EGFR modulates complement activation in head and neck squamous cell carcinoma.

BACKGROUND: The epidermal growth factor receptor (EGFR) is pivotal for growth of epithelial cells and is overexpressed in several epithelial cancers like head and neck squamous cell carcinoma (HNSCC). EGFR signalling is also involved in diverse innate immune functions in epithelia. We previously found a role for EGFR in modulating the complement system in skin, this prompted an investigation into EGFR role in complement modulation in HNSCC. METHODS: We used patient derived HNSCC cell lines with varying sensitivities to EGFR inhibitors, and generated EGFR inhibition resistant cell lines to study the role of EGFR in modulating complement in HNSCC. RESULTS: We found that HNSCC cell lines activate the complement system when incubated with human serum. This complement activation was increased in cell lines sensitive to EGFR inhibition following the use of the tyrosine kinase inhibitor Iressa. Sensitive cell line made resistant to EGFR-inhibitors displayed complement activation and a decrease in complement regulatory proteins even in the absence of EGFR-inhibitors. Complement activation did not cause lysis of HNSCC cells, and rather led to increased extracellular signal-regulated kinase (ERK) phosphorylation in one cell line. CONCLUSION: These data indicate that EGFR has a complement modulatory role in HNSCC, and that a prolonged EGFR-inhibition treatment in sensitive cancer cells increases complement activation. This has implications in understanding the response to EGFR inhibitors, in which resistance and inflammatory skin lesions are two major causes for treatment cessation.

The DNA damage response activates HPV16 late gene expression at the level of RNA processing.

We show that the alkylating cancer drug melphalan activated the DNA damage response and induced human papillomavirus type 16 (HPV16) late gene expression in an ATM- and Chk1/2-dependent manner. Activation of HPV16 late gene expression included inhibition of the HPV16 early polyadenylation signal that resulted in read-through into the late region of HPV16. This was followed by activation of the exclusively late, HPV16 splice sites SD3632 and SA5639 and production of spliced late L1 mRNAs. Altered HPV16 mRNA processing was paralleled by increased association of phosphorylated BRCA1, BARD1, BCLAF1 and TRAP150 with HPV16 DNA, and increased association of RNA processing factors U2AF65 and hnRNP C with HPV16 mRNAs. These RNA processing factors inhibited HPV16 early polyadenylation and enhanced HPV16 late mRNA splicing, thereby activating HPV16 late gene expression.

Short half-life of HPV16 E6 and E7 mRNAs sensitizes HPV16-positive tonsillar cancer cell line HN26 to DNA-damaging drugs.

Here we show that treatment of the HPV16-positive tonsillar cancer cell line HN26 with DNA alkylating cancer drug melphalan-induced p53 and activated apoptosis. Melphalan reduced the levels of RNA polymerase II and cellular transcription factor Sp1 that were associated with HPV16 DNA. The resulting inhibition of transcription caused a rapid loss of the HPV16 early mRNAs encoding E6 and E7 as a result of their inherent instability. As a consequence of HPV16 E6 and E7 down-regulation, the DNA damage inflicted on the cells by melphalan caused induction of p53 and activation of apoptosis in the HN26 cells. The BARD1-negative phenotype of the HN26 cells may have contributed to the failure to repair DNA damage caused by melphalan, as well as to the efficient apoptosis induction. Finally, nude mice carrying the HPV16 positive tonsillar cancer cells responded better to melphalan than to cisplatin, the chemotherapeutic drug of choice for tonsillar cancer. We concluded that the short half-life of the HPV16 E6 and E7 mRNAs renders HPV16-driven tonsillar cancer cells particularly sensitive to DNA damaging agents such as melphalan since melphalan both inhibits transcription and causes DNA damage.

A novel human in vitro papillomavirus type 16 positive tonsil cancer cell line with high sensitivity to radiation and cisplatin.

