Re-expression of CXCL14, a common target for epigenetic silencing in lung cancer, induces tumor necrosis - PubMed (original) (raw)

Re-expression of CXCL14, a common target for epigenetic silencing in lung cancer, induces tumor necrosis

M Tessema et al. Oncogene. 2010.

Abstract

Chemokines are important regulators of directional cell migration and tumor metastasis. A genome-wide transcriptome array designed to uncover novel genes silenced by methylation in lung cancer identified the CXC-subfamily of chemokines. Expression of 11 of the 16 known human CXC-chemokines was increased in lung adenocarcinoma cell lines after treatment with 5-aza-2'-deoxycytidine (DAC). Tumor-specific methylation leading to silencing of CXCL5, 12 and 14 was found in over 75% of primary lung adenocarcinomas and DAC treatment restored the expression of each of the silenced gene. Forced expression of CXCL14 in H23 cells, where this gene is silenced by methylation, increased cell death in vitro and dramatically reduced the in vivo growth of lung tumor xenografts through necrosis of up to 90% of the tumor mass. CXCL14 re-expression had a profound effect on the genome altering the transcription of over 1000 genes, including increased expression of 30 cell-cycle inhibitor and pro-apoptosis genes. In addition, CXCL14 methylation in sputum from asymptomatic early-stage lung cancer cases was associated with a 2.9-fold elevated risk for this disease compared with controls, substantiating its potential as a biomarker for early detection of lung cancer. Together, these findings identify CXCL14 as an important tumor suppressor gene epigenetically silenced during lung carcinogenesis.

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Figures

Figure 1

Transcription of the CXC-chemokines in lung cancer is regulated by methylation. a) Genes that are most likely regulated by promoter hypermethylation are expected in the top tier group (2-fold or higher change with DAC treatment (Y-axis > 1.4) and little or no change with TSA treatment (X-axis = -0.5 to 0.5). Treatment of H23 cells with DAC led to an 18.2, 2.4, and 2.2-fold increase in expression of CXCL5, CXCL11, and CXCL14 respectively on Agilent 44K expression array. In contrast, H23 cells treated with trichostatin A (TSA) showed little or no change in the expression of these genes suggesting DNA methylation is the primary regulator of the transcription of these genes in H23. Primary lung adenocarcinomas from current, former and never smokers showed tumor specific and highly prevalent methylation in the promoter CpG island of CXCL5 (b), CXCL12 (c), and CXCL14 (d).

Figure 2

DAC treatment restores expression of genes silenced by methylation. Expression of CXCL5 (a), CXCL12 (b), and CXCL14 (c) was silenced in untreated control (S) lung cancer cell lines with methylated promoter CpG islands and could be restored primarily with DAC (D) treatment. In some cell lines (CXCL5 in H1993 and CXCL14 in Calu-6) gene expression could also be restored by TSA (T) treatment. NHBEC and lung cancer cell lines with unmethylated promoter CpG islands, readily expressed these chemokines and their expression was not affected by treatment with either drug. CXCL14 expression in H2023 and H1568 cell lines was silenced in the absence of methylation and expression was primarily restored by TSA treatment. d) Dense methylation that corresponds with gene silencing was found in the promoter CpG island of CXCL14. Modified DNA was amplified using primers that do not discriminate between methylated and unmethylated alleles (the same used for COBRA), cloned, and five clones per sample were sequenced. The shaded area of each circle indicates the extent of methylation at that CpG and one clone represents 1/5th of the circle. CpGs 10-12 and 20-23 (placed in boxes) indicate the primer binding sites for the MSP assay and the CpGs recognized by BstU1 restriction enzyme used for COBRA are indicated by asterisks. Nearly all CpGs of samples that were unmethylated by COBRA and MSP assays (NHBEC 255, and 256, H1435, and primary lung adenocarcinoma sample #18) were free of methylation. Only 0-8% of the 260 CpGs (52 CpGs per clone and 5 clones per sample) were methylated in these samples. In contrast, 58-100% of the CpGs were methylated in samples that were strongly methylated by COBRA and MSP (H23, Calu-6 and primary tumor #54, #66, and #100). Primary adenocarcinoma #35 that was weakly methylated by the two assays was methylated for 33% of the CpGs.

Figure 3

Transient re-expression of CXCL14 induces cell death. Cell survival was evaluated by flow cytometry using a GFP containing expression vector. The normal level of cell death which occurs in 4-5% of H23 cells (a) was increased to 15-20% due to exposure to the transfection reagent alone (data not shown). GFP expression in the transfected cells increased cell death by 20-25% which was only marginally greater than seen with the transfection media (b). In contrast, expression of CXCL14-GFP led to a 40-45% cell death (c). This indicates that re-expression of CXCL14 in H23 cells where the endogenous expression is silenced by methylation increased cell death by 19.3 % compared to the GFP control (d).

Figure 4

Stable re-expression of CXCL14 significantly reduced tumor growth and induced necrosis of H23 xenografts in nude mice. a) Both the parental and CXCL14 expressing H23 cells formed detectable tumors within two wks post inoculation and showed comparable rates of tumor growth in the first four wks. After wk four, tumors from the parental H23 kept growing while those from CXCL14 expressing cells barely increased in size. The asterisks at wk 6, 9, and 10 indicate significant differences in tumor size. b) The size differences between the tumors in the two groups was obvious under the skin when the mice were sacrificed ten wks post-injection. c) Similarly, the size and weight of the tumors harvested from the CXCL14 expressing cells were significantly smaller than tumors from the parental H23 cells. d) Histological examination of H&E stained slides revealed tumors from the CXCL14 expressing cells contain large necrotic foci that involved up to 90% of the tumor mass as compared to tumor necrosis in the range of 20-30% of the tumor mass from the parental H23 cells.

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Re-expression of CXCL14, a common target for epigenetic silencing in lung cancer, induces tumor necrosis - PubMed (original) (raw)