Anti-EMMPRIN antibody treatment of head and neck squamous cell carcinoma in an ex-vivo model.

Targeting the molecular pathways associated with carcinogenesis remains the greatest opportunity to reduce treatment-related morbidity and mortality. Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as CD147, is a cell surface molecule known to promote tumor growth and angiogenesis in preclinical studies of head and neck carcinoma making it an excellent therapeutic target. To evaluate the feasibility of anti-EMMPRIN therapy, an ex-vivo human head and neck cancer model was established using specimens obtained at the time of surgery (n=22). Tumor slices were exposed to varying concentrations of anti-EMMPRIN monoclonal antibody and cetuximab for comparison purposes. Cetuximab is the only monoclonal antibody currently approved for the treatment of head and neck carcinoma. After treatment, tumor slices were assessed by immunohistochemistry and western blot analysis for apoptosis (TUNEL) and EMMPRIN expression. Of the tumor specimens 33% showed a significant reduction in mean ATP levels after treatment with cetuximab compared with untreated controls, whereas 58% of the patients responded to anti-EMMPRIN therapy (P<0.05). Samples, which showed reactivity to anti-EMMPRIN, also had greater EMMPRIN expression based on immunohistochemistry staining (49%) when compared with nonresponders (25%, P=0.06). In addition, TUNEL analysis showed a larger number of cells undergoing apoptosis in antibody-treated tumor slices (77%) compared with controls (30%, P<0.001) with activation of apoptotic proteins, caspase 3 and caspase 8. This study shows the potential of anti-EMMPRIN to inhibit proliferation and promote apoptosis and suggests its future role in the targeted treatment of head and neck carcinoma.

Transcriptomic dissection of tongue squamous cell carcinoma.

BACKGROUND: The head and neck/oral squamous cell carcinoma (HNOSCC) is a diverse group of cancers, which develop from many different anatomic sites and are associated with different risk factors and genetic characteristics. The oral tongue squamous cell carcinoma (OTSCC) is one of the most common types of HNOSCC. It is significantly more aggressive than other forms of HNOSCC, in terms of local invasion and spread. In this study, we aim to identify specific transcriptomic signatures that associated with OTSCC. RESULTS: Genome-wide transcriptomic profiles were obtained for 53 primary OTSCCs and 22 matching normal tissues. Genes that exhibit statistically significant differences in expression between OTSCCs and normal were identified. These include up-regulated genes (MMP1, MMP10, MMP3, MMP12, PTHLH, INHBA, LAMC2, IL8, KRT17, COL1A2, IFI6, ISG15, PLAU, GREM1, MMP9, IFI44, CXCL1), and down-regulated genes (KRT4, MAL, CRNN, SCEL, CRISP3, SPINK5, CLCA4, ADH1B, P11, TGM3, RHCG, PPP1R3C, CEACAM7, HPGD, CFD, ABCA8, CLU, CYP3A5). The expressional difference of IL8 and MMP9 were further validated by real-time quantitative RT-PCR and immunohistochemistry. The Gene Ontology analysis suggested a number of altered biological processes in OTSCCs, including enhancements in phosphate transport, collagen catabolism, I-kappaB kinase/NF-kappaB signaling cascade, extracellular matrix organization and biogenesis, chemotaxis, as well as suppressions of superoxide release, hydrogen peroxide metabolism, cellular response to hydrogen peroxide, keratinization, and keratinocyte differentiation in OTSCCs. CONCLUSION: In summary, our study provided a transcriptomic signature for OTSCC that may lead to a diagnosis or screen tool and provide the foundation for further functional validation of these specific candidate genes for OTSCC.

Dimension reduction and mixed-effects model for microarray meta-analysis of cancer.

The rapid advances in high-throughput microarray technologies greatly facilitate the disease biomarker discovery. However, the potential of these microarray data has not yet been fully utilized. This is partly due to the limited sample sizes of each individual study. Combining microarray data from multiple studies improves the statistical power of detecting differentially expressed genes. Here we present a method for combining the microarray datasets at array probeset level. Using datasets from two commonly used array platforms, the Affymetrix Human Genome U133A and Human Genome U133 Plus 2.0 arrays, we found laboratory effects may be more influential than the platform effect. A visualization scheme for merging the array data from different array platforms was proposed to qualitatively judge the degree of agreement between datasets. A mixed-effects model was applied to identify differentially expressed genes from the merged array data.

