Shaoyu Zhou

Frequency and phenotypic implications of mitochondrial DNA mutations in human squamous cell cancers of the head and neck.

Mitochondrial genomic mutations are found in a variety of human cancers; however, the frequency of mitochondrial DNA (mtDNA) mutations in coding regions remains poorly defined, and the functional effects of mitochondrial mutations found in primary human cancers are not well described. Using MitoChip, we sequenced the whole mitochondrial genome in 83 head and neck squamous cell carcinomas. Forty-one of 83 (49%) tumors contained mtDNA mutations. Mutations occurred within noncoding (D-loop) and coding regions. A nonrandom distribution of mutations was found throughout the mitochondrial enzyme complex components. Sequencing of margins with dysplasia demonstrated an identical nonconservative mitochondrial mutation (A76T in ND4L) as the tumor, suggesting a role of mtDNA mutation in tumor progression. Analysis of p53 status showed that mtDNA mutations correlated positively with p53 mutations (P < 0.002). To characterize biological function of the mtDNA mutations, we cloned NADH dehydrogenase subunit 2 (ND2) mutants based on primary tumor mutations. Expression of the nuclear-transcribed, mitochondrial-targeted ND2 mutants resulted in increased anchorage-dependent and -independent growth, which was accompanied by increased reactive oxygen species production and an aerobic glycolytic metabolic phenotype with hypoxia-inducible factor (HIF)-1α induction that is reversible by ascorbate. Cancer-specific mitochondrial mutations may contribute to development of a malignant phenotype by direct genotoxic effects from increased reactive oxygen species production as well as induction of aerobic glycolysis and growth promotion.

Increased Mitochondrial DNA Content in Saliva Associated with Head and Neck Cancer

Alterations of the mitochondrial DNA (mtDNA) have been described in human tumors and in other tissues in association with smoking exposure. We did quantitative PCR of cytochrome c oxidase I (Cox I) and cytochrome c oxidase II (Cox II) genes on oral rinse samples obtained from 94 patients with primary head and neck squamous cell carcinoma (HNSC) and a control group of 656 subjects. Mitochondrial DNA/nuclear DNA in saliva from HNSC patients and controls in relationship to smoking exposure, ethanol intake, and tumor stage were examined. Mean levels of Cox I and Cox II in saliva samples were significantly higher in HNSC patients: Cox I, 0.076 [95% confidence interval (95% CI), 0.06-0.09] and Cox II, 0.055 (95% CI, 0.04-0.07) in comparison with controls Cox I, 0.054 (95% CI, 0.05-0.06), P < 0.0001 and Cox II, 0.046 (95% CI, 0.04-0.05), P = 0.003 (t test). MtDNA levels were elevated in primary tumors when compared with matched, pretreatment saliva and significant correlation was noted (Cox I, r = 0.30, P = 0.005 and Cox II r = 0.33, P = 0.002, respectively, Pearsons correlation). On univariate analysis, smoking, age, HNSC diagnosis, and advanced stage of HNSC were associated with higher level of mtDNA content in saliva. Multivariate analysis showed a significant and independent association of HNSC diagnosis, age, and smoking with increasing mtDNA/nuclear DNA for Cox I and Cox II. mtDNA content alteration is associated with HNSC independently of age and smoking exposure, can be detected in saliva, and may be due to elevation in mtDNA content in primary HNSC.