BACKGROUND: Human papillomavirus (HPV) is an established risk factor for oropharyngeal squamous cell carcinoma (OSCC). The aim was to establish cell lines from HPV-positive tonsil carcinomas to be used for treatment development. METHODS: Fresh samples from 23 HPV-positive tonsil carcinomas were cultivated in vitro. The established cell line was analyzed for viral characteristics, cell karyotype, TP53 status, and growth capabilities in nude mice. In vitro studies of sensitivities to radiation, cisplatin and cetuximab were performed. RESULTS: After 19 months (eight passages), one cell line, LU-HNSCC-26, was established in vitro and also grew as xenografts. The tumor was from a 48 year old non-smoking man with non-keratinizing, p16 positive tonsil OSCC, stage T2N0M0 with HPV16. It contained 19.5 (CV% 3.7) HPV16 copies/cell (passage 8). The complete HPV16 genome sequence was obtained. Episomal HPV16 was present with an E2/E7 ratio of 1.1 (CV% 2.6). In addition, HPV16 mRNA specific for the intact E2 gene was detected. The viral expression manifested 1.0 (CV% 0.1) E7 mRNA copies per HPV16 genome. The karyotype was determined and the cell line demonstrated wild type TP53. The ID50 for radiation was 0.90 Gy and the IC50 for cisplatin was 0.99 µmol/L. The cell line was inhibited to a maximum of 18% by cetuximab. CONCLUSIONS: We established an in vitro tonsil carcinoma cell line containing episomal HPV16. This is an important step towards efficient treatment development.

Ultrasound Characterization of Microbead and Cell Suspensions by Speed of Sound Measurements of Neutrally Buoyant Samples.

We present an experimental method including error analysis for the measurement of the density and compressibility of cells and microbeads; these being the two central material properties in ultrasound-based acoustophoretic applications such as particle separation, trapping, and up-concentration. The density of the microparticles is determined by using a neutrally buoyant selection process that involves centrifuging of microparticles suspended in different density solutions, CsCl for microbeads and Percoll for cells. The speed of sound at 3 MHz in the neutrally buoyant suspensions is measured as a function of the microparticle volume fraction, and from this the compressibility of the microparticles is inferred. Finally, from the obtained compressibility and density, the acoustic scattering coefficients and contrast factor of the microparticles are determined, and in a sensitivity analysis, the impact of the measurement errors on the computed acoustic properties is reported. The determination of these parameters and their uncertainties allow for accurate predictions of the acoustophoretic response of the microparticles. The method is validated by determining the density (0.1-1% relative uncertainty) and compressibility (1-3% relative uncertainty) of previously well-characterized polymer microbeads and subsequently applied to determine the density (0.1-1% relative uncertainty), compressibility (1% relative uncertainty), scattering coefficients, and acoustic contrast factors for nonfixed and fixed cells, such as red blood cells, white blood cells, DU-145 prostate cancer cells, MCF-7 breast cancer cells, and LU-HNSCC-25 head and neck squamous carcinoma cells in phosphate buffered saline. The results show agreement with published data obtained by other methods.

Erythropoietin suppresses the activation of pro-apoptotic genes in head and neck squamous cell carcinoma xenografts exposed to surgical trauma.

BACKGROUND: Several studies on the use of erythropoietin (Epo) to treat anaemia in patients undergoing cancer treatment have shown adverse effects on tumour control and survival. Experimental studies indicate that this could be linked to an interaction with wound healing processes and not an effect on tumour cells per se. We have previously shown that erythropoietin in combination with surgical trauma stimulates tumour growth. In the present study, we investigated the effect of surgery and Epo on gene expression. METHODS: Human tumours from oral squamous cell cancer were xenotransplanted to nude mice treated with Epo. The tumours were then transected in a standardised procedure to mimic surgical trauma and the change in gene expression of the tumours was investigated by microarray analysis. qRT-PCR was used to measure the levels of mRNAs of pro-apoptotic genes. The frequency of apoptosis in the tumours was assessed using immunohistochemistry for caspase-3. RESULTS: There was little change in the expression of genes involved in tumour growth and angiogenesis but a significant down-regulation of the expression of genes involved in apoptosis. This effect on apoptosis was confirmed by a general decrease in the expression of mRNA for selected pro-apoptotic genes. Epo-treated tumours had a significantly lower frequency of apoptosis as measured by immunohistochemistry for caspase 3. CONCLUSIONS: Our results suggest that the increased tumour growth during erythropoietin treatment might be due to inhibition of apoptosis, an effect that becomes significant during tissue damage such as surgery.This further suggests that the decreased survival during erythropoietin treatment might be due to inhibition of apoptosis.

Cell-line-specific stimulation of tumor cell aggressiveness by wound healing factors - a central role for STAT3.

