Anti-inflammatory (M2) macrophage media reduce transmission of oligomeric amyloid beta in differentiated SH-SY5Y cells.

Neuroinflammation plays an influential role in Alzheimer's disease (AD), although the mechanisms underlying this phenomenon remain largely unknown. Microglia are thought to be responsible for the majority of these effects and can be characterized into resting (M0), proinflammatory (M1), or anti-inflammatory (M2) functional phenotypes. We investigated the effects of conditioned macrophage media, as an analogue to microglia, on the transfer of oligomeric amyloid beta (oAß) between differentiated SH-SY5Y cells. We also investigated how the different inflammatory environments related to intercellular and intracellular changes. We demonstrate that M2 products decrease interneuronal transfer of oAß, while recombinant interleukin (IL)-4, IL-10, and IL-13 increase transfer. There were no alterations to the mRNA of a number of AD-related genes in response to the combination of oAß and M0, M1, or M2, but several intracellular proteins, some relating to protein trafficking and the endosomal/lysosomal system, were altered. Stimulating microglia to an M2 phenotype may thus slow down the progression of AD and could be a target for future therapies.

Epidermal growth factor is a potential biomarker for poor cetuximab response in tongue cancer cells.

BACKGROUND: Head and neck squamous cell carcinoma is frequently associated with aberrant epidermal growth factor receptor (EGFR) signaling, which contributes to tumor growth. Here, the functional importance of EGFR ligands in relation to proliferation and sensitivity to the EGFR-targeted therapy cetuximab was investigated in three tongue cancer cell lines. METHODS: The influence of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumor cell proliferation and cetuximab response was evaluated by the addition of recombinant human (rh) proteins or by siRNA-mediated downregulation of the endogenous ligand production. The expression, activation and cellular distribution of EGFR were assessed by Western blot analysis and immunocytochemistry. RESULTS: EGF downregulation suppressed the proliferation of all investigated tumor cell lines, whereas the response to an increased level of EGF differed between EGFR-overexpressing and EGFR-non-overexpressing cell lines. Furthermore, tumor cells consistently displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing was associated with an improved cetuximab response. The data regarding AR and EPR were inconclusive. CONCLUSIONS: Our data suggest that the amount of EGF is a determinant of the tumor cell proliferation rate and the response to cetuximab treatment in tongue cancer. Thus, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

DNA repair genes XPC, XPD, XRCC1, and XRCC3 are associated with risk and survival of squamous cell carcinoma of the head and neck.

Head and neck squamous cell carcinomas (HNSCC) are a heterogenous group of tumors with a high rate of early recurrences, second primary tumors, and mortality. Despite advances in diagnosis and treatment over the past decades, the overall 5-year survival rate remains around 50%. Since the head-and neck-region is continuously exposed to potentially DNA-damaging exogenous and endogenous factors, it is reasonable to expect that the DNA repair genes play a part in the development, progression, and outcome of HNSCC. The aim of this study was to investigate the SNPs XPC A499V, XPD K751Q, XRCC1 R399Q, and XRCC3 T241M as potential risk factors and indicators of survival among Caucasian patients. One-hundred-sixty-nine patients as well as 344 healthy controls were included and genotyped with PCR-RFLP. We showed that XPC A499V was associated with increased risk of HNSCC, especially laryngeal carcinoma. Among women, XPD K751Q was associated with increased risk of oral SCC. Furthermore, XPD homozygous mutant individuals had the shortest survival time, a survival time that increased however after full dose radiotherapy. Wild-type individuals of XRCC3 T241M demonstrated an earlier age of onset. HPV-positive never smokers had lower frequencies of p53 mutation. Among HNSCC patients, HPV-positivity was significantly associated with XRCC1 R399Q homozygous mutant genotype. Moreover, combinations of putative risk alleles seemed to act synergistically, increasing the risk of HNSCC. In conclusion, our results suggest that SNPs of the DNA repair genes XPC, XPD, XRCC1, and XRCC3 may affect risk and survival of HNSCC.

