Expression and regulation of interleukins in human head and neck squamous cell carcinoma

Patients with head and neck squamous cell carcinoma (HNSCC) demonstrate abnormal cell-mediated immunity which is most pronounced at the primary tumor site. Therefore, we tested whether this aberrant immunity could be due to tumor-derived cytokines. We investigated the presence of cytokine mRNA and protein in 8 HNSCC-derived cell lines; RT-PCR results indicated mRNA's for IL-1$\alpha$ and TGF-$\alpha$ (8/8), TGF-$\beta$ (7/8), IL-1$\beta$ (7/8), IL-4 and IL-6 (4/8). IL-2, IFN-$\gamma,$ and TNF-$\alpha$ mRNA was not detected. Supernatants from 6 of these cell lines were analyzed by ELISA and IL-1$\alpha,$ IL-1$\beta,$ and IL-6 were markedly increased compared to HPV-16 immortalized human oral keratinocytes. IL-1$\alpha$ was found in the highest concentration $>$IL-6 $>$ IL-1$\beta.$^ To approach the mechanisms of cytokine regulation, 4 cell lines were compared for HPV DNA presence, p53 status, and cytokine expression. An association between HPV DNA and cytokine expression was not found. However, cell lines secreting the most IL-6 had mutant p53 and/or HPV 16 E6/E7 expression. Further regulatory investigations revealed that exogenous IL-1$\alpha$ and/or IL-1$\beta$ minimally stimulated the proliferation of 2/3 cell lines, as well as strongly induced IL-6 production in 3/3; this effect was completely abrogated by IL-1Ra. IL-1Ra also inhibited the secretion of IL-1$\alpha$ and IL-1$\beta$ in 2/3 cell lines. These data suggest an IL-1 autocrine loop in certain HNSCC cell lines. Because IL-2 induces IL-1 and is used in therapy of HNSCC, the expression of IL-2 receptor was also investigated; IL-2 $\alpha$ and $\beta$ subunits were detected in 3/3 cell lines and $\gamma$ subunits was detected in one. Exogenous IL-2 inhibited the proliferation, but stimulated the secretion of IL-1$\alpha$ in 2/3, and IL-1$\beta$ and IL-6 in 1/3 cell lines.^ To determine if our cell line findings were applicable to patients, immunohistochemistry was performed on biopsies from 12 invasive tumors. Unexpectedly, universal intracellular production of IL-1$\alpha,$ IL-1$\beta,$ and IL-6 protein was detected. Therefore, the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients. ^

Mechanism of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in head and neck squamous cell carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer and neuroblastoma in clinical trials. 4HPR induces growth inhibition and apoptosis in various cancer cells including head and neck squamous cell carcinoma (HNSCC) cells. 4HPR induces apoptosis by several mechanisms including increasing reactive oxygen species (ROS), or inducing mitochondrial permeability transition (MPT). 4HPR has also been shown to modulate the level of different proteins by transcriptional activation or posttranslational modification in various cellular contexts. However, the mechanism of its action is not fully elucidated. In this study, we explored the mechanism of 4HPR-induced apoptosis in HNSCC cells. ^ First, we identified proteins modulated by 4HPR by using proteomics approaches including: Powerblot western array and 2-dimensional polyacrylamide gel electrophoresis. We found that 4HPR modulated the levels of several proteins including c-Jun. Further analysis has shown that 4HPR induced activation of Activator Protein 1 (AP-1) components, c-Jun and ATF-2. We also found that 4HPR increased the level of Heat shock protein (Hsp) 70 and phosphorylation of Hsp27. ^ Second, we found that 4HPR induced prolonged activation of JNK, p38/MAPK and extracellular signal-regulated kinase (ERK). We also demonstrated that the activation of these kinases is required for 4HPR-induced apoptosis. JNK inhibitor SP600125 and siRNA against JNK1 and JNK2 suppressed, while overexpression of JNK1 enhanced 4HPR-induced apoptosis. p38/MAPK inhibitor PD169316 and MEK1/2 inhibitor PD98059 also suppressed 4HPR-induced apoptosis. We also demonstrated that activation of JNK, p38/MAPK and ERK is triggered by ROS generation induced by 4HPR. We also found that translation inhibitor, cycloheximide, suppressed 4HPR-induced apoptosis through inhibition of 4HPR-induced events (e.g. ROS generation, cytochrome c release, JNK activation and suppression of Akt). We also demonstrated that MPT is involved in 4HPR-induced apoptosis. ^ Third, we demonstrated the presence of NADPH oxidase in HNSCC 2B cells. We also found that 4HPR increased the level of the p67phox, a subunit of NADPH oxidase which participates in ROS production and apoptosis induced by 4HPR. ^ The novel insight into the mechanism by which 4HPR induces apoptosis can be used to improve design of future clinical studies with this synthetic retinoid in combination with specific MAPK modulators. ^

