Lovisa Farnebo

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. Todays 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.

WRAP53 promotes cancer cell survival and is a potential target for cancer therapy

We previously identified WRAP53 as an antisense transcript that regulates the p53 tumor suppressor. The WRAP53 gene also encodes a protein essential for Cajal body formation and involved in cellular trafficking of the survival of motor neuron complex, the telomerase enzyme and small Cajal body-specific RNAs to Cajal bodies. Here, we show that the WRAP53 protein is overexpressed in a variety of cancer cell lines of different origin and that WRAP53 overexpression promotes cellular transformation. Knockdown of the WRAP53 protein triggers massive apoptosis through the mitochondrial pathway, as demonstrated by Bax/Bak activation, loss of mitochondrial membrane potential and cytochrome c release. The apoptosis induced by WRAP53 knockdown could moreover be blocked by Bcl-2 overexpression. Interestingly, human tumor cells are more sensitive to WRAP53 depletion as compared with normal human cells indicating that cancer cells in particular depends on WRAP53 expression for their survival. In agreement with this, we found that high levels of WRAP53 correlate with poor prognosis of head and neck cancer. Together these observations propose a role of WRAP53 in carcinogenesis and identify WRAP53 as a novel molecular target for a large fraction of malignancies.

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.

Combining factors on protein and gene level to predict radioresponse in head and neck cancer cell lines.

BACKGROUND Radiotherapy is the main therapy for head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems, highlighting the need for predictive markers. In this study, we evaluated selected proteins, mutations, and single nucleotide polymorphisms (SNPs) involved in apoptosis, cell proliferation, and DNA repair alone or combined as predictive markers for radioresponse in 42 HNSCC cell lines. METHODS The expression of epidermal growth factor receptor, survivin, Bax, Bcl-2, Bcl-X(L) , cyclooxygenase-2 (COX-2), and heat shock protein 70 was analyzed by ELISA. Furthermore, mutations and SNPs in the p53 gene as well as SNPs in the MDM2, XRCC1, and XRCC3 genes were analyzed for their relation to radioresponse. To enable the evaluation of the predictive value of several factors combined, each cell line was allocated points based on the number of negative points (NNP) system, and the NNP sum was correlated with radioresponse. RESULTS Survivin was the only factor that alone was significantly correlated with the intrinsic radiosensitivity (IR; r = 0.36, P = 0.02). The combination of survivin, Bax, Bcl-2, Bcl-X(L) , COX-2, and the p53 Arg72Pro polymorphism was found to most strongly correlate with radioresponse (r = 0.553, P < 0.001). CONCLUSION These data indicate that the IR of 42 HNSCC cell lines can be predicted by a panel of factors on both the protein and gene levels. Moreover, among the investigated factors, survivin was the most promising biomarker of radioresponse.

Multiple genotypic aberrances associate to terminal differentiation‐deficiency of an oral squamous cell carcinoma in serum‐free culture

Oral squamous cell carcinoma (OSCC) lines proliferative in the serum-free conditions devised for normal oral keratinocytes (NOK) are virtually absent, complicating studies of carcinogenesis. A tongue squamous cell carcinoma generated under conditions for normal cell culture an apparently immortal line (termed LK0412) that has undergone >or=200 population doublings from over a year in culture. LK0412 exhibited epithelial morphology, intermediate filaments, desmosomes, and cytokeratin. Soft agar growth and tumorigenicity in athymic nude mice indicated the malignant phenotype. Compared with NOK, LK0412 exhibited increased indices for proliferation and apoptosis, and a decreased terminal differentiation index. Fetal bovine serum inhibited growth and increased apoptosis but failed to induce terminal differentiation of LK0412; the latter outcome differed clearly from that in NOK. Gene ontology assessment of transcript profiles implicated multiple alterations in biological processes, molecular functions, and cellular components in LK0412. Genetic changes, some that were confirmed to the protein level, included previously proposed OSCC markers, i.e., BAX, CDC2, and TP53, as well as multiple cancer-associated genes not considered for OSCC, e.g., BST2, CRIP1, ISG15, KLRC1, NEDD9, NNMT, and TWIST1. Elevation of p53 protein agreed with a missense mutation detectable in both the LK0412 line and the original tumor specimen. Moderate differentiation characterized the original tumor as well as tumors generated from inoculation of LK0412 in mice. Overall, the results suggest that the LK0412 cell line represent a subgroup of OSCC with unique genomic and phenotypic profiles. LK0412 should be useful to exploration of OSCC development, particularly the deregulated growth and differentiation responsiveness to serum factors.