Strong expression of survivin is associated with positive response to radiotherapy and improved overall survival in head and neck squamous cell carcinoma patients.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy that is associated with severe mortality despite advances in therapy. Today's standard treatment most commonly includes radiotherapy, often combined with chemotherapy or surgery. There are so far no established biomarkers to predict response to radiation, and thus the aim of this study was to investigate a series of markers that could potentially identify HNSCC patients who would benefit from radiotherapy. The selected markers, both proteins (epidermal growth factor receptor, survivin and p53), and single nucleotide polymorphisms (SNPs) in the genes of XRCC3, XRCC1, XPC, XPD, MDM2, p53 and FGFR4 were correlated to the response to radiotherapy and overall survival. Investigations were performed on pretreatment tumor biopsies from patients classified as responders or nonresponders to radiotherapy. Protein expression was examined using immunohistochemistry and the genotyping of specific SNPs was analyzed using PCR-RFLP or pyrosequencing. We found that survivin expression was significantly stronger in the responder group (p = 0.003) and that patients with a strong survivin expression had a significantly better overall survival (p < 0.001). Moreover, downregulation of survivin by siRNA in two HNSCC cell lines significantly decreased their sensitivity to radiation. Among the SNPs analyzed, patients with the XPD Lys751Gln SNP had a significantly shorter overall survival (p = 0.048), and patients with the FGFR4 Gly388Arg SNP had a significantly longer overall survival (p = 0.010). In conclusion, our results suggest that survivin plays an important role in the response to radiotherapy and may be a useful marker for predicting radiotherapy response in patients with HNSCC.

EGFR status and EGFR ligand expression influence the treatment response of head and neck cancer cell lines.

BACKGROUND: Combination treatment (chemoradiotherapy) is the standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems. A high level of epidermal growth factor receptor (EGFR) has been associated with a more aggressive phenotype as well as decreased responsiveness to radio- or chemotherapy. We examined the role of EGFR status and EGFR ligand expression for the treatment response. METHODS: Intrinsic sensitivity to radiotherapy, cisplatin, and cetuximab treatments was investigated in 25 HNSCC cell lines. EGFR gene copy number, mRNA and protein expression, EGFR and Akt phosphorylation status, and mRNA expression of the EGFR ligands were analyzed using quantitative PCR and ELISA and assessed for their impact on treatment sensitivity. RESULTS: Different treatment modalities yielded great diversity in outcome; of note, cetuximab treatment stimulated growth in one cell line. When treatments were combined primarily additive effects were observed. While radioresistance tended to be associated with a high level of phosphorylated EGFR (pEGFR; P = 0.09), cetuximab-resistant cells had low levels of pEGFR (P = 0.13). The three most cetuximab-sensitive cell lines had high EGFR gene copy numbers. Furthermore, cetuximab treatment response was significantly correlated with epiregulin mRNA expression (r = -0.408, P = 0.043). Cisplatin-resistant tumor cells expressed significantly lower levels of EGFR protein (P = 0.04) compared to cisplatin-sensitive cells and tended to have lower levels of phosphorylated Akt (pAkt; P = 0.13) and lower expression levels of amphiregulin (P = 0.18). CONCLUSIONS: Epidermal growth factor receptor status and ligand expression influence the treatment sensitivity of HNSCC cells and may be useful as predictive markers.

Cancer-associated fibroblasts induce matrix metalloproteinase-mediated cetuximab resistance in head and neck squamous cell carcinoma cells.

A growing body of evidence suggests that components of the tumor microenvironment, including cancer-associated fibroblasts (CAF), may modulate the treatment sensitivity of tumor cells. Here, we investigated the possible influence of CAFs on the sensitivity of head and neck squamous cell carcinoma (HNSCC) cell lines to cetuximab, an antagonistic epidermal growth factor receptor (EGFR) antibody. Cetuximab treatment caused a reduction in the proliferation rate of HNSCC cell lines, whereas the growth of HNSCC-derived CAF cultures was unaffected. When tumor cells were cocultured with CAFs in a transwell system, the cetuximab-induced growth inhibition was reduced, and a complete protection from growth inhibition was observed in one of the tumor cell lines investigated. Media that had been conditioned by CAFs offered protection from cetuximab treatment in a concentration-dependent manner, suggesting that the resistance to treatment was mediated by CAF-derived soluble factors. The coculture of HNSCC cell lines with CAFs resulted in an elevated expression of matrix metalloproteinase-1 (MMP-1) in both the tumor cells and CAFs. Moreover, the CAF-induced resistance was partly abolished by the presence of an MMP inhibitor. However, CAFs treated with siRNA targeting MMP-1 still protected tumor cells from cetuximab treatment, suggesting that several MMPs may cooperate to facilitate resistance or that the protective effect is mediated by another member of the MMP family. These results identify a novel CAF-dependent modulation of cetuximab sensitivity and suggest that inhibiting MMPs may improve the effects of EGFR-targeted therapy.

Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells as demonstrated by genome-wide microarray analysis.