Presence of HPV DNA in convalescent salivary rinses is an adverse prognostic marker in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) 16 is present in up to 60% of patients with head and neck squamous cell carcinoma (HNSCC) and confers a favorable prognosis in terms of recurrence and mortality. Previous reports demonstrated that HPV-16 DNA can be detected in the initial salivary rinses from these patients. In this study, we assessed the feasibility of post-treatment HPV DNA shed from the oral mucosa as a prognostic marker for persistent/recurrent head and neck cancer. Fresh tumor samples and pre- and post-treatment salivary rinses were collected from 59 patients with HNSCC. HPV-16 E6 and E7 DNA copy number in these samples were quantified by real time PCR. Twenty of 59 patients (33.9%) were HPV-16 positive in their tumors before treatment. Four of 20 HPV tumor positive patients ultimately developed recurrence, and two of these four patients were HPV-16 positive in surveillance salivary rinses (sensitivity=50%). Of the 39 (66.1%) HPV-16 negative patients on initial clinical presentation and the 16 HPV-16 positive patients who did not recur, none were HPV-16 positive in salivary rinses after treatment (specificity=100%). HPV-16 presence in follow-up salivary rinses preceded clinical detection of disease recurrence by an average of 3.5 months. Patients with presence of HPV-16 DNA in surveillance salivary rinses are at significant risk for recurrence. Quantitative measurement of salivary HPV-16 DNA has promise for surveillance and early detection of recurrence.

Mitochondrial mutations contribute to HIF1alpha accumulation via increased reactive oxygen species and up-regulated pyruvate dehydrogenease kinase 2 in head and neck squamous cell carcinoma.

Purpose: Mitochondrial mutations have been identified in head and neck squamous cell carcinoma (HNSCC), but the pathways by which phenotypic effects of these mutations are exerted remain unclear. Previously, we found that mitochondrial ND2 mutations in primary HNSCC increased reactive oxygen species (ROS) and conferred an aerobic, glycolytic phenotype with HIF1α accumulation and increased cell growth. The purpose of the present study was to examine the pathways relating these alterations. Experimental Design: Mitochondrial mutant and wild-type ND2 constructs were transfected into oral keratinocyte immortal cell line OKF6 and head and neck cancer cell line JHU-O19 and established transfectants. The protein levels of HIF1α, pyruvate dehydrogenease (PDH), phosphorylated PDH, and pyruvate dehydrogenease kinase 2 (PDK2), together with ROS generation, were compared between the mutant and the wild type. Meanwhile, the effects of small molecule inhibitors targeting PDK2 and mitochondria-targeted catalase were evaluated on the ND2 mutant transfectants. Results: We determined that ND2 mutant down-regulated PDH expression via up-regulated PDK2, with an increase in phosphorylated PDH. Inhibition of PDK2 with dichloroacetate decreased HIF1α accumulation and reduced cell growth. Extracellular treatment with hydrogen peroxide, a ROS mimic, increased PDK2 expression and HIF1α expression, and introduction of mitochondria-targeted catalase decreased mitochondrial mutation-mediated PDK2 and HIF1α expression and suppressed cell growth. Conclusions: Our findings suggest that mitochondrial ND2 mutation contributes to HIF1α accumulation via increased ROS production, up-regulation of PDK2, attenuating PDH activity, thereby increasing pyruvate, resulting in HIF1α stabilization. This may provide insight into a potential mechanism, by which mitochondrial mutations contribute to HNSCC development.

Mitochondrial DNA Content Increase in Response to Cigarette Smoking

An increase in mitochondrial DNA (mtDNA) content and decline in mitochondrial function occurs with aging and in response to DNA-damaging agents, including tobacco smoke. We did a cross-sectional study and quantified changes in mtDNA content in a population of individuals with varied smoking and alcohol exposure. Age, smoking history, ethanol intake, and other demographic data were characterized for 604 individuals participating in a screening study for smoking-related upper aerodigestive malignancy. Total DNA was extracted from exfoliated cells in saliva. DNA from a nuclear gene, β-actin, and two mitochondrial genes, cytochrome c oxidase I and II (Cox I and Cox II), were quantified by real-time PCR. mtDNA content was correlated with age, exposure history, and other variables using multivariate regression analyses. A significant increase (P < 0.001) in mtDNA content was noted in smokers (31% and 29% increase for Cox I and Cox II, respectively) and former smokers (31% and 34%) when compared with never smokers. This association persisted after adjustment for other significant factors including age, alcohol drinking, and income (P < 0.001). Increased mtDNA content was positively associated with pack-years of smoking (P = 0.02). Despite an average smoking cessation interval of 21 years in former smokers, tobacco cessation interval was not statistically significantly associated with mtDNA content. Smoking is associated with increased mtDNA content in a dose-dependent fashion. Mitochondrial DNA alterations in response to smoking persist for several decades after smoking cessation, consistent with long-term, smoking-related damage. (Cancer Epidemiol Biomarkers Prev 2006;15(1):19–24)