Nuclear expression of WRAP53β is associated with a positive response to radiotherapy and improved overall survival in patients with head and neck squamous cell carcinoma

OBJECTIVES Today there are no reliable predictive markers for radiotherapy response in head and neck squamous cell carcinoma (HNSCC), leading to both under- and over-treatment of patients, personal suffering, and negative socioeconomic effects. Inherited mutation in WRAP53β (WD40 encoding RNA Antisense to p53), a protein involved in intracellular trafficking, dramatically increases the risk of developing HNSCC. The purpose of this study was to investigate whether WRAP53β can predict response to radiotherapy in patients with HNSCC. MATERIALS AND METHODS Tumor biopsies from patients with HNSCC classified as responders or non-responders to radiotherapy were examined for the expression of the WRAP53β protein and single nucleotide polymorphisms in the corresponding gene employing immunohistochemistry and allelic discrimination, respectively. In addition, the effect of RNAi-mediated downregulation of WRAP53β on the intrinsic radiosensitivity of two HNSCC cell lines was assed using crystal violet and clonogenic assays. RESULTS Nuclear expression of WRAP53β was significantly associated with better response to radiotherapy and improved patient survival. Downregulation of WRAP53β with siRNA in vitro enhanced cellular resistance to radiation. CONCLUSIONS Our findings suggest that nuclear expression of WRAP53β promotes tumor cell death in response to radiotherapy and is a promising predictor of radiotherapy response in patients with HNSCC.

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.

Targeting aldehyde dehydrogenase activity in head and neck squamous cell carcinoma with a novel small molecule inhibitor

Chemoresistant cancer cells express high levels of aldehyde dehydrogenases (ALDHs), particularly in head and neck squamous cell carcinoma (HNSCC). The ALDH family of enzymes detoxify both exogenous and endogenous aldehydes. Since many chemotherapeutic agents, such as cisplatin, result in the generation of cytotoxic aldehydes and oxidative stress, we hypothesized that cells expressing high levels of ALDH may be more chemoresistant due to their increased detoxifying capacity and that inhibitors of ALDHs may sensitize them to these drugs. Here, we show that overall ALDH activity is increased with cisplatin treatment of HNSCC and that ALDH3A1 protein expression is particularly enriched in cells treated with cisplatin. Activation of ALDH3A1 by a small molecule activator (Alda-89) increased survival of HNSCC cells treated with cisplatin. Conversely, treatment with a novel small molecule ALDH inhibitor (Aldi-6) resulted in a marked decrease in cell viability, and the combination of Aldi-6 and cisplatin resulted in a more pronounced reduction of cell viability and a greater reduction in tumor burden in vivo than what was observed with cisplatin alone. These data indicate that ALDH3A1 contributes to cisplatin resistance in HNSCC and that the targeting of ALDH, specifically, ALDH3A1, appears to be a promising strategy in this disease.Chemoresistant cancer cells express high levels of aldehyde dehydrogenases (ALDHs), particularly in head and neck squamous cell carcinoma (HNSCC). The ALDH family of enzymes detoxify both exogenous and endogenous aldehydes. Since many chemotherapeutic agents, such as cisplatin, result in the generation of cytotoxic aldehydes and oxidative stress, we hypothesized that cells expressing high levels of ALDH may be more chemoresistant due to their increased detoxifying capacity and that inhibitors of ALDHs may sensitize them to these drugs. Here, we show that overall ALDH activity is increased with cisplatin treatment of HNSCC and that ALDH3A1 protein expression is particularly enriched in cells treated with cisplatin. Activation of ALDH3A1 by a small molecule activator (Alda-89) increased survival of HNSCC cells treated with cisplatin. Conversely, treatment with a novel small molecule ALDH inhibitor (Aldi-6) resulted in a marked decrease in cell viability, and the combination of Aldi-6 and cisplatin resulted in a more pronounced reduction of cell viability and a greater reduction in tumor burden in vivo than what was observed with cisplatin alone. These data indicate that ALDH3A1 contributes to cisplatin resistance in HNSCC and that the targeting of ALDH, specifically, ALDH3A1, appears to be a promising strategy in this disease.