Activated Vav2 modulates cellular invasion through Rac1 and Cdc42 in oral squamous cell carcinoma.

The Rho family of GTPases regulates cellular adhesion and motility. Guanine nucleotide exchange factors (GEFs) in turn regulate GTPases by promoting nucleotide exchange from GDP to GTP. We have determined that GTP-bound Rac1 is elevated in invasive oral squamous cell carcinoma (OSCC) cell lines. Because of the critical role of invasion in the progression and metastasis of OSCC, we investigated if the GEF Vav2 modulated Rac1 and Cdc42 activation and thus influenced OSCC invasion. Expression levels of Vav2 did not correlate with invasion but phosphorylated or activated Vav2 was associated with the more invasive cell lines. Transfection of activated Vav2 into the immortalized keratinocyte cell line HaCat and a low-level expressing Vav2 invasive OSCC cell line resulted in increased GTP-bound Rac1 and Cdc42 and increased invasion. Thus, activation of Vav2 appears to modulate cellular invasion through specific regulation of Rac1 and Cdc42 activity in OSCC.

Lab-on-a-chip technologies for oral-based cancer screening and diagnostics: capabilities, issues, and prospects.

The design of a microfluidic lab-on-a-chip system for point-of-care cancer screening and diagnosis of oral squamous cell carcinoma (OSCC) is presented. The chip is based on determining a approximately 30-gene transcription profile in cancer cells isolated from oral fluid samples. Microfluidic cell sorting using magnetic beads functionalized with an antibody against cancer-specific cell-surface antigens (e.g., epithelial cell adhesion molecule [EpCAM]) is described. A comprehensive cancer diagnostics chip will integrate microfluidic components for cell lysis, nucleic acid extraction, and amplification and detection of a panel of mRNA isolated from a subpopulation of cancer cells contained in a clinical specimen.

Identification of a gene signature for rapid screening of oral squamous cell carcinoma.

PURPOSE: Oral cancer is a major health problem worldwide and in the U.S. The 5-year survival rate for oral cancer has not improved significantly over the past 20 years and remains at approximately 50%. Patients diagnosed at an early stage of the disease typically have an 80% chance for cure and functional outcome, however, most patients are identified when the cancer is advanced. Thus, a convenient and an accurate way to detect oral cancer early will decrease patient morbidity and mortality. The ability to noninvasively monitor oral cancer onset, progression, and treatment outcomes requires two prerequisites: identification of specific biomarkers for oral cancers as well as noninvasive access to and monitoring of these biomarkers that could be conducted at the point of care (i.e., practitioner's or dentist's office) by minimally trained personnel. EXPERIMENTAL DESIGN: Here, we show that DNA microarray gene expression profiling of matched tumor and normal specimens can identify distinct anatomic site expression patterns and a highly significant gene signature distinguishing normal from oral squamous cell carcinoma (OSCC) tissue. RESULTS: Using a supervised learning algorithm, we generated a 25-gene signature for OSCC that can classify normal and OSCC specimens. This 25-gene molecular predictor was 96% accurate on cross-validation, averaging 87% accuracy using three independent validation test sets and failing to predict non-oral tumors. CONCLUSION: Identification and validation of this tissue-specific 25-gene molecular predictor in this report is our first step towards developing a new, noninvasive, microfluidic-based diagnostic technology for mass screening, diagnosis, and treatment of pre-OSCC and OSCC.

The beta1 cytoplasmic domain regulates the laminin-binding specificity of the alpha7X1 integrin.

