John H. Lee

Age, sexual behavior and human papillomavirus infection in oral cavity and oropharyngeal cancers.

There are few well‐established patient risk factors associated with human papillomavirus (HPV) infection in cancers of the oral cavity and oropharynx. The purpose of this study was to determine if there were significant different risk factors and tumor characteristics between HPV‐positive and HPV‐negative cancer cases. HPV was evaluated in cancer tissue and exfoliated oral cells of 193 oral cavity/oropharynx cancer patients using PCR and direct DNA sequencing. A patient questionnaire collected information about risk factors, sexual practices and medical history. The prevalence of HPV high‐risk (HR) types was 20% in cancer cases. Three types were identified: HPV‐16 (87%), HPV‐18 (3%) and HPV‐33 (11%). Risk factors for HPV‐HR included younger age (≤ 55 years vs. > 55 years; adjusted OR = 3.4; 95% CI = 1.6–7.3) and younger‐age cases who had more lifetime sex partners (adjusted OR = 3.8; 95% CI = 1.4–10.1), practiced oral‐genital sex (adjusted OR = 4.3; 95% CI = 1.8–10.4) or oral‐anal sex (adjusted OR = 19.5; 95% CI = 3.4–113). Compared to HPV‐negative cancers, HPV‐HR cancers were more likely to have a positive HPV‐HR exfoliated oral cytology test (adjusted OR = 7.8; 95% CI = 3.4–18.4), later stage (adjusted OR = 3.0), nodal involvement (adjusted OR = 4.1) and advanced grade (adjusted OR = 3.0). This study shows new evidence that the prevalence of oncogenic mucosal HPV is higher in younger‐age oral cavity/oropharynx cancer cases whose sexual practices are typically associated with sexual transmission of the virus. HPV detection also appears to be an indicator of advanced disease characteristics that may require different clinical treatment for this subset of patients. An exfoliated oral cytology test for HPV was a significant predictor of HR types in the cancers, suggesting that an oral rinse may provide an early biomarker of infected tumors.

National Cancer Data Base Report on Malignant Paragangliomas of the Head and Neck

Malignant paragangliomas of the head and neck are rare, with previous reports limited to nine or fewer patients. The current review included 59 cases extracted from the National Cancer Data Base that were diagnosed between 1985–1996.

Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo.

Adenovirus (Ad)-mediated gene transfer to airway epithelia is inefficient because the apical membrane lacks the receptor activity to bind adenovirus fiber protein. Calcium phosphate (CaPi) precipitates have been used to deliver plasmid DNA to cultured cell lines. However, such precipitates are not effective in many primary cultures or in vivo. Here we show that incorporating recombinant adenovirus into a CaPi coprecipitate markedly enhances transgene expression in cells that are resistant to adenovirus infection. Enhancement requires that the virus be contained in the precipitate and viral proteins are required to increase expression. Ad: CaPi coprecipitates increase gene transfer by increasing fiber-independent binding of virus to cells. With differentiated cystic fibrosis (CF) airway epithelia in vitro, a 20-min application of Ad:CaPi coprecipitates that encode CF transmembrane conductance regulator produced as much CF transmembrane conductance regulator Cl- current as a 24-h application of adenovirus alone. We found that Ad:CaPi coprecipitates also increased transgene expression in mouse lung in vivo; importantly, expression was particularly prominent in airway epithelia. These results suggest a new mechanism for gene transfer that may be applicable to a number of different gene transfer applications and could be of value in gene transfer to CF airway epithelia in vivo.

Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer

BACKGROUND Human papillomavirus (HPV) is the most identifiable cause of head and neck squamous cell cancer (HNSCC). Compared with HPV-negative HNSCC, HPV-positive HNSCC presents at an advanced stage but with significantly better survival. We created a syngeneic mouse model of HPV-positive and HPV-negative HNSCC by transforming mouse primary tonsil epithelial cells with either HPV oncogenes or a nonantigenic RNA interference strategy that affects similar oncogenic pathways. OBJECTIVES To examine the effect of radiation therapy on HPV-positive and HPV-negative tumors in immune-competent and immune-incompetent mice and to examine responses in human cancer cell lines. DESIGN Prospective in vivo murine model. MAIN OUTCOME MEASURES Survival and tumor growth. RESULTS For human and murine transformed cell lines, HPV-positive cells were more resistant to radiation and cisplatin therapy compared with HPV-negative cells. In vivo, HPV-positive tumors were more sensitive to radiation, with complete clearance at 20 Gy, compared with their HPV-negative counterparts, which showed persistent growth. Cisplatin in vivo cleared HPV-positive tumors but not HPV-negative tumors. However, neither radiation or cisplatin therapy cured immune-incompetent mice. Adoptive transfer of wild-type immune cells into immune-incompetent mice restored HPV-positive tumor clearance with cisplatin therapy. CONCLUSIONS The HPV-positive tumors are not more curable based on increased epithelial sensitivity to cisplatin or radiation therapy. Instead, radiation and cisplatin induce an immune response to this antigenic cancer. The implications of these results may lead to novel therapies that enhance tumor eradication for HPV-positive cancers.

Human Papillomavirus Type 16 E6 Activates NF-κB, Induces cIAP-2 Expression, and Protects against Apoptosis in a PDZ Binding Motif-Dependent Manner

ABSTRACT Infection with human papillomavirus (HPV) is a critical factor in the pathogenesis of most cervical cancers and some aerodigestive cancers. The HPV E6 oncoprotein from high-risk HPV types contributes to the immortalization and transformation of cells by multiple mechanisms, including degradation of p53, transcriptional activation of human telomerase reverse transcriptase (hTERT), and degradation of several proteins containing PDZ domains. The ability of E6 to bind PDZ domain-containing proteins is independent of p53 degradation or hTERT activation but does correlate with oncogenic potential (R. A. Watson, M. Thomas, L. Banks, and S. Roberts, J. Cell Sci. 116:4925-4934, 2003) and is essential for induction of epithelial hyperplasia in vivo (M. L. Nguyen, M. M. Nguyen, D. Lee, A. E. Griep, and P. F. Lambert, J. Virol. 77:6957-6964, 2003). In this study, we found that HPV type 16 E6 was able to activate NF-κB in airway epithelial cells through the induction of nuclear binding activity of p52-containing NF-κB complexes in a PDZ binding motif-dependent manner. Transcript accumulation for the NF-κB-responsive antiapoptotic gene encoding cIAP-2 and binding of nuclear factors to the proximal NF-κB binding site of the cIAP-2 gene promoter are induced by E6 expression. Furthermore, E6 is able to protect cells from TNF-induced apoptosis. All of these E6-dependent phenotypes are dependent on the presence of the PDZ binding motif of E6. Our results imply a role for targeting of PDZ proteins by E6 in NF-κB activation and protection from apoptosis in airway epithelial cells.

The PDZ binding motif of human papillomavirus type 16 E6 induces PTPN13 loss, which allows anchorage-independent growth and synergizes with ras for invasive growth.

ABSTRACT The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Rasv12, resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.

Engineering Novel Cell Surface Receptors for Virus-mediated Gene Transfer

The absence of viral receptors is a major barrier to efficient gene transfer in many cells. To overcome this barrier, we developed an artificial receptor based on expression of a novel sugar. We fed cells an unnatural monosaccharide, a modified mannosamine that replaced the acetyl group with a levulinate group (ManLev). ManLev was metabolized and incorporated into cell-surface glycoconjugates. The synthetic sugar decorated the cell surface with a unique ketone group that served as a foundation on which we built an adenovirus receptor by covalently binding biotin hydrazide to the ketone. The artificial receptor enhanced adenoviral vector binding and gene transfer to cells that are relatively resistant to adenovirus infection. These data are the first to suggest the feasibility of a strategy that improves the efficiency of gene transfer by using the biosynthetic machinery of the cell to engineer novel sugars on the cell surface.