Decreased mitochondrial DNA content in posttreatment salivary rinses from head and neck cancer patients

Purpose and Experimental Design: Alterations in mitochondrial DNA (mtDNA) sequence and content have been described in human tissues and tumors in association with smoking exposure. We did quantitative PCR analysis of cytochrome c oxidase (Cox) I and Cox II genes to measure changes in mtDNA content in pretreatment and posttreatment salivary rinses obtained from 76 patients undergoing surgical resection for primary head and neck squamous cell carcinoma. We also examined the relationship between changes in mtDNA content and postoperative radiation therapy, smoking exposure, alcohol intake, and other clinical characteristics. Results: Overall, mtDNA content in posttreatment saliva was significantly decreased. The mean change for Cox I was −0.21 [95% confidence interval (95% CI), −0.44 to 0.01, P = 0.06] and for Cox II was −0.31 (95% CI, −0.55 to −0.08, P = 0.01). Patients in the radiation therapy group exhibited a significant decrease compared with the nonradiated group (P = 0.03 for Cox I; P = 0.05 for Cox II). In addition, significant decreases in Cox I (−0.71; 95% CI, −1.17 to −0.25, P = 0.005) and Cox II (−0.65; 95% CI, −1.17 to −0.13, P = 0.02) were found in never-smoking patients but not in former or current smokers. Conclusion: Our data suggest that salivary mtDNA content is decreased in never smokers and in response to radiation therapy after primary surgical resection.

Mitochondrial Mutations Are a Late Event in the Progression of Head and Neck Squamous Cell Cancer

Purpose: To determine the timing of mitochondrial mutations in the progression of head and neck squamous cell carcinoma. Experimental Design: Twenty-three mitochondrial mutations were identified in 12 tumors using a high-throughput mitochondrial sequencing array. Areas of adjacent dysplastic and normal epithelium adjacent to tumors were sequenced using conventional methods for the presence of mutations that occurred in the corresponding tumor. Results: Two of 23 (8.7%) tumor mitochondrial mutations (2 of 12 tumors) were present in both the areas of adjacent dysplasia and normal epithelium. Five of 23 (21.7%) tumor mitochondrial mutations (4 of 12 tumors) were present in areas of adjacent dysplasia. Eleven of 12 tumors contained nonsynonymous mutations that resulted in protein coding alterations. A significant difference (P < 0.01, χ2) was found in the incidence of mitochondrial mutation that occurred after development of cancer compared with adjacent areas dysplasia and normal epithelium. Conclusions: The majority of mitochondrial mutations occur during or after the transition of preneoplastic epithelium to cancer in head and neck squamous cell carcinoma, indicating that these are a late event in head and neck carcinogenesis.

Mitochondrial Resequencing Arrays Detect Tumor-Specific Mutations in Salivary Rinses of Patients with Head and Neck Cancer

Purpose: Alterations of the mitochondrial genome have been identified in multiple solid tumors and in many head and neck squamous cell carcinomas (HNSCC). Identification of mitochondrial mutations in the salivary rinses of patients with HNSCC has potential application in disease detection. In this study, we used the MitoChip v2.0 mitochondrial genome resequencing array to detect minor populations of mitochondrial DNA in salivary rinses of patients with HNSCC. Experimental Design: Salivary rinses from 13 patients with HNSCC, whose tumors carried mitochondrial mutations, were collected before surgical resection. DNA isolated from salivary rinses and serial dilutions of DNA derived from HNSCC-derived cell lines with known mitochondrial mutations were sequenced using the MitoChip, and analyzed using a quantitative algorithm which we developed to detect minor populations of mitochondrial DNA from MitoChip probe intensity data. Results: We detected heteroplasmic populations of mitochondrial DNA up to a 1:200 dilution using MitoChip v2.0 and our analysis algorithm. A logarithmic relationship between the magnitude of assay intensity and concentration of minor mitochondrial populations was shown. This technique was able to identify tumor-specific mitochondrial mutations in salivary rinses from 10 of 13 (76.9%) patients with head and neck cancer. Conclusions: Minor populations of mitochondrial DNA and disease-specific mitochondrial mutations in salivary rinses of patients with HNSCC can be successfully identified using the MitoChip resequencing array and the algorithm which we have developed. This technique has potential application in the surveillance of patients after resection and may have applicability in the surveillance of body fluids in other tumor types.