During muscle development, the laminin-specific alpha7 integrin is alternatively spliced in the putative ligand-binding domain to yield either the alpha7X1 or the alpha7X2 variant. The relative level of alpha7X1 and alpha7X2 is developmentally regulated. Similarly, the partner beta1 integrin cytoplasmic domain is converted from the beta1A to the beta1D splice variant. To determine whether beta1D modulates the activity of the alpha7 receptor, cells were transfected with alpha7X1 and beta1D cDNA. alpha7X1 coupled with beta1A failed to adhere to laminin-1, whereas cotransfectants expressing alpha7X1 and beta1D showed strong adhesion. Interestingly, alpha7X1 complexed with beta1A and beta1D displayed the same level of poor adhesion to laminin-2/4 or strong adhesion to laminin-10/11. These findings indicate that alpha7 function is regulated not only by X1/X2 in its extracellular domain but also by beta1 cytoplasmic splice variants. It is likely that expression of beta1D alters alpha7X1 binding to laminin isoforms by a process related to ligand affinity modulation. Functional regulation of alpha7beta1 by developmentally regulated splicing events may be important during myogenic differentiation and repair because the integrin mediates adhesion, motility, and cell survival.

Gene expression signature predicts lymphatic metastasis in squamous cell carcinoma of the oral cavity.

Metastasis via the lymphatics is a major risk factor in squamous cell carcinoma of the oral cavity (OSCC). We sought to determine whether the presence of metastasis in the regional lymph node could be predicted by a gene expression signature of the primary tumor. A total of 18 OSCCs were characterized for gene expression by hybridizing RNA to Affymetrix U133A gene chips. Genes with differential expression were identified using a permutation technique and verified by quantitative RT-PCR and immunohistochemistry. A predictive rule was built using a support vector machine, and the accuracy of the rule was evaluated using crossvalidation on the original data set and prediction of an independent set of four patients. Metastatic primary tumors could be differentiated from nonmetastatic primary tumors by a signature gene set of 116 genes. This signature gene set correctly predicted the four independent patients as well as associating five lymph node metastases from the original patient set with the metastatic primary tumor group. We concluded that lymph node metastasis could be predicted by gene expression profiles of primary oral cavity squamous cell carcinomas. The presence of a gene expression signature for lymph node metastasis indicates that clinical testing to assess risk for lymph node metastasis should be possible.

The role of transforming growth factor alpha in determining growth factor independence.

Growth factor independence is a hallmark of malignancy that is attributed to the development of autocrine growth factor loops in cancer cells. However, growth factor-dependent normal cells also exhibit autocrine activity, thus raising the issue of how endogenously produced activity in cancer cells differs in a manner that leads to growth factor independence. We have examined this issue by comparing growth factor-independent HCT116 human colon carcinoma cells with a growth factor-dependent subcompartment of malignant cells designated HCT116b that was isolated from the same patient tumor. Therefore, the development of the growth factor-independent phenotype represents clonal progression within the tumor in vivo. The growth factor independence of HCT116 cells was shown to be dependent on autocrine transforming growth factor (TGF)-alpha activity, yet the isoparental HCT116b subcompartment showed similar levels of TGF-alpha expression as HCT116 when cells were in exponential growth. When both cell lines were growth arrested by nutrient deprivation, HCT116b cells required nutrient replenishment and growth factors for reinitiation of DNA synthesis, whereas HCT116 cells required only nutrient replenishment. In contrast to growth factor-dependent HCT116b cells, the HCT116 cells showed up-regulation of TGF-alpha expression during growth arrest as a result of enhanced transcription. This increased TGF-alpha expression in quiescent HCT116 cells was associated with constitutive epidermal growth factor receptor (EGFR) activation in the growth-arrested state, whereas growth-arrested HCT116b cells did not show EGFR activation. TGF-alpha antisense transfection of HCT116 cells showed that EGFR activation was due to increased TGF-alpha expression. Pretreatment of growth-arrested HCT116 cells with AG1478, a selective inhibitor of EGFR tyrosine kinase activity, blocked the reinitiation of DNA synthesis, demonstrating that growth factor independence was due to the increased TGF-alpha expression and EGFR activation of these cells in growth arrest relative to growth factor-dependent HCT116b cells. Importantly, the level of EGFR activation in growth-arrested HCT116 cells was only slightly higher than that of exponential cells, indicating that it was inappropriate EGFR activation in growth arrest rather than the amplitude of activation that generated growth factor independence.

VEGF-A and alphaVbeta3 integrin synergistically rescue angiogenesis via N-Ras and PI3-K signaling in human microvascular endothelial cells.