Characterization of Jak, STAT, and Src interactions in Head and Neck Squamous Cell Carcinoma

Recurrence of Head and Neck Squamous Cell Carcinoma (HNSCC) is common; thus, it is essential to improve the effectiveness and reduce toxicity of current treatments. Proteins in the Src/Jak/STAT pathway represent potential therapeutic targets, as this pathway is hyperactive in HNSCC and it has roles in cell migration, metastasis, proliferation, survival, and angiogenesis. During short-term Src inhibition, Janus kinase (Jak) 2, and signal transducer and activator of transcription (STAT) 3 and STAT5 are dephosphorylated and inactivated. Following sustained Src inhibition, STAT5 remains inactive, but Jak2 and STAT3 are reactivated following their early inhibition. To further characterize the mechanism of this novel feedback pathway we performed several experiments to look at the interactions between Src, Jak2, STAT5 and STAT3. We attempted to develop a non-radioactive kinase assay using purified recombinant Jak2 and Src proteins, but found that phospho-tyrosine antibodies were non-specifically binding to purified recombinant proteins. We then performed in vitro kinase assays (IVKAs) using purified recombinant Jak2, Src, STAT3, and STAT5 proteins with and without Src and Jak2 pharmacologic inhibitors. We also examined the interactions of these proteins in intact HNSCC cells. We found that recombinant Jak2, STAT3, and STAT5 are direct substrates of Src and that recombinant Src, STAT3, and STAT5 are direct substrates of Jak2 in the IVKA. To our knowledge, the finding that Src is a Jak substrate is novel and has not been shown before. In intact HNSCC cells we find that STAT3 can be reactivated despite continuous Src inhibition and that STAT5 continues to be inhibited despite Jak2 reactivation. Also, Jak2 inhibition did not affect Src or STAT5 activity but it did cause STAT3 inhibition. We hypothesized that the differences between the intact cells and the IVKA assays were due to a potential need for binding partners in intact HNSCC cells. One potential binding partner that we examined is the epidermal growth factor receptor (EGFR). We found that EGFR activation caused increased activation of Src and STAT5 but not Jak2. Our results demonstrate that although STAT3 and STAT5 are capable of being Src and Jak2 substrates, in intact HNSCC cells Src predominantly regulates STAT5 and Jak2 regulates STAT3. Regulation of STAT5 by Src may involve interactions between Src and EGFR. This knowledge along with future studies will better define the mechanisms of STAT regulation in HNSCC cells and ultimately result in an ideal combination of therapeutic agents for HNSCC.

The risk prediction for SPM and its impact on the survival in patients with head and neck squamous cell carcinoma

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth common malignancy in the world, with high rates of developing second primary malignancy (SPM) and moderately low survival rates. This disease has become an enormous challenge in the cancer research and treatments. For HNSCC patients, a highly significant cause of post-treatment mortality and morbidity is the development of SPM. Hence, assessment of predicting the risk for the development of SPM would be very helpful for patients, clinicians and policy makers to estimate the survival of patients with HNSCC. In this study, we built a prognostic model to predict the risk of developing SPM in patients with newly diagnosed HNSCC. The dataset used in this research was obtained from The University of Texas MD Anderson Cancer Center. For the first aim, we used stepwise logistic regression to identify the prognostic factors for the development of SPM. Our final model contained cancer site and overall cancer stage as our risk factors for SPM. The Hosmer-Lemeshow test (p-value= 0.15>0.05) showed the final prognostic model fit the data well. The area under the ROC curve was 0.72 that suggested the discrimination ability of our model was acceptable. The internal validation confirmed the prognostic model was a good fit and the final prognostic model would not over optimistically predict the risk of SPM. This model needs external validation by using large data sample size before it can be generalized to predict SPM risk for other HNSCC patients. For the second aim, we utilized a multistate survival analysis approach to estimate the probability of death for HNSCC patients taking into consideration of the possibility of SPM. Patients without SPM were associated with longer survival. These findings suggest that the development of SPM could be a predictor of survival rates among the patients with HNSCC.^

From genes to radioresistance in head and neck squamous cell carcinoma

Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.

