HPV-16 E6, hDlg and Connexin 43 in cervical carcinogenesis

Sun, Peng (2005) HPV-16 E6, hDlg and Connexin 43 in cervical carcinogenesis. PhD thesis, University of Glasgow.

Abstract

Disruption of gap junctional intercellular communication and/or Connexins (the
channel proteins of gap junctions) is frequently reported in malignant cell lines and
tumours. Many tumor cells exhibit aberrant gap junctional intercellular
communication (GJIC), which can be restored by transfection with Connexin genes.
Of the Connexin family, Connexin 43 has attracted the most attention as it is widely
expressed in many tissues and Connexin 43 gap junctions correlate with various
physiological functions. Connexin 43 is a short-lived protein (half-life of 1-3 h in
cultured cells), both lysosomes and proteasomes having been reported to be involved
in its degradation.
Certain human papillomaviruses (HPV) associated with the development of cancers,
especially of the cervix, have been reported to downregulate GJIC in vitro. There is
also evidence for reduced gap junctions in cervical dysplasia. The association
between HPV and GJIC, and the mechanism and consequence of deregulated GJIC in
cervical tumour progression, remains unclear. In HPV-16 associated cervical cancer,
the viral oncogene E6 inactivates the tumour suppressor p53, but also has p53-
independent functions in tumour progression. One of these may involve interaction
with hDlg (the human homologue of Drosophila Discs Large), a tumour suppressor
present in epithelial cells at sites of cell-cell contact and which regulates cell polarity
and attachment. hDlg contains three PDZ protein-protein interaction domains, the
second PDZ domain of which binds E6. Connexin 43 also has a PDZ binding domain in its C-terminus.
Previously, it was demonstrated that Connexin 43 relocates from the membrane to the
cytoplasm in a novel HPV-16 E6-containing cervical epithelial cell line (named
WI2GPXY) that is fully transformed and invasive and deficient in gap-junctional
intercellular communication (Aasen T et aI., 2003 Oncogene 22, 7969- 7980).
The overall aim of this project was to investigate the relationship of loss of gap
junctions to malignant progression by comparing the properties of W12GPXY with
those of the non-malignant parental cell line, WI2G, from which W12GPXY was
derived.First, microarray was used to identify global differences in RNA expression between
the two cell lines, large differences were seen in expression of angiogenesis-related
genes and they were confirmed by Real-Time RT-PCR for three genes, IL-8, VEGF
and FGF-2. No significant differences were noted for connexin genes but there were
differences in MAGUK family members including hDlg.
However, protein expression studies by western blot and immunofluorescence
staining showed a significant increase (2.9 fold) of HPV-16 E6 in W12GPXY cells,
which co-localises with hDlg in the cytoplasm. Connexin 43 also binds hDlg.
Treatment ofW12GPXY cells with Leptomycin B to trap E6 in the nucleus or siRNA
knockdown of E6 abrogate the relocation and co-location of hDlg and endogenous
wild type Connexin 43 and restore Connexin 43 gap junction at points of cell-cell
contact. Further, when C33a cells (HPV -negative cervical tumour cells which
normally retain large Connexin 43 gap junctions) are transfected with HPV -18 wild
type E6, changes in localisation of Connexin 43 and hDlg are consistent with those in
W12GPXY cells. However, C33a cells transfected with a mutant E6 lacking the hDlg binding site retain Connexin 43 gap junction plaques. Finally, Connexin 43 associates
with hDlg through its PDZ-binding domain and this is required for its relocalisation to
the cytoplasm in W12GPXY cells. The results suggest that increased cytoplasmic E6
associated with malignant progression of W12GPXY cells redistributes Connexin 43
from membrane junctions into the cytoplasm by a mechanism involving binding of
hDlg to the Connexin 43 C-tenninal tail. The findings have uncovered a new role for
hDlg in trafficking of Connexin 43. It also provides a novel mechanism for the loss
of gap junctional intercellular communication during malignant progression of
squamous epithelial cells.
The specific roles played by lysosomes and proteasomes in the degradation of
Connexin 43 in W12GPXY cells were also studied. The results suggest the
involvement of both proteolytic pathways, although the lysosome seems to be the
major compartment for Connexin 43 degradation. Association with HPV-16 E6 and
hDlg together with proteasome activity seems to be required for Connexin 43
redirection from the cell membrane and transport into the lysosomal degradation
pathway. Taken together, these results suggest that Connexin 43 gap junction
intercellular communication was lost from the cell membrane requiring maintenance
of E6 and hDlg complexes for proteasomal internalization and consequently transport
into lysosomal compartment for degradation in W12GPXY cells.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology > QR355 Virology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Supervisor's Name: Hodgins, Dr. Malcolm B., Edward, Dr. Mike and Graham, Dr. Sheila V.
• Date of Award: 2005
• Depositing User: Ms Dawn Pike
• Unique ID: glathesis:2005-5003
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 06 Mar 2014 12:59
• Last Modified: 01 Aug 2022 08:39
• URI: https://theses.gla.ac.uk/id/eprint/5003