectly upregulates expression of the oncogenes AXL, PVT1, and ELK1 by downregulating NCOR2 and ZBTB7A expression.

(A‒C) Kaplan-Meier survival analysis of AXL (A), PVT1 (B), and ELK1 (C) levels in LUAD datasets. The auto cutoff was used to divide samples into low and high groups in the KM Plotter database (http://kmplot.com/analysis). AXL cutoff, 136 in the range between 5 and 677; PVT1 cutoff, 454 in the range between 12 and 15881; ELK1 cutoff, 13 in the range between 2 and 524; AXL probe set, 202685_s_at; PVT1 probe set, 1558290_a_at; ELK1, 220802_at. (D‒F) The effect of NCOR2 (D), ZBTB7A (E), or EHF (F) knockdown on AXL, PVT1, ELK1 and ARF1 mRNA levels in H1792 cells. (G‒I) Chromatin levels of ZBTB7A at the AXL (G) and PVT1 (H) genes and of NCOR2 at the ELK1 gene (I). Schematic representations of individual genes are shown (top panels). Arrows indicate the primer sites for PCR amplification of ChIP-enriched DNAs. TSS, transcription start site. (J‒L) The effect of NCOR2 or ZBTB7A knockdown on AXL (J), PVT1 (K), and ELK1 (L) mRNA levels in HP1γ-depleted H1792 cells. H1792 cells were treated with the following four groups of shRNA-containing viruses: 1) shLuc, 2) shHP1γ−16, 3) shHP1γ−16 and shNCOR2–1, and 4) shHP1γ−16 and shZBTB7A-1. shLuc-treated cells were used as controls. For analysis of mRNA levels in J‒L, quantitative RT-PCR was used. (M) A hypothetical model for the molecular mechanism underlying the tumor-promoting function of HP1γ in LUAD. HP1γ interacts with H3K9me3 at the NCOR2 and ZBTB7A promoters and represses NCOR2 and ZBTB7A expression. NCOR2 downregulates ELK1 expression, and ZBTB7A represses expression of AXL and PVT1. HP1γ-mediated repression of NCOR2 and ZBTB7A increases expression levels of tumor-promoting factors (e.g., AXL, PVT1, and ELK1) to enhance the proliferation, migration, and tumorigenic growth of LUAD cells.