Review: Head and neck squamous cell carcinoma in sub-Saharan Africa

Aim Review the literature from 1990 to 2013 to determine known anatomic sites, risk factors, treatments, and outcomes of head and neck squamous cell carcinoma (HNSCC) in sub-Saharan Africa. Methods Using a systematic search strategy, literature pertaining to HNSCC in sub-Saharan Africa was reviewed and patient demographics, anatomic sites, histology, stage, treatment, and outcomes were abstracted. The contributions of human immunodeficiency virus (HIV), human papillomavirus (HPV) and behavioural risk factors to HNSCC in the region were assessed. Results Of the 342 papers identified, 46 were utilized for review, including 8611 patients. In sub-Saharan Africa, the oropharyngeal/oral cavity was found to be the most common site, with 7750 cases (90% of all cases). Few papers distinguished oropharyngeal from oral cavity, making identification of possible HPV-associated oropharyngeal squamous cell carcinoma (SCC) difficult. SCC of the nasopharynx, nasal cavity, or paranasal sinuses was identified in 410 patients (4.8% of all cases). Laryngeal SCC was found in 385 patients (4.5% of all cases), and only 66 patients (0.8% of all cases) with hypopharyngeal SCC were identified. In 862 patients with data available, 43% used tobacco and 42% used alcohol, and reported use varied widely and was more common in laryngeal SCC than that of the oropharyngeal/oral cavity. Toombak and kola nut use was reported to be higher in patients with HNSCC. Several papers reported HIV-positive patients with HNSCC, but it was not possible to determine HNSCC prevalence in HIV-positive compared to negative patients. Reports of treatment and outcomes were rare. Conclusions The oropharyngeal/oral cavity was by far the most commonly reported site of HNSCC reported in sub-Saharan Africa. The roles of risk factors in HNSCC incidence in sub-Saharan Africa were difficult to delineate from the available studies, but a majority of patients did not use tobacco and alcohol.

ERCC1 protein, mRNA expression and T19007C polymorphism as prognostic markers in head and neck squamous cell carcinoma patients treated with surgery and adjuvant cisplatin-based chemoradiation

Adjuvant cisplatin-based chemoradiation improves survival in HNSCC patients presenting with risk features. ERCC1 (excision repair cross-complementation group 1) is associated with resistance to chemo- and radiation therapy and may have a prognostic value in HNSCC patients. Here we studied ERCC1 expression and the polymorphism T19007C as prognostic markers in these patients. This is a retrospective and translational analysis, where ERCC1 protein expression was evaluated by immunohistochemistry, using an H-score, and mRNA expression was determined by RT-PCR. T 19007C genotypes were detected by PCR-RFLP carried out using DNA template extracted from normal lymph nodes. A high H-score was seen in 32 patients (54%), who presented better 5-year overall survival (5-y OS: 50% vs. 18%, HR 0.43, p=0.026). Fifteen out of 45 patients (33%), with high mRNA expression, presented better 5-year overall survival (OS) (86% vs. 30%, HR 0.26, p=0.052). No OS difference was detected among T 19007C genotypes. High H-score and mRNA expression remained significant as favorable prognostic factors in a multivariate analysis. Collectively, our results suggest that high ERCC1 expression seems to be associated with better OS rates in HNSCC patients submitted to adjuvant cisplatin-based chemoradiation.

Analysis of naturally acquired CD4+T-cell responses to MAGE-A3 and MAGE-A4 cancer/testis antigens in patients with resected head and neck squamous cell carcinoma

