Identification of DBCCR1 as a suppressor in the development of lung cancer that is associated with increased DNA methyltransferase 1

Abstract

Accumulating evidence has pointed to a role of the CpG island hypermethylation in the regulation of cancer-related genes in tumor progression. However, the biological impacts in cancer pathogenesis associated with down-regulation of such gene targets remains elusive. Here we focused on a potential target of hypermethylation, DBCCR1 (deleted in bladder cancer chromosome region 1), a gene encoding a candidate tumor suppressor. We found that the expression of DBCCR1 is significantly lower in the lung cancer tissues compared with adjacent non-tumor tissues of patients. Importantly, the decreased DBCCR1 was found correlated with more advanced stages of cancer, and with a significantly shorter survival of patients. Genetic silencing DBCCR1 in human lung cancer cell line A549 resulted in an enhanced proliferation, migration, and invasion capacity. Conversely, restoring DBCCR1 expression blocked the growth and inhibited the ability of cancer cell in migration and invasion. Interestingly, DBCCR1 attenuates the expression of DNMT1 (DNA methyltransferase 1), suggesting a reciprocal regulation between genetic silencing of cancer suppressor genes and activating DNA methylation. Our data thus implicates DBCCR1 downregulation as a potential module in the pathogenesis of lung cancer through DNA methylation.

Keywords: DBCCR1; DNA methyltransferase; epigenetics; lung cancer; tumor suppressor.

Figure 1. DBCCR1 expression was low in both patient tissue and lung cancer cell lines

(A) The mRNA levels of DBCCR1 were low in 12 representative lung cancer patient tissues compared with adjacent non-tumor tissues by PCR. Especially the mRNA levels of DBCCR1 decreased followed the increase of cancer stages (I, II, III and IV) (p<0.01). (B) The DBCCR1 protein levels in 4 lung cancer cell lines were normalized to the β-actin protein level and plotted. The data were mean ± SD of three independent experiments. Quantitation by densitometry was shown on below (**P<0.01, compared with normal Human bronchial epithelium cell line-BEAS-2B).

Figure 2. Kaplan-Meier survival curves of DBCCR1 expression status

Patients were divided into high and low DBCCR1 expressers according to the basis score of DBCCR1. Patients with high expression group of DBCCR1 had longer overall survival.

Figure 3. Knockdown and over-expressed of DBCCR1 effected the growth in A549 cell line

A549 cells were transfected with DBCCR1-shRNA or control-shRNA for 48 h for knockdown of DBCCR1. A549 cells were infected with Lenti-virus with DBCCR1 or Lenti-virus control for 48 h for over-expression of DBCCR1. DBCCR1 expression was detected by PCR (A) and Western blot (B). Quantitation by densitometry was shown on below (**P<0.01, compared with normal cell line). (C) The cell growth statuses were observed of DBCCR1-off, over-expressed and normal A549 cells after culture for 5 days by microscopy. The original cell number were the same of 1×10⁵ in 6-well plate. The down-expressed DBCCR1 promoted the tumor cell growth and over-expressed DBCCR1 suppressed the growth. -off, over-expressed and normal A549 cells DBCCR1-off, over-expressed and normal A549 cells. Scale bar 0.5 μm. Quantitation by counting the cell number was shown on below (**P<0.01, compared with normal cell line).

Figure 4. Change the expression of DBCCR1 effected the proliferation, migration and invasion in A549 cell line

(A) CCK-8 assay was used to detect cell viability of A549 cells treated with DBCCR1-shRNA (compared with control shRNA) and Lenti-virus with DBCCR1. A549 cells were placed on 96-well plates (5×10³ cells/well) and incubated with fresh medium. Growth curves were detected. Points and range lines at different day (1, 3, 5, 7 and 14 days) represent mean and SD of at least three independent experiments in triplicate. OD value was measured at 450 nm and data demonstrated a significant growth induction by knockdown of DBCCR1 (p<0.01). (B) Migration kit assay with DBCCR1-shRNA (compared with control shRNA) and Lenti-virus with DBCCR1 was tested. Migration of the cells to the blank area was visualized at 72 h with an inverted Leica phase-contrast microscope (9200 magnification). Quantitation was shown on below (**P<0.01, compared with normal cell line). Data demonstrated a significant migration capacity induction by knockdown of DBCCR1. (C) The relation of DBCCR1 expression and invasion capacity was tested at 72 h after culturing cells by transwell assays. DBCCR1-shRNA cells showed lower penetration rate through the membrane compared with control-shRNA and mock cells. Scale bar 0.5 μm. Quantitation was shown on below (**P<0.01, compared with normal cell line).

Figure 5. Change of DBCCR1 expression in A549 cells results in reduced counter-trend change of methylation

(A) The mRNA levels of DNMT1 were high-rich in 12 representative lung cancer patient tissues compared with adjacent non-tumor tissues by PCR (p<0.01). (B) The protein levels of DNMT1 were high-rich in A549 compared BEAS-2B by WB. Quantitation by densitometry was shown on below (**P<0.01, compared with non-tumor tissues). A549 cells were transfected with DBCCR1-shRNA or control-shRNA for 48 h for knockdown of DBCCR1. A549 cells were infected with Lenti-virus with DBCCR1 or Lenti-virus control for 48 h for over-expression of DBCCR1. DNMT1 expression was detected by PCR (C) and Western blot (D). Quantitation by densitometry was shown on below (**P<0.01, compared with normal cell line).