Most of my research (conducted in close collaboration with
Dr. Kim E. Creek) centers on cervical cancer, from the mechanisms
that determine immortalization and progression to malignancy in
HPV16-transformed human cells, to the epidemiology of HPV infection
and cervical neoplasia in South Carolina women. A parallel project
in the laboratory investigates the biology of ErbB2-positive breast
cancer cells and their responses to Herceptin.
Role of the EGF receptor in HPV16-mediated human cell carcinogenesis:
The transforming ability of oncogenic HPVs resides primarily
in the oncoproteins E6 and E7. These proteins have a variety of
activities and interact with many cellular proteins, however,
the best described activities of E6 and E7 are also required for
transformation in most cell systems: E6 degrades p53, and E7 binds
and inactivates RB. Thus, the combined actions of these two oncoproteins
promote genomic instability and transformation. We determined
that the epidermal growth factor receptor (EGFR) plays a key role
in the mechanisms by which HPV16 oncoproteins transform human
epithelial cells. EGFR levels increase dramatically in human keratinocytes
early after transfection with HPV16 DNA, and increase again (up
to 10-fold) in growth factor independent HPV16-transformed cells.
We determined that the early increase in EGFR levels is linked
to the expression of the oncoprotein E6, which can induce EGFR
mRNA in normal human keratinocytes. We also determined that normal
human keratinocytes tolerate only relatively modest increases
in EGFR signaling, and that E6 alone cannot overcome the mechanisms
that prevent marked overexpression of the EGFR in these cells.
E7 appears to abolish the senescence response to EGFR overexpression,
and allow for cells overexpressing the EGFR to proliferate. We
are investigating the molecular mechanisms by which E6 induces
the EGFR and the contribution of E7 to this effect, using site-directed
mutants of E6 and E7, and also RNA interference-based approaches.
Gene expression profiling of HPV-mediated transformation:
We are using DNA microarrays to explore the gene expression profiles
associated with HPV16-mediated transformation, with a particular
emphasis on differential gene expression between low-passage cells
– which are sensitive to TGF-beta and require exogenous
growth factor to proliferate in culture – and differentiation-resistant
cells –which are not growth inhibited by TGF-beta and are
growth factor-independent. We aim at extending the same analysis
to cervical specimens, in order to identify potential biomarkers
of progression that may be useful to identify women at the highest
risk for cancer, among the many who present with abnormal Pap
smears.
Epidemiology of HPV infection and cervical cancer in South Carolina:
Ann L. Coker (formerly at the Department of Epidemiology and Biostatistics
of the University of South Carolina School of Public Health) and
I have been following over time a population of low-income, primarily
minority women for progression to high grade squamous intraepithelial
lesions, in the attempt to learn more about the factors (in addition
to persistent HPV infection, which we confirmed is a major determinant)
that contribute to progression of HPV-mediated lesions to malignancy.
These factors include life-style factors, smoking, contraceptive
methods, and the adeno-associated virus. The latter appears to
protect against progression in women infected with oncogenic HPV.
More recently, in collaboration with Dr. Kathy Luchok, also of
the USC School of Public Health we have investigated the role
of stress in facilitating persistence of HPV infection. We are
now initiating a new study of the determinants of HPV persistence
in Caucasian and African-American female college students, aimed
at identifying the immunological, HPV-associated, and life-style
factors that contribute to persistent HPV infection, and how these
may vary in different populations. Along the same lines, in collaboration
with Subbi and Rajesh Mathur, at the Medical University of South
Carolina, we are conducting a study of promising biomarkers of
progression in tissue and serum samples from patients with cervical
neoplasia.
Variant TGF-alpha precursors produced by alternative splicing
differentially interact with ErbBs and modify Herceptin responses
in ErbB2-positive breast cancer cells.
We discovered two variant forms of transforming growth factor-alpha
precursor (proTGF-alpha), produced by alternative splicing of
the proTGF-alpha mRNA. The variants are widely expressed in normal
human keratinocytes as well as in cell lines derived from human
tumors of epithelial origin, and some cancer cell lines that have
lost expression of wild type (wt) proTGF-alpha still express the
variant forms. These novel proTGF-alpha variants differ from wt
proTGF-alpha only at the C-terminus, where two valine residues
are replaced by other non- branched chain amino acids. The TVV
motif at the C-terminus of wt proTGF-alpha is recognized by PDZ
domain proteins and is believed to be necessary for correct trafficking
and maturation of proTGF-alpha. Therefore, replacement of the
valine residues with other amino acids is bound to profoundly
affect interactions of proTGF-alpha with intracellular proteins.
We went on to discover that the C-termini of wt and variant proTGF-alpha
precursors mediate specific interactions with members of the ErbB
family of receptors: while wt proTGF-alpha interacts with ErbB4,
the two variants interact with ErbB2. These interactions lead
to activation of ErbB2 in the absence of serum, in CHO cells expressing
wt or variant proTGF-alpha. We recently determined that exogenous
variant proTGF-alpha precursors decrease sensitivity of breast
cancer cells to the growth inhibitory effects of Herceptin, a
selective antibody directed against ErbB2 commonly used in the
treatment of ErbB2-positive breast cancer. |