We recently showed that normal fibroblasts mediate capillary-like differentiation of human microvascular endothelial cells (HMVEC) in a 3-D angiogenesis model. Here, we show that a collaborative effect of VEGF-A and alphaVbeta3 integrin is critical in fibroblast-mediated angiogenesis because enhancement of both VEGF production by fibroblasts and beta3 integrin expression in HMVEC can rescue capillary-like endothelial differentiation under reduced serum conditions. To investigate the downstream signaling mechanisms, we compared N-Ras and Rho/Rac/Cdc42, as well as phosphatidylinositol 3-kinase (PI3-K) and Akt, for their involvement in the capillary-like network formation. The dominant-negative mutant of N-Ras (N-RasN17), but not the mutants of Rho/Rac/Cdc42, suppressed network formation. Overexpression of a constitutively active form of PI3-K rescued the network formation, which was inhibited by a dominant-negative >beta3 integrin; however, an active form of Akt failed to rescue the inhibition but induced a phenotypic change in HMVEC. Moreover, PI3-K is a downstream target of N-Ras because it could be co-immunoprecipitated with N-Ras, and its active form could rescue the inhibitory effect of N-Ras N17. Thus, our data indicate the existence of N-Ras- and PI3-K-dependent but Rho/Rac/Cdc42- and Akt-independent signaling mechanisms for the synergistic effect of VEGF-A and alphaVbeta3 on fibroblast-mediated microvascular network formation.

Dysregulation of hypoxia inducible factor-1alpha in head and neck squamous cell carcinoma cell lines correlates with invasive potential.

OBJECTIVES/HYPOTHESIS: Tumor hypoxia appears to be closely associated with tumor propagation, malignant progression, and resistance to radiotherapy. Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor that is upregulated under hypoxic conditions and activates hypoxic adaptation pathways which include neovascularization, erythropoiesis, and glycolysis. Hypoxia inducible factor-1alpha is under tight regulation with undetectable levels of expression in normoxia and robust expression in hypoxia. Mutations that activate oncogenes or inactivate tumor suppressor genes increase the expression of HIF-1alpha. Furthermore, it has been demonstrated that HIF-1alpha is overexpressed in head and neck squamous cell carcinoma and that the degree of expression has predictive and prognostic significance for patients undergoing radiotherapy. The study investigated whether overexpression of HIF-1alpha in head and neck squamous cell carcinoma results from a physiological response to local hypoxia or from oncogenic mutational progression. STUDY DESIGN: Expression of HIF-1alpha under normoxic and hypoxic conditions was evaluated in cell lines derived from head and neck squamous cell carcinoma. Cell lines that were used displayed varying degrees of in vitro invasiveness. METHODS: Hypoxia inducible factor-1alpha expression was detected by Western blot analysis. Cells were treated for 3 hours in 1% oxygen, then re-exposed to normoxia for varying times before lysis and detection of HIF-1alpha. RESULTS: Under normoxic conditions, HIF-1alpha expression was upregulated in invasive cells compared with noninvasive cells, and the degradation of HIF-1alpha following a hypoxic stimulus was blunted in invasive cells as compared with noninvasive cells. CONCLUSION: The authors presented evidence that dysregulation of HIF-1alpha may play a role in the malignant progression of head and neck squamous cell carcinoma. It is likely that dysregulated expression of the transcription factor HIF-1alpha contributes to the invasive properties associated with hypoxia and advanced head and neck squamous cell carcinoma.

Integrins in head and neck squamous cell carcinoma invasion.

OBJECTIVE: To relate the invasive properties of different squamous cell cancer cell lines to the function and expression of the integrins. STUDY DESIGN: A series of in vitro and in vivo experiments were designed to assess and compare integrin expression and function in two different head and neck squamous cell carcinoma cell lines. METHODS: Invasive properties of two squamous cell carcinoma cell lines (UM-SCC-1 and JHU-022-SCC) were assessed using an in vitro artificial matrix assay as well as an in vivo system with orthotopically implanted tumor cells in mice. Whole cell and surface expression levels of integrin subunits (alpha2, alpha3, alpha5, alpha6, beta1, and beta4) were determined for each cell line using Western blot analysis and flow cytometry. We compared the ability of JHU-022-SCC and UM-SCC-1 cells to bind the extracellular matrix elements collagen IV, fibronectin, laminin 5, and laminin10 using an in vitro adhesion assay. Contributions of the different integrins to the adhesive properties were determined by selective antibody blocking of different subunits. RESULTS: The UM-SCC-1 cell line is 50% more invasive in vitro and displays a greater propensity for perineural and lymphatic invasion in vivo. The UM-SCC-1 cells exhibited greater adherence to fibronectin than JHU-022-SCC cells. Alpha6 and beta4 expression is approximately twofold greater in the JHU-022-SCC cells. Alpha2, alpha3, and beta1 expression appears to be upregulated in UM-SCC-1 cells. CONCLUSION: The UM-SCC-1 carcinoma cells are more invasive than JHU-022-SCC cells and may be related to differential expression of the integrins alpha6beta4, alpha3beta1, and alpha2beta1.

