James Lang

Aberrant CpG-island methylation has non-random and tumour-type-specific patterns

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Optimization of an enrichment process for circulating tumor cells from the blood of head and neck cancer patients through depletion of normal cells

The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patients blood from 4.05 × 109 to 8.04 × 103 cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patients blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log10) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominately epithelial or mesenchymal characteristics?). Biotechnol. Bioeng. 2009;102: 521–534.

Genome-Wide Hypomethylation in Head and Neck Cancer Is More Pronounced in HPV-Negative Tumors and Is Associated with Genomic Instability

Loss of genome-wide methylation is a common feature of cancer, and the degree of hypomethylation has been correlated with genomic instability. Global methylation of repetitive elements possibly arose as a defense mechanism against parasitic DNA elements, including retrotransposons and viral pathogens. Given the alterations of global methylation in both viral infection and cancer, we examined genome-wide methylation levels in head and neck squamous cell carcinoma (HNSCC), a cancer causally associated with human papilloma virus (HPV). We assayed global hypomethylation levels in 26 HNSCC samples, compared with their matched normal adjacent tissue, using Pyrosequencing-based methylation assays for LINE repeats. In addition, we examined cell lines derived from a variety of solid tumors for LINE and SINE (Alu) repeats. The degree of LINE and Alu hypomethylation varied among different cancer cell lines. There was only moderate correlation between LINE and Alu methylation levels, with the range of variation in methylation levels being greater for the LINE elements. LINE hypomethylation was more pronounced in HPV-negative than in HPV-positive tumors. Moreover, genomic instability, as measured by genome-wide loss-of-heterozygosity (LOH) single nucleotide polymorphism (SNP) analysis, was greater in HNSCC samples with more pronounced LINE hypomethylation. Global hypomethylation was variable in HNSCC. Its correlation with both HPV status and degree of LOH as a surrogate for genomic instability may reflect alternative oncogenic pathways in HPV-positive versus HPV-negative tumors.

Dysregulation of MicroRNA-34a Expression in Head and Neck Squamous Cell Carcinoma Promotes Tumor Growth and Tumor Angiogenesis

Background MicroRNAs (miRs) are small non-coding RNAs that play an important role in cancer development where they can act as oncogenes or as tumor-suppressors. miR-34a is a tumor-suppressor that is frequently downregulated in a number of tumor types. However, little is known about the role of miR-34a in head and neck squamous cell carcinoma (HNSCC). Methods and Results miR-34a expression in tumor samples, HNSCC cell lines and endothelial cells was examined by real time PCR. Lipofectamine-2000 was used to transfect miR-34a in HNSCC cell lines and human endothelial cells. Cell-proliferation, migration and clonogenic survival was examined by MTT, Xcelligence system, scratch assay and colony formation assay. miR-34a effect on tumor growth and tumor angiogenesis was examined by in vivo SCID mouse xenograft model. Our results demonstrate that miR-34a is significantly downregulated in HNSCC tumors and cell lines. Ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation and migration. miR-34a overexpression also markedly downregulated E2F3 and survivin levels. Rescue experiments using microRNA resistant E2F3 isoforms suggest that miR-34a-mediated inhibition of cell proliferation and colony formation is predominantly mediated by E2F3a isoform. In addition, tumor samples from HNSCC patients showed an inverse relationship between miR-34a and survivin as well as miR-34a and E2F3 levels. Overexpression of E2F3a completely rescued survivin expression in miR-34a expressing cells, thereby suggesting that miR-34a may be regulating survivin expression via E2F3a. Ectopic expression of miR-34a also significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft model. Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell functions. Conclusions Taken together, these findings suggest that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of HNSCC.

Multiparameter Analysis, including EMT Markers, on Negatively Enriched Blood Samples from Patients with Squamous Cell Carcinoma of the Head and Neck

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.

Application of immunomagnetic cell enrichment in combination with RT‐PCR for the detection of rare circulating head and neck tumor cells in human peripheral blood

Detection of rare, circulating tumor cells (CTCs) in human peripheral blood is a potential indicator of prognosis and diagnosis in oncology. Typical methods to detect these CTCs are either by immunocytochemistry (ICCS) or RT‐PCR. However without accurate, rapid, and reproducible enrichment processes, these detection techniques are labor intensive and/or unreliable. In this article, a repeatable enrichment process that included a flow‐through immunomagnetic cell separation system, the quadrupole magnetic sorter (QMS) was optimized with the aid of a statistical analysis software package. The QMS was operated in a negative mode of operation by immunomagnetically targeting normal human peripheral blood lymphocytes (PBL) through the CD45 surface marker. Three head and neck squamous carcinoma cell lines (HNSCC), Detroit‐562, SCC‐4, and CAL‐27, were used to determine the sensitivity of RT‐PCR for the epidermal growth factor receptor (EGFR) in spiked PBL. The detection purity needed for detection was found to be one cell in 104, one cell in 103, and one cell in 105 for the Detroit‐562, SCC‐4, and CAL‐27, respectively. The actual number of cancer cells needed for RT‐PCR detection ranged from 30 to 1 cell. To mimic the potential concentration of rare CTC present in peripheral blood of cancer patients, the spiking concentration was chosen to be one cancer cell per 105 total leukocytes from healthy donors. Using a single step immunomagnetic labeling, the final, optimized enrichment process produced a 57.6 ± 30.3‐fold (n = 6) enrichment of the rare cancer cells with a final cancer cell recovery of (77.8 ± 6.6)%.

