Faculty and Staff
|Title:||Professor of Pathology, Microbiology & Immunology
|Department:||Pathology, Microbiology & Immunology
School of Medicine
Pathology, Microbiology & Immunology
- Fogarty Visiting Fellow, National Cancer Institute, NIH, Bethesda, MD (1985-87)
- Postdoctoral fellow, Istituto di Patologia Generale and Istituto di Microbiologia,
Universita` degli Studi di Sassari, Sassari, Italy (1983-84)
- M.D. Universita` degli Studi di Sassari, Sassari, Italy (1983)
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.
- Coker AL, Bond SM, Williams A, Gerasimova T, Pirisi L.
Cancer Detect Prev. 2002; 26(2): 121-8.
Active and passive smoking, high-risk human papillomaviruses and cervical neoplasia.
Few studies have evaluated the role of passive smoke exposure and cervical neoplasia risk. We assessed the role of active and passive cigarette smoke exposure and risk of cervical squamous intraepithelial lesion (SIL) in a case-control study based in a South Carolina Health Department; 59 high-grade SIL (HSIL) cases, 313 low-grade SIL (LSIL) cases and 427 controls were recruited and interviewed. Passive cigarette smoke exposure was significantly (P < 0.05) associated with high grade SIL (adjusted odds ratio (aOR) = 2.2) and low-grade SIL (aOR = 1.4). Active smoking was associated with SIL only among White women (aOR = 1.8). High-risk human papillomaviruses (HR-HPVs) appear to interact with active cigarette smoking to increase HSIL risk. HSIL cases compared with LSIL cases were significantly more likely to be HR-HPV positive current smokers (aOR = 3.0; 95% CI: (1.2, 7.7)). These data suggest that active and perhaps passive smoke exposure may be important co-factors in HSIL development among HR-HPV positive women.
- Walters JJ, Muhammad W, Fox KF, Fox A, Xie D, Creek KE, Pirisi L.
Rapid Commun Mass Spectrom 2001;15(18):1752-9
Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry
Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.
- Akerman GS, Tolleson WH, Brown KL, Zyzak LL, Mourateva E, Engin TS, Basaraba A, Coker
AL, Creek KE, Pirisi L.
Cancer Res 2001 May 1;61(9):3837-43
Human papillomavirus type 16 E6 and E7 cooperate to increase epidermal growth factor receptor (EGFR) mRNA levels, overcoming mechanisms by which excessive EGFR signaling shortens the life span of normal human keratinocytes
Epidermal growth factor receptor (EGFR) levels are dramatically increased in human keratinocytes (HKc) immortalized with full-length human papillomavirus type 16 (HPV16) DNA (HKc/HPV16), but increases in EGFR levels actually precede immortalization. In some normal HKc strains, acute expression of HPV16 E6 (but not HPV16 E5, HPV16 E7, or HPV6 E6) from LXSN retroviral vectors produced an increase in EGFR mRNA levels detectable at 24 h and stable for up to 10 days after infection. However, about one-half of the individual normal HKc strains we analyzed proved unresponsive to E6 induction of EGFR mRNA despite the robust expression of E6 and degradation of p53. E6 responsiveness of normal HKc strains correlated inversely with initial EGFR levels: although HKc strains expressing relatively low basal EGFR levels grew poorly and tolerated the infection protocol with difficulty, they responded to E6 with an increase in EGFR mRNA and protein and with robust proliferation. However, those HKc strains expressing high basal EGFR levels grew well, but did not respond to E6 with increased EGFR levels or with proliferation. Immunostaining of paraffin-embedded foreskin tissue for the EGFR confirmed that there is an intrinsic interindividual variability of EGFR expression in HKC: These results prompted us to investigate the effects of overexpression of the EGFR in normal HKC: Infection of normal HKc with a LXSN retrovirus expressing the full-length human EGFR cDNA resulted in a dramatic reduction in growth rate and a shorter life span. Although acute expression (1-10 days after infection) of HPV16 E7 alone did not induce the EGFR, acute expression of E6 and E7 together increased EGFR levels in normal HKc unresponsive to E6 alone. Also, HKc infected with E7 alone expressed increased EGFR levels at early stages of extended life span (at passage 9 after infection), and HKc immortalized by HPV16 E7 alone expressed EGFR levels comparable with those of E6/E7-immortalized cells. These results support a key role of the EGFR in HPV16-mediated transformation of HKC: In addition, these data show that normal HKc do not tolerate excessive EGFR levels/signaling, and such intolerance must be overcome in order for HKc to become immortalized by HPV16. We conclude that both E6 and E7 contribute to increasing EGFR levels, but with different mechanisms: although E6 can increase EGFR levels, it cannot overcome the resistance of normal HKc to excessive EGFR signaling. On the other hand E7, which alone does not acutely increase EGFR mRNA or protein, allows for EGFR overexpression in normal HKC.