BACKGROUND: Local recurrence is a major factor affecting survival after treatment for head and neck squamous cell carcinoma (HNSCC). It is possible that the normal processes involved in wound healing after surgical removal of a primary tumor can boost the regrowth of residual cancer cells, thereby contributing to the recurrent growth. In this work, we collected human wound fluids and used them to investigate the effect of wound healing factors on HNSCC cell lines in vitro. METHODS: Wound fluids were collected from thyroidectomized patients diagnosed with benign disease and were included in assays of cell proliferation, migration, cell scattering, and invasion. The involvement of intracellular signaling pathways and membrane receptors were investigated by western blotting and the inclusion of specific inhibitors. RESULTS: One out of four cell lines was greatly stimulated in proliferation, migration, cell scattering, and invasion by the addition of wound fluid as compared with addition of fetal bovine or human serum. These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 activation abolished the wound fluid response, showing that STAT3 plays an important role in the wound healing response. Several of the observed phenotypic changes were epithelial-to-mesenchymal transition (EMT)-like, but the appropriate changes were not seen in any of the EMT markers investigated. The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3. CONCLUSIONS: In conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

Coomassie staining as loading control in Western blot analysis.

In Western blotting, immunodetection of housekeeping proteins is routinely performed to detect differences in electrophoresis loading. The present work describes a much faster and simpler protein staining method, which is compatible with ordinary blocking conditions. In addition, the method can be used after immunodetection with superior linearity compared to ordinary staining methods. After immunoblotting and staining, protein bands can be further identified using peptide mass fingerprinting.

Reduced drug accumulation is more important in acquired resistance against oxaliplatin than against cisplatin in isogenic colon cancer cells.

Preclinical studies have indicated that there is only partial cross-resistance between cisplatin and oxaliplatin. The molecular background for this is incompletely known. To investigate the differences in resistance, we rendered a colon cancer cell line (S1) resistant against cisplatin and oxaliplatin and characterized the subclones with regard to cross-resistance, platinum uptake, and gene expression profiles. Four oxaliplatin and four cisplatin-resistant cell lines were produced from S1 by step-wise increasing the concentrations of the drugs in the growth medium. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and platinum accumulation in cell lysates and DNA preparations by inductively coupled plasma mass spectroscopy. Gene expression was investigated by cDNA microarrays. The protein expression of the ATP-binding cassette B1 (ABCB1) was measured by immunohistochemistry. The cisplatin-resistant cell lines were 1.5-6.2-fold resistant against cisplatin and the oxaliplatin-resistant sublines 2.6-17-fold resistant against oxaliplatin. There was a limited degree of cross-resistance. Oxaliplatin resistance could be explained to a larger degree by reduced drug accumulation whereas mechanisms for increased tolerance against platinum incorporation in DNA seemed to be of higher importance for resistance against cisplatin. A greater number of ABC transporters were upregulated in the oxaliplatin-resistant cell lines compared with those selected for cisplatin resistance. ABCB1 was highly overexpressed in the three most oxaliplatin-resistant sublines, but significantly underexpressed in the two most cisplatin-resistant cell lines. This was also confirmed by immunohistochemistry. However, functional tests did not show any increase in ABCB1 transport activity in the oxaliplatin-resistant sub-lines compared with S1.

Substantial intrinsic variability in chemoradiosensitivity of newly established anaplastic thyroid cancer cell-lines.

Background: Well characterized human cell lines are needed for preclinical treatment studies of anaplastic thyroid cancer (ATC).Aims/Objectives: The aim was to establish, verify and characterize a panel of ATC cell lines.Material and methods: Cell lines were established from ATC fine-needle aspiration biopsies and characterized genetically and functionally regarding treatment sensitivities.Results: Eight cell lines were established in vitro and the anaplastic thyroid origin was verified. Seven of the cell lines were also grown as xenografts. The cell lines harboured complex karyotypes with modal numbers in hyperdiploid to near-pentaploid range. Five were TP53 mutated and three carried the BRAFV600E mutation. None had rearrangements of RET. For doxorubicin, IC50 ranged from 0.42 to 46 nmol/L and for paclitaxel from 1.6 to 196 nmol/L. Radiation sensitivity varied between 2.6 and 6.3 Gy. Two of the BRAF mutated cell lines displayed high sensitivity to vemurafenib, while the third was similar to the wild-type ones.Conclusions and significance: We describe a series of new ATC cell lines demonstrating large heterogeneity in the response to cytostatic drugs and the BRAF inhibitor vemurafenib. The observations are relevant to future attempts to optimize treatment combinations for ATC.

No direct effects of erythropoietin beta on a head and neck squamous cell carcinoma cell line which is growth stimulated in vivo.