A Nordic survey on the management of head and neck CUP.

Abstract Conclusion: The management of Head and Neck Cancer of Unknown Primary (HNCUP) patients varies both between centres within and also between the Nordic countries. This study contributes to a continuing discussion of how to improve the accuracy of diagnosis and quality of treatment of HNCUP patients. Objectives: The initiative for this study was based on the lack of common guidelines for diagnostic procedures and for treatment of HNCUP patients in the Nordic countries constituting a region having a rather homogeneous population. Method: A structured questionnaire was sent to all university hospitals in the five Nordic countries. Results: Four of the five Nordic countries use either national guidelines or specific protocols when handling HNCUP. The main diagnostic tools are PET-CT, fine needle aspiration, endoscopic evaluation with biopsies, and most often bilateral tonsillectomy. At 21 of 22 university hospitals the treatment decision is made at a multidisciplinary conference. Three of seven Swedish centres use only radiotherapy or chemoradiotherapy to treat N+ HNCUP patients. Robotic surgery for biopsy of the tongue base is beginning to become an alternative to targeted biopsies in Sweden and Finland. Narrow Band Imaging is used only in Finland.

WRAP53β, survivin and p16INK4a expression as potential predictors of radiotherapy/chemoradiotherapy response in T2N0-T3N0 glottic laryngeal cancer

The current treatment recommendation for T2-3N0M0 glottic squamous cell carcinoma (SCC) in the Nordic countries comprises of radiotherapy (RT) and chemoradiotherapy (CRT). Tumor radiosensitivity varies and another option is primary surgical treatment, which underlines the need for predictive markers in this patient population. The aim of the present study was to investigate the relation of the proteins WRAP53β, survivin and p16INK4a to RT/CRT response and ultimate outcome of patients with T2-T3N0 glottic SCC. Protein expression was determined using immunohistochemistry on tumors from 149 patients consecutively treated with RT or CRT at Helsinki University Hospital, Karolinska University Hospital, and Linköping University Hospital during 1999–2010. Our results demonstrate a significantly better 5-year relapse-free survival, disease-free survival (DFS), disease-specific survival and overall survival of patients with T3N0 tumors treated with CRT compared with RT alone. Patients with tumors showing a cytoplasmic staining of WRAP53β revealed significantly worse DFS compared with those with nuclear staining. For survivin, we observed a trend towards better 5-year DFS in patients with strong nuclear survivin expression compared with those with weak nuclear survivin expression (P=0.091). Eleven (7%) tumors showed p16 positivity, with predilection to younger patients, and this age group of patients with p16-positive SCC had a significantly better DFS compared with patients with p16-negative SCC. Taken together, our results highlight WRAP53β as a potential biomarker for predicting RT/CRT response in T2-T3N0 glottic SCC. p16 may identify a small but distinct group of glottic SCC with favorable outcome. Furthermore, for T3N0 patients better outcome was observed following CRT compared to RT alone.