Despite advances in the medical and surgical treatment of Head and Neck (HN) squamous cell carcinoma (HNSCC), long term survival has remained unchanged in the last 20 years. The obvious limitations of traditional therapeutic options strongly urge the development of novel therapeutic approaches. The molecular cloning of tumor antigens recognized by T lymphocytes in recent years has provided targets for specific immunotherapy. In this regard, frequent expression of Cancer Testis Antigens (CTA) has been repeatedly observed among HN tumors. We analyzed CTA expression in 46 HNSCC patients and found that MAGE-A3 and/or -A4 CTA were positive in over 70% of samples, regardless of the anatomical site of primary tumors in the upper aerodigestive tract. Still, immune responses against these CTA in HNSCC patients have not yet been investigated in detail. In this study we assessed the responsiveness of HNSCC patient's lymphocytes against overlapping peptides spanning the entire MAGE-A3 and -A4 proteins. After depletion of CD4+CD25+ regulatory T cells, and following three rounds of in vitro stimulation with pools of overlapping peptides, peripheral blood mononuclear cells (PBMCs) of HNSCC patients were screened by IFN-g and TNF-a intracellular cytokine staining for reactivity against MAGE-A3 or -A4 derived peptides. Cytokine secreting CD4+ T cells, specific for several peptides, were detected in 7/7 patients. In contrast, only 2/5 PBMC from healthy donors showed weak T cell responses against 2 peptides. CD4+ T cells specific for one epitope MAGE-A3(281-295), previously described as an HLA-DR11 restricted epitope naturally processed and presented by dendritic cells and tumor cells, were detected in two patients. MAGE-A3(161-175) specific CD4+ T cells were found in one patient. Six MAGE-A3 and -A4 new epitopes are being characterized. Together, these data suggest that naturally acquired CD4+ T cell responses against CT antigens occur in vivo in HNSCC patients, providing a rational basis for the use of the identified peptides in vaccination protocols.

Prognostic value of arginase-II expression and regulatory T-cell infiltration in head and neck squamous cell carcinoma.

Tumor-infiltrating lymphocytes are present in a variety of tumors and play a central role in antitumor immune responses. Nevertheless, most cancers progress probably because tumors are only weakly immunogenic and develop multiple immunosuppressive mechanisms. In the present study, on head and neck squamous cell carcinoma, we found high intraepithelial infiltration of regulatory FOXP3(+) T cells, and relatively high levels of BDCA2(+) and FOXP3(+) cells in stromal (peripheral) regions of the tumors. Tumor-infiltrating (intraepithelial) FOXP3(+) T cells were significantly more frequent in patients with oropharynx and oral cavity squamous cell carcinoma and in patients without lymph node metastasis. Furthermore, arginase-II (ARG2) was expressed by 60%, inducible nitric oxide synthetase by 9%, cyclooxygenase-2 by 43%, and B-cell lymphoma 2 (BCL2) by 26% of tumors. Interestingly, the absence of ARG2 expression, enhanced stromal infiltration of CD11c(+) myeloid dendritic cells, and high numbers of FOXP3(+) T cells were each significantly associated with prolonged overall survival, and the latter two parameters were also confirmed by multivariate analysis. For disease-free survival, multivariate analysis revealed significant negative correlations with BCL2 and ARG2 expression by tumor cells. These findings shed new light on mechanisms of cancer progression, and provide rationales for therapeutic inhibition of immunosuppressive mechanisms in head and neck squamous cell carcinoma.

Pattern and clinical significance of cancer-testis gene expression in head and neck squamous cell carcinoma

Les cancer-testis antigènes appartiennent à la famille des antigènes tumoraux spécifiques. Ils ont montré un pouvoir immunogène chez les patients porteurs de différents cancers. En effet, ils stimulent sélectivement les lymphocytes cytotoxiques, et leur expression spécifique dans les tissus tumoraux en fait une cible idéale pour une vaccination antitumorale. Le but de cette étude est d'identifier l'expression de certains de ces antigènes, d'analyser leur valeur pronostique et de déterminer la meilleure cible antigénique pour permettre une immunothérapie spécifique dans les carcinomes épidermoïdes des voies aérodigestives supérieures. Le profil et le taux d'expression de 12 cancer-testis antigènes (MAGE-A1, MAGE-A3, MAGE-A4, MAGEA10, MAGE-C2, NY-ESO-1, LAGE-1, SSX-2, SSX-4, BAGE, GAGE-1/2, GAGE-3/4) et de 3 autres antigènes tumoraux spécifiques (PRAME, HERV-K-MEL, NA-17A) ont été évalués par RT-PCR sur 57 échantillons de cancers ORL primaires. Les paramètres tumoraux et cliniques ont été prospectivement collectés afin de corréler ces données avec le résultat de nos investigations immunobiologiques. Quatre-vingt-huit pour cent des tumeurs expriment au moins 1 antigène. Une co-expression de 3 gènes ou plus est détectée chez 59% des patients. MAGE-A4 (60%), MAGE-A3 (51%), PRAME (49%) et HERV-K-MEL (42%) sont les gènes le plus fréquemment exprimés. Ils sont totalement absents des muqueuses saines avoisinantes. La présence de MAGE-A et NY-ESO-1 à la surface des cellules a été vérifiée par immunohistochimie. Nos analyses statistiques ont permis d'identifier une diminution de la survie liée au cancer chez les patients porteurs d'une tumeur exprimant de multiples cancer-testis antigènes et notamment MAGE-A4 dont l'expression indépendante d'autres éléments cliniques s'associe statistiquement à un taux de survie diminué. Nos résultats ont permis d'identifier un rôle pronostique de l'expression des gènes associés aux tumeurs dont l'expression est apparemment liée à un phénotype de malignité plus élevé. Cette constatation, corroborée par l'identification parallèle d'un infiltrat lymphocytaire spécifique confirme l'utilité potentielle de certains cancer-testis antigènes comme cible pour une immunothérapie ciblée dans les carcinomes des voies aérodigestives supérieures