INTRODUCTION: Erythropoiesis-stimulating agents (ESAs) are used in cancer therapy to reverse anaemia. It has been suggested that ESAs might improve treatment outcome by reducing tumour hypoxia, but ESAs might also increase tumour growth. In this work, the effect of recombinant human erythropoietin (rHuEpo) beta was investigated on a human head and neck squamous carcinoma cell (HNSCC) line in vitro. The cell line was previously growth stimulated in combination with surgery in a xenograft model and the investigation was initiated to see if rHuEpo directly affects the tumour cell line, alone or in combination with cell stress, or if the in vivo effect should be attributed to secondary effects. MATERIAL AND METHODS: The cell line LU-HNSCC-7 was grown in vitro and treated with rHuEpo alone or in combination with radiation, cisplatin, hypoxia or tumour extracts. The expression of the Epo receptor (EpoR) was investigated by western blotting after one- and two-dimensional electrophoresis, RT-PCR and through analysis of the effect on EpoR signalling. RESULTS: The cell line was shown not to express EpoR. Furthermore, it was only possible to detect a minor effect on cell growth (1.4 times over control, p < 0.001) under specific conditions and at supra-pharmacological concentrations of rHuEpo beta. No effect was detected on cell migration. None of the cell stressing treatments could enhance the minor growth stimulatory effect of rHuEpo beta. DISCUSSION: The conclusion is that rHuEpo beta does not stimulate tumour growth of the investigated cell line through a direct interaction with tumour cells. We hypothesise that interactions with stromal cells and the stimulation of wound healing responses might, at least partly, explain the negative effects of ESA administration during cancer treatment. We propose that EpoR expression in HNSCC tumour cells might not be a good marker for prediction of ESA induced worsening of outcomes after cancer treatment.

Identification of Targetable Lesions in Anaplastic Thyroid Cancer by Genome Profiling.

Anaplastic thyroid cancer (ATC) is a rare and extremely malignant tumor with no available cure. The genetic landscape of this malignancy has not yet been fully explored. In this study, we performed whole exome sequencing and the RNA-sequencing of fourteen cases of ATC to delineate copy number changes, fusion gene events, and somatic mutations. A high frequency of genomic amplifications was seen, including 29% of cases having amplification of CCNE1 and 9% of CDK6; these events may be targetable by cyclin dependent kinase (CDK) inhibition. Furthermore, 9% harbored amplification of TWIST1, which is also a potentially targetable lesion. A total of 21 fusion genes in five cases were seen, none of which were recurrent. Frequent mutations included TP53 (55%), the TERT promoter (36%), and ATM (27%). Analyses of mutational signatures showed an involvement of processes that are associated with normal aging, defective DNA mismatch repair, activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) activity, failure of DNA double-strand break repair, and tobacco exposure. Taken together, our results shed new light on the tumorigenesis of ATC and show that a relatively large proportion (36%) of ATCs harbor genetic events that make them candidates for novel therapeutic approaches. When considering that ATC today has a mortality rate of close to 100%, this is highly relevant from a clinical perspective.

GABPA inhibits invasion/metastasis in papillary thyroid carcinoma by regulating DICER1 expression.

The ETS family transcription factor GABPA is suggested as an oncogenic element, which is further supported by the recent reporting of it as the sole ETS member to activate the mutant TERT promoter in thyroid carcinomas (TC). However, it remains unclear how GABPA contributes to TC pathogenesis. The present study is designed to address this issue. TERT expression was significantly diminished in TERT promoter-mutated TC cells upon GABPA inhibition. Surprisingly, GABPA depletion led to robustly increased cellular invasion independently of TERT promoter mutations and TERT expression. DICER1, a component of the microRNA machinery, was identified as a downstream effector of GABPA. GABPA facilitated Dicer1 transcription while its depletion reduced Dicer1 expression. The mutation of the GABPA binding site in the DICER1 promoter led to diminished basal levels of DICER1 promoter activity and abolishment of GABPA-stimulated promoter activity as well. The forced DICER1 expression abrogated the invasiveness of GABPA-depleted TC cells. Consistently, the analyses of 93 patients with papillary thyroid carcinoma (PTC) revealed a positive correlation between GABPA and DICER1 expression. GABPA expression was negatively associated with TERT expression and promoter mutations, in contrast to published observations in cancer cell lines. Lower GABPA expression was associated with distant metastasis and shorter overall/disease-free survival in PTC patients. Similar results were obtained for PTC cases in the TCGA dataset. In addition, a positive correlation between GABPA and DICER1 expression was seen in multiple types of malignancies. Taken together, despite its stimulatory effect on the mutant TERT promoter and telomerase activation, GABPA may itself act as a tumor suppressor rather than an oncogenic factor to inhibit invasion/metastasis in TCs and be a useful predictor for patient outcomes.