ssDNA-Binding Protein 2 Is Frequently Hypermethylated and Suppresses Cell Growth in Human Prostate Cancer

Purpose: Prostate cancer is a major cause of cancer death among men and the development of new biomarkers is important to augment current detection approaches. Experimental Design: We identified hypermethylation of the ssDNA-binding protein 2 (SSBP2) promoter as a potential DNA marker for human prostate cancer based on previous bioinformatics results and pharmacologic unmasking microarray. We then did quantitative methylation-specific PCR in primary prostate cancer tissues to confirm hypermethylation of the SSBP2 promoter, and analyzed its correlation with clinicopathologic data. We further examined SSBP2 expression in primary prostate cancer and studied its role in cell growth. Results: Quantitative methylation-specific PCR results showed that the SSBP2 promoter was hypermethylated in 54 of 88 (61.4%) primary prostate cancers versus 0 of 23 (0%) in benign prostatic hyperplasia using a cutoff value of 120. Furthermore, we found that expression of SSBP2 was down-regulated in primary prostate cancers and cancer cell lines. Hypermethylation of the SSBP2 promoter and its expression were closely associated with higher stages of prostate cancer. Reactivation of SSBP2 expression by the demethylating agent 5-aza-2′-deoxycytidine in prostate cancer cell lines confirmed epigenetic inactivation as one major mechanism of SSBP2 regulation. Moreover, forced expression of SSBP2 inhibited prostate cancer cell proliferation in the colony formation assay and caused cell cycle arrest. Conclusion: SSBP2 inhibits prostate cancer cell proliferation and seems to represent a novel prostate cancer–specific DNA marker, especially in high stages of human prostate cancer.

Head and Neck Cancer Cell Lines Are Resistant to Mitochondrial-Depolarization-Induced Apoptosis

Purpose: Mitochondrial dysfunction has been linked to defects in the apoptotic pathway, and solid tumors, including head and neck squamous cell carcinoma (HNSCC), exhibit defects in apoptosis. Loss of mitochondrial membrane potential (ΔΨm) is an early initiating event in the mitochondrial apoptotic pathway. We investigated the apoptotic response of 3 head and neck cancer cell lines treated with a mitochondrial-membrane-depolarizing agent, valinomycin, and studied the ability of depolarization to induce release of cytochrome c in these cell lines. Experimental Design: HNSCC cell lines JHU-011, -012 and -019, and a leukemia control cell line HL-60 were assayed for ΔΨm after valinomycin treatment by staining with mitochondrial-membrane-potential-specific probe JC-1 and stained with apoptosis-specific probe annexin-V to measure their rate of apoptosis by FACS. Western blotting was also applied to detect cytoplasmic cytochrome c release. Results: ΔΨm in head and neck cell lines started to show slight loss of ΔΨm, while HL-60 showed significant loss of ΔΨm after 30 min of treatment. All cell lines demonstrated complete mitochondrial depolarization within 24 h, however, only the control cell line HL-60 underwent apoptosis. In addition, HNSCC cell lines did not demonstrate cytoplasmic cytochrome c release despite significant mitochondrial membrane depolarization, while HL-60 cell initiated apoptosis and cytochcrome c release after 24 h of treatment. Conclusions: Head and neck cancer cell lines exhibit defects in mitochondrial-membrane-depolarization-induced apoptosis as well as impaired release of cytochrome c despite significant mitochondrial membrane depolarization. Proximal defects in the mitochondrial apoptosis pathway are a feature of HNSCC.