Correlation between subjective evaluation of symptoms and objective findings in early recurrent head and neck squamous cell carcinoma.

IMPORTANCE: This study addresses the value of patients' reported symptoms as markers of tumor recurrence after definitive therapy for head and neck squamous cell carcinoma. OBJECTIVE: To evaluate the correlation between patients' symptoms and objective findings in the diagnosis of local and/or regional recurrences of head and neck squamous cell carcinomas in the first 2 years of follow-up. DESIGN: Retrospective single-institution study of a prospectively collected database. SETTING: Regional hospital. PARTICIPANTS: We reviewed the clinical records of patients treated for oral cavity, oropharyngeal, laryngeal, and hypopharyngeal carcinomas between January 1, 2008, and December 31, 2009, with a minimum follow-up of 2 years. MAIN OUTCOMES AND MEASURES: Correlation between symptoms and oncologic status (recurrence vs remission) in the posttreatment period. RESULTS: Of the 101 patients included, 30 had recurrences. Pain, odynophagia, and dysphonia were independently correlated with recurrence (odds ratios, 16.07, 11.20, and 5.90, respectively; P < .001). New-onset symptoms had the best correlation with recurrences. Correlation was better between 6 to 12 and 18 to 21 months after therapy and in patients initially treated unimodally (P < .05). Primary stage and tumor site had no effect. CONCLUSIONS AND RELEVANCE: The correlation between symptoms and oncologic status is low during substantial periods within the first 2 years of follow-up. New-onset symptoms, especially pain, odynophagia, or dysphonia, better correlate with tumor recurrence, especially in patients treated unimodally.

Concurrent use of cisplatin or cetuximab with definitive radiotherapy for locally advanced head and neck squamous cell carcinomas. Strahlentherapie in Kombination mit platinbasierter Chemotherapie oder Cetuximab zur Behandlung von lokal fortgeschrittenen Plattenepithelkarzinomen im Kopf-Hals-Bereich.

AIM: The goal of the present work was to compare outcomes of definitive concurrent cisplatin-based chemoradiotherapy (CRT) with cetuximab-based bioradiotherapy (BRT) in locally advanced head-and-neck squamous cell carcinoma (HNSCC). PATIENTS AND METHODS: Between 2006 and 2012, 265 patients with locally advanced HNSCC were treated at our institution with CRT (n = 194; 73 %) with three cycles of cisplatin (100 mg/m(2), every 3 weeks) or BRT (n = 71; 27 %) with weekly cetuximab. Patients receiving BRT had more pre-existing conditions (Charlson index ≥ 2) than the CRT group (p = 0.005). RESULTS: Median follow-up was 29 months. In all, 56 % of patients treated with CRT received the planned three cycles (92 % at least two cycles) and 79 % patients treated with BRT received six cycles or more. The 2-year actuarial overall survival (OS) and progression-free survival (PFS) were 72 % and 61 %, respectively. In the multivariate analysis (MVA), T4 stage, N2-3 stage, smoking status (current smoker as compared with never smoker), and non-oropharyngeal locations predicted for OS, whereas BRT association with OS was of borderline significance (p = 0.054). The 2-year actuarial locoregional control (LRC) and distant control (DC) rates were 73 and 79 %, respectively. CRT was independently associated with an improved LRC (2-year LRC: 76 % for CRT vs. 61 % for BRT) and DC (2-year LRC: 81 % for CRT vs. 68 % for BRT) in comparison with BRT (p < 0.001 and p = 0.01 in the MVA). Subgroup analyses showed that T4 patients benefited significantly from CRT (vs. BRT) in LRC, while T1-3 did not. BRT patients had more G3-4 skin complications (p < 0.001) and CRT patients had higher rates of feeding tube placement (p = 0.006) and G3-4 gastrointestinal toxicities (p < 0.001). CONCLUSION: This retrospective analysis showed a better LRC in locally advanced HNSCC treated by cisplatin-based CRT than cetuximab-based BRT, and a nonsignificant trend towards an improved OS.