Regression analysis and modelling of data acquisition for SELDI-TOF mass spectrometry.

MOTIVATION: Pre-processing of SELDI-TOF mass spectrometry data is currently performed on a largel y ad hoc basis. This makes comparison of results from independent analyses troublesome and does not provide a framework for distinguishing different sources of variation in data. RESULTS: In this article, we consider the task of pooling a large number of single-shot spectra, a task commonly performed automatically by the instrument software. By viewing the underlying statistical problem as one of heteroscedastic linear regression, we provide a framework for introducing robust methods and for dealing with missing data resulting from a limited span of recordable intensity values provided by the instrument. Our framework provides an interpretation of currently used methods as a maximum-likelihood estimator and allows theoretical derivation of its variance. We observe that this variance depends crucially on the total number of ionic species, which can vary considerably between different pooled spectra. This variation in variance can potentially invalidate the results from naive methods of discrimination/classification and we outline appropriate data transformations. Introducing methods from robust statistics did not improve the standard errors of the pooled samples. Imputing missing values however-using the EM algorithm-had a notable effect on the result; for our data, the pooled height of peaks which were frequently truncated increased by up to 30%.

Rescue Effect Inherited in Colony Formation Assays Affects Radiation Response.

It is well known that nonirradiated cells can exhibit radiation damage (bystander effect), and recent findings have shown that nonirradiated cells may help protect irradiated cells (rescue effect). These findings call into question the traditional view of radiation response: cells cannot be envisioned as isolated units. Here, we investigated traditional colony formation assays to determine if they also comprise cellular communication affecting the radiation response, using colony formation assays with varying numbers of cells, modulated beam irradiation and media transfer. Our findings showed that surviving fraction gradually increased with increasing number of irradiated cells. Specifically, for DU-145 human prostate cancer cells, surviving fraction increased 1.9-to-4.1-fold after 5-12 Gy irradiation; and for MM576 human melanoma cells, surviving fraction increased 1.9-fold after 5 Gy irradiation. Furthermore, increased surviving fraction was evident after modulated beam irradiation, where irradiated cells could communicate with nonirradiated cells. Media from dense cell culture also increased surviving fraction. The results suggest that traditional colony formation assays comprise unavoidable cellular communication affecting radiation outcome and the shape of the survival curve. We also propose that the increased in-field surviving fraction after modulated beam irradiation is due to the same effect.

Cell line dependent expression of EpCAM influences the detection of circulating tumor cells with CellSearch.

OBJECTIVES: The existence of circulating tumor cells has emerged as an important factor for prognosis and survival. The CellSearch method is the only circulating tumor cell detection method approved by the US Food and Drug Administration for clinical use. It relies on the detection of EpCAM (epithelial cell adhesion molecule) and is approved for colon cancer, breast cancer and prostate cancer. We now investigated whether CellSearch can be used to quantify circulating tumor cells in head and neck squamous cell cancer. STUDY DESIGN AND METHODS: In this study, we investigated the expression of EpCAM in 12 head and neck squamous cell cancer cell lines using Western blot and how this affected their detectability with CellSearch in peripheral blood. RESULTS: We found a great variation in the expression of EpCAM between our head and neck squamous cell cancer cell lines. This was accompanied by variations in counting efficiency. CONCLUSION: We suggest that for reliable quantification of circulating tumor cells in blood from patients with head and neck squamous cell cancer cell, an epitope independent method is preferable. LEVEL OF EVIDENCE: NA.

Genomic complexity and targeted genes in anaplastic thyroid cancer cell lines.

Anaplastic thyroid cancer (ATC) is a highly malignant disease with a very short median survival time. Few studies have addressed the underlying somatic mutations, and the genomic landscape of ATC thus remains largely unknown. In the present study, we have ascertained copy number aberrations, gene fusions, gene expression patterns, and mutations in early-passage cells from ten newly established ATC cell lines using single nucleotide polymorphism (SNP) array analysis, RNA sequencing and whole exome sequencing. The ATC cell line genomes were highly complex and displayed signs of replicative stress and genomic instability, including massive aneuploidy and frequent breakpoints in the centromeric regions and in fragile sites. Loss of heterozygosity involving whole chromosomes was common, but there were no signs of previous near-haploidisation events or chromothripsis. A total of 21 fusion genes were detected, including six predicted in-frame fusions; none were recurrent. Global gene expression analysis showed 661 genes to be differentially expressed between ATC and papillary thyroid cancer cell lines, with pathway enrichment analyses showing downregulation of TP53 signalling as well as cell adhesion molecules in ATC. Besides previously known driver events, such as mutations in BRAF, NRAS, TP53 and the TERT promoter, we identified PTPRD and NEGR1 as putative novel target genes in ATC, based on deletions in six and four cell lines, respectively; the latter gene also carried a somatic mutation in one cell line. Taken together, our data provide novel insights into the tumourigenesis of ATC and may be used to identify new therapeutic targets.