microRNA-107 functions as a candidate tumor-suppressor gene in head and neck squamous cell carcinoma by downregulation of protein kinase Cɛ

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600 000 new cases diagnosed each year. Understanding the molecular pathways that lead to HNSCC is crucial to identify new targets for anti-cancer drug development. Protein kinase Cɛ (PKCɛ) is elevated in HNSCC and regulates the activation of Akt, Stat3 and Rho GTPases. To date, the molecular mechanism of PKCɛ dysregulation in HNSCC remains to be elucidated. In silico analysis identified three putative microRNA-107 (miR-107) binding sites in the 3′-untranslated region (UTR) of PKCɛ. An inverse relationship was revealed between miR-107 and PKCɛ in HNSCC cell lines. Delivery of miR-107 reduced PKCɛ levels in SCC15, SCC25 and CAL27, three HNSCC cell lines with high PKCɛ and low miR-107. The activity of a luciferase reporter construct containing the 3′-UTR of PKCɛ was downregulated by miR-107 and mutations in the three cognate miR-107 binding sites completely ablated the regulation by miR-107. Treatment with miR-107 significantly blocked cell proliferation, DNA replication, colony formation and invasion in SCC25 and CAL27 cells. Ectopic expression of miR-resistant PKCɛ was sufficient to partially rescue the loss-of-function phenotype in miR-107-overexpressing SCC25 cells. Tumor growth in nude mice was retarded by 93±7% in CAL27/miR-107 cells compared with CAL27/miR-control cells. Last, human primary HNSCC tumors with elevated PKCɛ had reduced miR-107 expression. Our results demonstrate that PKCɛ is directly regulated by miR-107 and, moreover, suggest that miR-107 may be a potential anti-cancer therapeutic for HNSCC.

YM155 Reverses Cisplatin Resistance in Head and Neck Cancer by Decreasing Cytoplasmic Survivin Levels

Cisplatin is one of the commonly used chemotherapeutic drugs for the treatment of head and neck squamous cell carcinoma (HNSCC). However, acquisition of cisplatin resistance is common in patients with HNSCC, and it often leads to local and distant failure. In this study, we showed that survivin expression is significantly upregulated in HNSCC primary tumors and cell lines. In addition, survivin levels were significantly higher in human papilloma virus–negative patients that normally respond poorly to cisplatin treatment. Survivin expression was further increased in cisplatin-resistant cells (CAL27-CisR) as compared with its parent cells (CAL27). Therefore, we hypothesized that targeting of survivin in HNSCC could reverse the resistant phenotype in tumor cells, thereby enhancing the therapeutic efficacy of cisplatin. We used both in vitro and in vivo models to test the efficacy of YM155, a small molecule survivin inhibitor, either as a single agent or in combination with cisplatin. YM155 significantly decreased survivin levels and cell proliferation in a dose-dependent manner. In addition, YM155 pretreatment significantly reversed cisplatin resistance in cancer cells. Interestingly, YM155 treatment altered the dynamic localization of survivin in cells by inducing a rapid reduction in cytoplasmic survivin, which plays a critical role in its antiapoptotic function. In a severe combined immunodeficient mouse xenograft model, YM155 significantly enhanced the antitumor and antiangiogenic effects of cisplatin, with no added systemic toxicity. Taken together, our results suggest a potentially novel strategy to use YM155 to overcome the resistance in tumor cells, thereby enhancing the effectiveness of the chemotherapy in HNSCC. Mol Cancer Ther; 11(9); 1988–98. ©2012 AACR.

Confocal Images of Circulating Tumor Cells Obtained Using a Methodology and Technology That Removes Normal Cells

A completely negative enrichment technology was used to detect circulating tumor cells, CTCs, in the peripheral blood of head and neck cancer patients. Of 32 blood samples, 63% contained CTCs and the number of CTCs identified per mL of blood collected ranged from 0 to 214. The final purity ranged from 1 CTC in 9 total cells to 1 CTC in 20,000 total cells, the final purity being both a function of the number of CTCs and the performance of the specific enrichment. Consistent with previous reports, CTC were positively identified if: (1) they contained a nucleus based on DAPI stain, (2) stained positive for cytokeratins, and (3) have a high nuclei to cytoplasmic ratio. In addition, for a blood sample to be considered positive for CTCs, the enriched sample must be positive for epithelial growth factor receptor, EGFR, as measured by RT-PCR. While most of the blood samples were obtained during surgery, a number were taken prior to and during surgery. In all of the pre- and postsurgery paired samples, significant numbers of CTCs were detected. A number of these enriched samples were observed under confocal microscope in addition to the microscopic observations under traditional wide-field fluorescent microscope. As expected, the FITC stained cytokeratins appeared in the cytoplasm and the average size of these positively stained cells, on the cytospin, was in the range of 8-12 mum. Future studies will involve the investigation if cancer stem cell and mesenchymal markers are present on these CTCs and correlations of patient outcome to the number and type of CTC present.

Clinical, genomic, and metagenomic characterization of oral tongue squamous cell carcinoma in patients who do not smoke.

Evidence suggests the incidence of oral tongue squamous cell carcinoma is increasing in young patients, many who have no history of tobacco use.