Impaired aldehyde dehydrogenase 1 subfamily member 2A-dependent retinoic acid signaling is related with a mesenchymal-like phenotype and an unfavorable prognosis of head and neck squamous cell carcinoma.

BACKGROUND: An inverse correlation between expression of the aldehyde dehydrogenase 1 subfamily A2 (ALDH1A2) and gene promoter methylation has been identified as a common feature of oropharyngeal squamous cell carcinoma (OPSCC). Moreover, low ALDH1A2 expression was associated with an unfavorable prognosis of OPSCC patients, however the causal link between reduced ALDH1A2 function and treatment failure has not been addressed so far. METHODS: Serial sections from tissue microarrays of patients with primary OPSCC (n = 101) were stained by immunohistochemistry for key regulators of retinoic acid (RA) signaling, including ALDH1A2. Survival with respect to these regulators was investigated by univariate Kaplan-Meier analysis and multivariate Cox regression proportional hazard models. The impact of ALDH1A2-RAR signaling on tumor-relevant processes was addressed in established tumor cell lines and in an orthotopic mouse xenograft model. RESULTS: Immunohistochemical analysis showed an improved prognosis of ALDH1A2(high) OPSCC only in the presence of CRABP2, an intracellular RA transporter. Moreover, an ALDH1A2(high)CRABP2(high) staining pattern served as an independent predictor for progression-free (HR: 0.395, p = 0.007) and overall survival (HR: 0.303, p = 0.002), suggesting a critical impact of RA metabolism and signaling on clinical outcome. Functionally, ALDH1A2 expression and activity in tumor cell lines were related to RA levels. While administration of retinoids inhibited clonogenic growth and proliferation, the pharmacological inhibition of ALDH1A2-RAR signaling resulted in loss of cell-cell adhesion and a mesenchymal-like phenotype. Xenograft tumors derived from FaDu cells with stable silencing of ALDH1A2 and primary tumors from OPSCC patients with low ALDH1A2 expression exhibited a mesenchymal-like phenotype characterized by vimentin expression. CONCLUSIONS: This study has unraveled a critical role of ALDH1A2-RAR signaling in the pathogenesis of head and neck cancer and our data implicate that patients with ALDH1A2(low) tumors might benefit from adjuvant treatment with retinoids.

Targeting Adenoviral Gene Therapy Vectors to Head and Neck Squamous Cell Carcinoma and Heart

Gene therapy aims to treat diseases by introducing genetic material to the diseased tissue. For cancer treatment it is important to destroy cancerous cells; this can be achieved by introducing a gene, which induces cell death or by allowing viral vectors to replicate, which also results in destruction of cancerous cells. For cardiac diseases the approach is more like the former, except the gene produces beneficial effects, like angiogenesis. Adenoviruses have many beneficial qualities, which make the virus an interesting gene therapy vector; it can be produced relatively easily, its manipulation is quite easy and it has naturally broad tropism. By removing or replacing certain genes in the adenoviral genome, it can be made non-replicative. In this study, adenoviral receptor expression patterns were characterized in both head and neck squamous cell carcinoma and the human heart. Adenovirus serotype 5 receptor expression in head and neck cancer cell lines was found to be highly variable between cell lines and overall at lower levels, while Ad35 receptor expression was more uniform and at higher levels in all analyzed cell lines. It was also shown that a hybrid virus Ad5/35 is able to infect cells refractory to Ad5, which correlates with receptor expression in these cells. Furthermore, this difference in infection properties extends to cell killing efficiency in case of conditionally replicative viruses. Expression levels of adenoviral receptors CAR, CD46, CD86 and αv-integrins were found to be high both in normal and dilated cardiomyopathy heart tissue. The receptor levels also correlate with transduction efficiency after intracardiac injection. Ad5 showed superior transduction ability compared with Ad5/35, but evoked also a more profound immune reaction when administered this way. Adenoviral gene therapy vectors are the most used delivery vehicles in clinical trials to date. These vectors have proven to be well tolerated and positive results have been obtained when combined with traditional treatments, although poor transduction efficiency has often been reported due to low-level expression of viral receptors on target cells. In spite of this, the results are encouraging and merit for further research.