Wound-healing factors can prime head and neck cancer cells to increase their tumor-forming capacity.

OBJECTIVES/HYPOTHESIS: We investigated whether exposing a wound-healing-sensitive cell line to human wound fluid (HWF) could prime the cells to increase their tumor-forming ability in nude mice and, if so, whether this ability can be inhibited by pharmacological substances. STUDY DESIGN: Experimental animal model. METHODS: Take rate was measured in BALB/c nude mice after pretreatment of the cells with HWF using human serum and fetal bovine serum as controls. Inhibition of signal transducer and activator of transcription 3 (STAT3) with S3I-201 tocilizumab, and of interleukin 6 receptor (IL6R) with tocilizumab was performed. RESULTS: Preincubation with HWF resulted in a significant increase in take rate compared to controls. The increase in take rate could be decreased by both STAT3 and IL6R inhibition. CONCLUSIONS: The results indicate that head and neck squamous cell cancer cells might be stimulated to increase their tumor-forming ability both close to a surgical wound and at more distant locations, as a consequence of the wound-healing response. The work also suggests new treatment modalities aimed at decreasing these stimulatory effects. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E213-E217, 2016.

Establishment and characterization of a human papillomavirus type 16-positive tonsillar carcinoma xenograft in BALB/c nude mice.

BACKGROUND: Among head and neck cancers, human papillomavirus type 16 (HPV16) is associated with tonsillar carcinomas. Despite this, no HPV16-positive tonsillar cancer cell line has been established in nude mice. METHODS: Fresh tonsillar carcinoma biopsies were obtained from 23 patients and implanted subcutaneously into nude mice (BALB/c, nu/nu). RESULTS: After 7 months, one xenograft was established. The primary tumor harbored 2.7 copies (95% confidence interval = 2.4-2.9) of HPV16/cell and displayed 99.9% (7904/7906) nucleotide identity to HPV16 (EU118173.1). The xenograft showed increased methylation in two E2-binding sites of the HPV16 genome. Both episomal and integrated HPV16 were detected in the original tumor and in 14 xenografts from the second passage. From this passage, a viral load of 6.4 copies/cell (range = 4.6-9.6) and 3.7 (range = 1.0-5.5) E7-mRNA transcripts/HPV16-genome were detected. CONCLUSION: This xenograft represents the first established HPV16-positive tonsillar tumor in nude mice and could provide an experimental system of HPV16-positive tonsillar cancers.

Anti- or pro-proliferation­conditional options for TGF-α and cetuximab in head and neck squamous cell carcinoma.

OBJECTIVES: Cetuximab is an epidermal growth factor receptor (EGFR)-targeting drug that has shown effects in head and neck squamous cell carcinoma (HNSCC). The effects are, however, small and have mainly been proven in a subset of patients, and the cost-effectiveness has been questioned. For this reason, we need to know more about the basic mechanisms controlling the effect of EGFR signalling on tumour growth. MATERIALS AND METHODS: We investigated the effect of the EGFR ligand transforming growth factor alpha (TGF-α) and cetuximab, alone and in combination, on HNSCC cell lines, measuring proliferation and the activity of intracellular signalling pathways. RESULTS: In line with previous reports we found the majority of the cell lines to be growth-inhibited by TGF-α. Surprisingly, two of the cell lines, which were more thoroughly investigated, were either growth-inhibited or stimulated by both cetuximab and TGF-α, depending on the presence or absence of the other substance. We also present data indicating the existence of two different receptor activities emanating from the EGFR protein. CONCLUSION: We therefore show that TGF-α can have both growth-stimulating and growth-inhibiting effects in the same cell line and that EGFR-targeting drugs can be similarly double-edged. The implication for such drugs is that the micro-environment within a tumour, and possibly within portions of a tumour, may influence the growth-inhibiting effect of the drug. There may also be important implications for our understanding of EGFR signalling and its influence on growth and development.