Correlation of dyskerin expression with active proliferation independent of telomerase.

BACKGROUND: Dyskerin, which is an important component of the telomerase complex and is needed for normal telomerase activity, is frequently overexpressed in neoplasia. Dyskerin also plays an essential role in ribosome biogenesis. Because protein synthesis increases during tumorigenesis, this led us to hypothesize that dyskerin expression would be upregulated independently of the cell immortalization mechanism. METHODS: Dyskerin and telomerase reverse transcriptase (TERT) expression were examined in oral squamous cell carcinomas (OSCC) and patient-matched controls, as well as in a panel of telomerase-positive and telomerase-negative cells. Antisense inhibition of TERT was used to test the effects of downregulation of telomerase on dyskerin expression. RESULTS: Dyskerin was frequently overexpressed in OSCC and in immortalized and transformed keratinocytes relative to primary cells, independently of TERT and telomerase activity. Instead, dyskerin expression strongly correlated with cell proliferation rates. CONCLUSIONS: The role of dyskerin in tumorigenesis does not correlate with its function within the telomerase complex.

Is gene expression profiling of head and neck cancers ready for the clinic?

One of the major challenges in the head and neck oncology clinic is the need to identify biomarkers and/or gene expression signatures that complement, strengthen and increase the sensitivity and specificity of the current clinicopathologic analyses. Microarray analysis of head and neck tumors has demonstrated that the combined influence of many genes or biomarkers can make superior identifiers and/or predictors of tumor behavior and patient outcome. Here, an update of the recent literature on the prognostic and predictive value of microarrays for patients with head and neck squamous cell carcinomas is presented. Microarray technology has the potential for improved decision-making and corroboration within the clinical setting. However, further integration, standardization, validation and research are required before the use of microarray analysis is ready for routine clinical management of head and neck squamous cell carcinomas.

Lab-on-a-chip for oral cancer screening and diagnosis.

Oral squamous cell carcinoma (OSCC) is a disfiguring and deadly cancer. Despite advances in therapy, many patients continue to face a poor prognosis. Early detection is an important factor in determining the survival of patients with OSCC. No accurate, cost-efficient, and reproducible method exists to screen patients for OSCC. As a result, many patients are diagnosed at advanced stages of the disease. Early detection would identify patients, facilitating timely treatment and close monitoring. Mass screening requires a rapid oral cancer diagnostic test that can be used in a clinical setting. Current diagnostic techniques for OSCC require modern laboratory facilities, sophisticated equipment, and elaborate and lengthy processing by skilled personnel. The lab-on-chip technology holds the promise of replacing these techniques with miniaturized, integrated, automated, inexpensive diagnostic devices. This article describes lab-on-chip devices for biomarker-based identification of oral cancer. Similar methods can be employed for the screening of other types of cancers.

Carbon nanopipettes for cell probes and intracellular injection.

We developed integrated, carbon-based pipettes with nanoscale dimensions (CNP) that can probe cells with minimal intrusion, inject fluids into the cells, and concurrently carry out electrical measurements. Our manufacturing technique does not require cumbersome nanoassembly and is amenable to mass production. Using CNPs, we demonstrate the injection of reagents into cells with minimal intrusion and without inhibiting cell growth.

Modulation of satellite cell adhesion and motility following BMP2-induced differentiation to osteoblast lineage.