RADIATION RESPONSE IN TWO HPV INFECTED HEAD AND NECK CANCER CELL LINES IN COMPARISON TO A NON-HPV INFECTED CELL LINE AND RELATIONSHIP TO SIGNALING THROUGH AKT

PURPOSE Human papilloma virus (HPV)-associated cancers of the head and neck (H&N) are increasing in frequency and are often treated with radiation. There are conflicting data in the literature regarding the radiation response in the presence of HPV infection, with some data suggesting they may be more sensitive to radiation. There are few studies looking at in vitro effects of HPV and further sensitization by inhibitors of specific signaling pathways. We are in the process of starting a clinical trial in H&N cancer patients using nelfinavir (NFV) (which inhibits Akt) and it would be important to know the effect of HPV on radiation response +/- NFV. METHODS AND MATERIALS Two naturally infected HPV-16 cell lines (UPCI-SCC90 and UMSCC47) and the HPV-negative SQ20B H&N squamous carcinoma cells were used. Western blots with or without 10 uM NFV were done to evaluate signaling from the PI3K-Akt pathway. Clonogenic assays were done in the three cell lines with or without NFV. RESULTS Both UPCI-SCC90 and UMSCC47 cells were sensitive to radiation as compared with SQ20B and the degree corresponded to Akt activation. The SQ20B cell line has an activating mutation in EGFR resulting in phosphorylation (P) of Akt; UMSCC47 has decreased P-phosphatase and TENsin (PTEN), resulting in increased P-Akt; UPCI-SCC90 had overexpression of P-PTEN and decreased P-Akt. NFV resulted in downregulation of Akt in all three cell lines, resulting in sensitization to radiation. CONCLUSIONS HPV-infected H&N cancers are sensitive to radiation. The degree of sensitivity correlates to Akt activation and they can be further sensitized by NFV.

Reinstating Aberrant mTORC1 Activity in Huntington’s Disease Mice Improves Disease Phenotypes

Huntingtons disease (HD) is caused by a polyglutamine tract expansion in huntingtin (HTT). Despite HTTs ubiquitous expression, there is early and robust vulnerability in striatum, the cause of which is poorly understood. Here, we provide evidence that impaired striatal mTORC1 activity underlies varied metabolic and degenerative phenotypes in HD brain and show that introducing the constitutively active form of the mTORC1 regulator, Rheb, into HD mouse brain, alleviates mitochondrial dysfunction, aberrant cholesterol homeostasis, striatal atrophy, impaired dopamine signaling, and increases autophagy. We also find that the expression of Rhes, a striatum-enriched mTOR activator, is reduced in HD patient and mouse brain and that exogenous addition of Rhes alleviates motor deficits and improves brain pathology in HD mice. Our combined work indicates that impaired Rhes/mTORC1 activity in HD brain may underlie the notable striatal susceptibility and thus presents a promising therapeutic target for HD therapy.

HPV16-E6 associated hTERT promoter acetylation is E6AP dependent, increased in later passage cells and enhanced by loss of p300

The E6 oncoprotein from high‐risk HPV types activates human telomerase reverse transcriptase (hTERT) transcription in human keratinocytes. Studies on how E6 regulates hTERT have implicated E‐box or X‐box elements in the hTERT promoter (Veldman et al., Proc Natl Acad Sci USA 2003;100:8211–14; Oh et al., J Virol 2001;75:5559–66; Gewin et al., Genes Dev 2004;18:2269–82), but the mechanism of activation by E6 is still controversial and not well defined. Here, we demonstrate that induction of both hTERT expression and telomerase activity by HPV‐16 E6 in early passage keratinocytes is associated with acetylation of histone H3 at the hTERT promoter, is dependent on the E6 associated protein (E6AP) and is not exclusively reliant on E‐box or X‐box elements. Further increases in histone acetylation of the hTERT promoter and hTERT transcriptional activity in E6 expressing cells that had been passaged extensively in culture were found to occur only with the endogenous promoter and not with an exogenously introduced hTERT promoter construct. Telomerase activity at both early and late passages, however, was dependent on E6AP expression, implying a continued reliance on E6 function for telomerase activity. Our results demonstrate that E6 induces hTERT promoter acetylation, but that further increases in telomerase activity and histone acetylation in later passage E6 expressing cells are independent of E6 activation of the core hTERT promoter. We also provide evidence that the transcription factor p300 is a potential repressor of telomerase activation and histone acetylation in the context of E6 expression. These studies give insight into how immortalization by HPV results in upregulation of hTERT and furthers our understanding of how telomerase is activated during the process of malignant transformation.