Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of human cancer and frequently metastasizes to LNs. Identifying metastasis-promoting factors is of immense clinical interest, as the prognosis for patients with even a single unilateral LN metastasis is extremely poor. Here, we report that p90 ribosomal S6 kinase 2 (RSK2) promotes human HNSCC cell invasion and metastasis. We determined that RSK2 was overexpressed and activated in highly invasive HNSCC cell lines compared with poorly invasive cell lines. Expression of RSK2 also correlated with metastatic progression in patients with HNSCC. Ectopic expression of RSK2 substantially enhanced the invasive capacity of HNSCC cells, while inhibition of RSK2 activity led to marked attenuation of invasion in vitro. Additionally, shRNA knockdown of RSK2 substantially reduced the invasive and metastatic potential of HNSCC cells in vitro and in vivo in a xenograft mouse model, respectively. Mechanistically, we determined that cAMP-responsive element-binding protein (CREB) and Hsp27 are phosphorylated and activated by RSK2 and are important for the RSK2-mediated invasive ability of HNSCC cells. Our findings suggest that RSK2 is involved in the prometastatic programming of HNSCC cells, through phosphorylation of proteins in a putative signaling network. Moreover, targeting RSK2 markedly attenuates in vitro invasion and in vivo metastasis of HNSCC cells, suggesting that RSK2 may represent a therapeutic target in the treatment of metastatic HNSCC.
It remains unclear how cancer cells coordinate glycolysis and biosynthesis to support rapidly growing tumors. We found that glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, contributes to biosynthesis regulation in part by controlling intracellular levels of its substrate 3-phosphoglycerate (3-PG) and product 2-phosphoglycerate (2-PG). 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP), while 2-PG activates 3-phosphoglycerate dehydrogenase to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA or a small molecule inhibitor PGMI-004A results in increased 3-PG and decreased 2-PG levels in cancer cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth.
Background
The AT-motif Binding Factor 1 (ATBF1) gene is frequently altered at the genetic level in several types of cancer, but its protein expression and subcellular localization have not been well studied in human cancers including head and neck squamous cell carcinomas (HNSCCs).
Methods
ATBF1 expression and localization were examined in five cell lines and 197 clinical specimens of HNSCC, and correlated with pathological and clinical characteristics.
Results
ATBF1 was predominantly localized in the nucleus of hyperplastic squamous epithelium. Whereas nuclear ATBF1 dramatically decreased in invasive tumors (p=0.0012), cytoplasmic ATBF1 levels progressively increased from dysplasia to invasive tumors (p<0.0001), and the increase correlated with poor survival. Reduced nuclear ATBF1 level was also detected in HNSCC cell lines.
Conclusions
Nuclear localization of ATBF1 is frequently interrupted in HNSCC, and the interruption is significantly associated with the progression of HNSCC. The cytoplasmic ATBF1 level could be useful for predicting patient survival.
Inhibition of cyclooxygenase-2 (COX-2) pathways may have significant implications for the prevention and treatment of head and neck squamous cell carcinoma (HNSCC). COX-2 is overexpressed in both premalignant lesions and invasive HNSCC. We examined COX-2 expression by immunohistochemistry in normal tissues, different stages of premalignant lesions, and carcinoma in-situ (CIS). We also evaluated the correlation between COX-2 expression and clinical characteristics of HNSCC patients. Tissue specimens were obtained from: premalignant lesions from 25 subjects enrolled in a biochemoprevention trial; tumor samples collected at diagnosis from 38 HNSCC patients enrolled in an induction chemotherapy trial; and normal control tissues from 10 non-cancer, non-smoking subjects. COX-2 was expressed in early and intermediate stages of premalignant lesions, increasing first in the basal and parabasal layers, then lower spinous, and upper spinous layers. This correlation was noted in normal epithelium (p<0.0001), histologically normal in-field samples (p<0.0001), low-grade dysplasia (p=0.024), and moderate-grade dysplasia (p=0.009), but was lost in the majority of high-grade dysplasia/CIS (p=0.896). COX-2 expression was also noted to increase progressively through the early stages of premalignancy, and to decrease in severe/CIS stage and invasive carcinoma. COX-2 expression in tumors from patients treated with induction chemotherapy was correlated with overall survival after controlling for clinical variables. These findings elucidate the differential expression pattern of COX-2 in stages of head and neck premalignant lesions and invasive carcinoma, supporting the rationale for COX-2 inhibition as an important strategy for cancer chemoprevention. Further validation of COX-2 expression is needed in prospective ongoing chemoprevention trials.