Quiescent satellite cells represent pluripotent stem cells capable of differentiating into other lineages. To define the potential changes in adhesion and motility in these differentiating cells, we utilized an established model system of murine-derived satellite cells induced with BMP2 to undergo osteoblastic differentiation. When mouse myogenic satellite cells were treated with BMP2, myogenesis was inhibited, and interaction with extracellular matrix ligands was altered. alpha7 integrin expression was rapidly downregulated with attenuation of adhesion and migration on laminin substrates. BMP2 also induced alpha2 integrin expression with increased adhesion and motility on collagen substrates as the pluripotent myoblasts develop into the osteogenic lineage. We examined the effect of BMP2 on alpha7 promoter activity in myoblasts using a CAT reporter gene. BMP2 was found to suppress integrin expression through a transcriptional mechanism. The results identify a novel role for BMP2 in modulating satellite cell integrin expression and altering their interactions with the microenvironment during osteoblastic differentiation.

The extracellular matrix in oral squamous cell carcinoma: friend or foe?

Oral squamous cell carcinoma is a disfiguring, highly invasive and metastatic cancer. Despite advances in detection and therapy, many patients will continue to face a poor prognosis. It is well established that the predominate factor determining overall survival in patients with oral squamous cell carcinoma is lymph node involvement. Tumor growth and progression to invasive cancer requires tumor cell interactions with the extracellular matrix. An understanding of how the extracellular matrix influences tumor development and invasion is fundamental in the development of new prognostic indicators and treatment strategies for oral squamous cell carcinoma. In this review, we summarize how changes in the extracellular matrix contribute to oral cancer development.

Suppression of laminin-5 expression leads to increased motility, tumorigenicity, and invasion.

Laminin-5 (Ln-5) is expressed in several human carcinomas and hypothesized to contribute to tumor invasion. To understand the role of Ln-5 in human cancers, we stably delivered small interfering RNAs (siRNAs) directed against the Ln-5 gamma2 chain into JHU-022-SCC cells (022), a non-invasive oral squamous cell carcinoma (OSCC) cell line which secretes Ln-5. Lysates from gamma2 siRNA cells (022-sigamma2) had nearly undetectable levels of the gamma2 chain while the alpha3 and beta3 subunits of Ln-5 remained unchanged compared to parental and control. In conditioned medium from 022-sigamma2 cells, the gamma2 chain and the Ln-5 heterotrimer were barely detectable, similar to an invasive OSCC cell line. Conditioned medium from 022-sigamma2 cells contained less alpha3 and beta3 subunits than both parental and control. Although the proliferation and adhesive properties of the 022-sigamma2 cells remained similar to parental and control cells, 022-sigamma2 cells showed increased detachment and a fibroblastic morphology similar to invasive cells. Moreover, migration, in vitro invasion, and in vivo tumorigenicity were enhanced in 022-sigamma2 cells. Our results suggest that the Ln-5 gamma2 chain regulates the secretion of the alpha3 and beta3 subunits. More importantly, suppression of Ln-5 results in a phenotype that is representative of invasive tumor cells.

Regulation of alpha7 integrin expression during muscle differentiation.

Expression of the laminin-binding alpha7 integrin is tightly regulated during myogenic differentiation, reflecting required functions that range from cell motility to formation of stable myotendinous junctions. However, the exact mechanism controlling alpha7 expression in a tissue- and differentiation-specific manner is poorly understood. This report provides evidence that alpha7 gene expression during muscle differentiation is regulated by the c-Myc transcription factor. In myoblasts, alpha7 is expressed at basal levels, but following conversion to myotubes the expression of the integrin is strongly elevated. The increased alpha7 mRNA and protein levels following myogenic differentiation are inversely correlated with c-Myc expression. Transfection of myoblasts with the c-Myc transcription factor down-regulated alpha7 expression, whereas overexpression of Madmyc, a dominant-negative c-Myc chimera, induced elevated alpha7 expression. Functional analysis with site-specific deletions identified a specific double E-box sequence in the upstream promoter region (-2.0 to -2.6 kb) that is responsible for c-Myc-induced suppression of alpha7 expression. DNA-protein binding assays and supershift analysis revealed that c-Myc forms a complex with this double E-box sequence. Our results suggest that the interaction of c-Myc with this promoter region is an important regulatory element controlling alpha7 integrin expression during muscle development and myotendinous junction formation.