Platelets contain numerous growth factors essential for wound and fracture healing. We investigated the gene expression in human osteoblast-like cells stimulated with lysed platelets prepared in acidic, neutral, or alkaline buffers. Lysed platelets prepared in buffers at pH 5.4, 7.4, and 7.9, were added after neutralization to hFOB 1.19 cells. Genome-wide microarray analysis was performed using the Affymetrix GeneChip 7G Scanner. Biometric, cluster, and pathway analyses were performed with GeneSpring GX. Biometric analyses demonstrated that 53 genes were differentially regulated (p ≤ 0.005, ≥2-fold increase). Pathway analysis revealed 10 significant pathways of which eight are common ones regulating bone formation and cancer growth. Eleven genes were selected for quantitative real-time polymerase chain reaction (PCR) based on the microarray analysis of the lysed platelets prepared in the pH 5.4 experiments. In conclusion, acidic preparations of lysed platelet concentrates release factors essential for cell proliferation and particularly cell metabolism under hypoxic conditions. The genetic response from these factors was dominated by genes associated with the same pathways observed in bone formation and cancer growth. Activation of TGF-ß in the acidic preparation could be a stimulatory key factor of cell proliferation. These results support the hypothesis that acidification of platelets modifies the stimulatory response of mesenchymal cells in vitro, which is analogous with the observed milieu of a low pH present in wound and fracture sites, as well as in growing tumors.

Proteins and single nucleotide polymorphisms involved in apoptosis, growth control, and DNA repair predict cisplatin sensitivity in head and neck cancer cell lines.

The present study was undertaken to evaluate the possibility of using a panel of proteins and single nucleotide polymorphisms (SNPs) involved in apoptosis, growth control, and DNA repair as predictive markers for cisplatin sensitivity. For this purpose the intrinsic cisplatin sensitivity (ICS) was determined in 39 cell lines derived from squamous cell carcinomas of the head and neck using a colony-forming assay. In these cell lines and in normal oral keratinocytes (NOK), the expression of epidermal growth factor receptor (EGFR), Hsp70, Bax, Bcl-2, Bcl-XL, survivin, and COX-2 was determined. Moreover, the p53, MDM2, FGFR4, XPC, XPD, XRCC1, and XRCC3 genes were analyzed for the presence of specific single nucleotide polymorphisms (SNPs). Pearson's correlation test showed that EGFR was the only protein that was significantly correlated to the ICS (r=0.388, p=0.015). The combination of EGFR, Hsp70, Bax, and Bcl-2 gave the strongest correlation (r=0.566, p

Matrix metalloproteinase-7 and -13 expression associate to cisplatin resistance in head and neck cancer cell lines.

Concomitant chemoradiotherapy is a common treatment for advanced head and neck squamous cell carcinomas (HNSCC). Cisplatin is the backbone of chemotherapy regimens used to treat HNSCC. Therefore, the aim of this study was to identify predictive markers for cisplatin treatment outcome in HNSCC. The intrinsic cisplatin sensitivity (ICS) was determined in a panel of tumour cell lines. From this panel, one sensitive and two resistant cell lines were selected for comparative transcript profiling using microarray analysis. The enrichment of Gene Ontology (GO) categories in sensitive versus resistant cell lines were assessed using the Gene Ontology Tree Machine bioinformatics tool. In total, 781 transcripts were found to be differentially expressed and 11 GO categories were enriched. Transcripts contributing to this enrichment were further analyzed using Ingenuity Pathway Analysis (IPA) for identification of key regulator genes. IPA recognized 20 key regulator genes of which five were differentially expressed in sensitive versus resistant cell lines. The mRNA level of these five genes was further assessed in a panel of 25 HNSCC cell lines using quantitative real-time PCR. Among these key regulators, MMP-7 and MMP-13 are implicated as potential biomarkers of ICS. Taken together, genome-wide transcriptional analysis identified single genes, GO categories as well as molecular networks that are differentially expressed in HNSCC cell lines with different ICS. Furthermore, two novel predictive biomarkers for cisplatin resistance, MMP-7 and MMP-13, were identified.

Polymorphism of FGFR4 in cancer development and sensitivity to cisplatin and radiation in head and neck cancer.

The aim of this study was to investigate the predisposition of the FGFR4 Gly/Arg polymorphism for development of head and neck squamous cell carcinoma (HNSCC) and, furthermore, to examine if the FGFR4 Arg(388) allele can be associated with resistance to chemo- and radiotherapy. When analysing 110 tumour biopsies a significant 1.7-fold increased risk to develop HNSCC in individuals carrying the Gly(388) allele (p=0.026) was found. Moreover a 2-fold increased risk for males harbouring the Gly(388) allele (p=0.031) to develop HNSCC was detected. In 39 HNSCC cell lines the role of the Arg(388) allele for radiation and cisplatin sensitivity was investigated. Our results show no role of the Arg(388) allele for the radiosensitivity (p=0.996) but indicate a tendency to increased cisplatin sensitivity (p=0.141). When screening the transmembrane and kinase domains in the FGFR4 gene a novel mutation, probably generating a truncated protein lacking exons 14-18, was found in six of eight selected cell lines. Taken together, we have here identified a marker that predicts the risk to develop HNSCC and possibly the sensitivity to cisplatin as well as a novel mutation in the FGFR4 gene.