Epigenetic repression of E-cadherin by human papillomavirus 16 E7 protein

Abstract

A common feature shared between several human cancer-associated viruses, such as Epstein-Barr virus, Hepatitis B virus and Hepatitis C virus, and Human papillomavirus (HPV) is the ability to reduce the expression of cellular E-cadherin. Since E-cadherin is used by Langerhans cells to move through the stratified epithelium, its reduction may affect the efficiency by which the immune system responds to HPV infection and the length of persistent HPV infections. We observed that the E7 protein of this virus (HPV16) is most efficient at reducing E-cadherin levels. This E7 activity is independent of retinoblastoma protein or AP-2alpha degradation. Instead it is associated with augmentation of cellular DNA methyltransferase I (Dnmt1) activity. Significantly, inhibition of Dnmt activity re-established E-cadherin levels of the cells, presenting the possibility that similar epigenetic intervention clinically may be a way to re-establish the influx of Langerhans cells into infected epithelium to counteract HPV persistence.

Fig. 1.

Southern blot analyses of DNA extracted from various passages (p) of (a) NIKS cells that harbour wild-type HPV16 DNA and (b) NIKS cells that harbour E7null HPV16 episomes. The blots were probed with full-length HPV16 DNA. The size marker used was a 1 kb supercoiled DNA ladder.

Fig. 2.

(a) Western blot of proteins extracted from NIKS and NIKS-bearing episomal HPV16 DNA. Lysates were prepared from either confluent or subconfluent cells. The membrane was probed with antibodies against E-cadherin or actin. (b) Lysates of NIKS cell, NIKS carrying wild-type episomal HPV16 DNA and two independently derived NIKS lines harbouring E7null HPV16 episomes were analysed by western blotting using antibodies against HPV16 E7, p53, E-cadherin and actin. Two independent analyses of E-cadherin of these cells are shown. (c) Western blots of protein lysates from NIKS cells that were infected with LXSN retrovirus (vector), LXSN E6, LXSN E7 or LXSN E6/7, after selection with neomycin. Antibodies against E-cadherin, p53 and actin were used.

Fig. 3.

(a) Analyses of E-cadherin protein in NIKS and NIKS harbouring wild-type HPV16 episomes, with (+) and without (−) treatment with the proteasome inhibitor, ALLN. Actin served as loading control and p53 served to indicate that the proteasome was indeed inhibited by the compound. (b) Quantitative PCR measurements of E-cadherin transcripts in NIKS (ctrl), NIKS containing HPV16 episomes (HPV16), NIKS infected with empty retroviral vector (LXSN) or E7-contaning retroviral vector (LXSNE7). E-cadherin transcripts are normalized against beta-actin transcript levels.

Fig. 4.

(a) Western blot analyses of E-cadherin, pRb and AP-2α proteins from NIKS and NIKS + HPV16wt DNA. (b) Analyses of E-cadherin and pRb in cells infected with retrovirus-expressing shGFP or retrovirus-expressing short hairpin RNA against pRb (shpRb). Beta-actin was used as loading control. (c) Western blot analyses of Dnmt1, Dnmt3a, p53 and actin in protein extracts of NIKS (Ctrl) and NIKS harbouring episomal HPV16 DNA. (d) Analyses of Dnmt1 protein in extracts from NIKS cells infected with LXSN (vector), LXSNE6 or LXSNE7 retroviruses, after selection with neomycin. Actin serves as loading control. (e) DNA methyltransferase assay of proteins extracted from NIKS cells (Ctrl), NIKS cells harbouring HPV16 episomes, NIKS cells infected with empty retroviral vector (LXSN) or E7-bearing retroviruses (LXSNE7). Bkg is the background counts of the assay.

Fig. 5.

Treatment of NIKS and various NIKS-derived cells with inhibitor of DNA methyltransferase, 5-aza-deoxycytidine (DAC). (a) Protein lysates were prepared and analysed on a western blot with antibodies against E-cadherin and beta-actin (as loading control). (b) RNAs were extracted from treated (+) or untreated (−) cells and E-cadherin and beta-actin transcripts were extracted and their relative amounts measured using quantitative PCR and normalized against beta-actin transcripts.

Fig. 6.

Methylation status of CpGs within the E-cadherin promoter. The methylation state of each CpG within the E-cadherin promoter (position −319 to +16) was analyzed and obtained from nucleotide sequences of 17 individual clones each of bisulphite-converted DNA that were isolated from NIKS cells and NIKS cells harbouring HPV16DNA.