Purpose
We investigated the efficacy and underlying molecular mechanism of a novel chemopreventive strategy combining epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with cyclooxygenase-2 inhibitor (COX-2I).
Experimental Design
We examined the inhibition of tumor cell growth by combined EGFR-TKI (erlotinib) and COX-2I (celecoxib) treatment using head and neck cancer (HNC) cell lines and a preventive xenograft model. We studied the antiangiogenic activity of these agents and examined the affected signaling pathways by immunoblotting analysis in tumor cell lysates and immunohistochemistry (IHC) and enzyme immunoassay (EIA) analyses on the mouse xenograft tissues and blood, respectively. Biomarkers in these signaling pathways were studied by IHC, EIA, and an antibody array analysis in samples collected from participants in a phase I chemoprevention trial of erlotinib and celecoxib.
Results
The combined treatment inhibited HNC cell growth significantly more potently than either single agent alone in cell line and xenograft models, and resulted in greater inhibition of cell cycle progression at G1 phase than either single drug. The combined treatment modulated the EGFR and mTOR signaling pathways. A phase I chemoprevention trial of combined erlotinib and celecoxib revealed an overall pathologic response rate of 71% at time of data analysis. Analysis of tissue samples from participants consistently showed downregulation of EGFR, pERK and pS6 levels after treatment, which correlated with clinical response.
Conclusion
Treatment with erlotinib combined with celecoxib offers an effective chemopreventive approach through inhibition of EGFR and mTOR pathways, which may serve as potential biomarkers to monitor the intervention of this combination in the clinic.
FAS-associated death domain (FADD) is a key adaptor protein that bridges a death receptor (e.g., death receptor 5; DR5) to caspase-8 to form the death-inducing signaling complex during apoptosis. The expression and prognostic impact of FADD in head and neck squamous cell carcinoma (HNSCC) have not been well studied. This study focuses on detecting FADD expression and analyzing its prognostic impact in primary and metastatic HNSCCs. We found a significant increase in FADD expression in primary tumors with lymph node metastasis (LNM) in comparison with primary tumors with no LNM. This increase was significantly less in the matched LNM tissues. Both univariate and multivariable analyses indicated that lower FADD expression was significantly associated with better disease-free survival and overall survival in HNSCC patients with LNM although FADD expression did not significantly affect survival of HNSCC patients without LNM . When combined with DR5 or caspase-8 expression, patients with LNM expressing both low FADD and DR5 or both low FADD and caspase-8 had significantly better prognosis than those expressing both high FADD and DR5 or both high FADD and caspase-8. However, the expression of both low FADD and caspase-8 was significantly linked to worse overall survival compared with both high FADD and caspase-8 expression in HNSCC patients without LNM. Hence, we suggest that FADD alone or together with DR5 and caspase-8 participates in metastatic process of HNSCC.
Purpose
This study aimed to understand the prognostic value of integrin β1 expression in head and neck squamous cell carcinoma (HNSCC) and the mechanism underlying its association with metastatic HNSCC.
Experimental Design
Archival HNSCC tissues including 99 non-metastatic primary tumors and 101 metastatic primary tumors were examined for the association of integrin β1 expression with metastasis and disease prognosis by appropriate statistical methods. Fluorescence activated cell sorting was used to separate the integrin β1high/+ cell population from the integrin β1low/− population in HNSCC cell lines. These two populations and integrin β1 shRNA knock-down HNSCC cells were examined for the effect of integrin β1 on invasion in vitro and on lymph node and lung metastases in a xenograft mouse model. Expression and activation of matrix metalloproteinases (MMPs) were examined by zymography.
Results
Statistical analysis showed that integrin β1 expression was significantly higher in the metastatic primary tumors than in the non-metastatic tumors (42.6% vs 24.8%, p<0.0001 and p<0.0001 by univariate and multivariate analyses, respectively). In patients with lymph node metastasis, integrin β1 expression was inversely correlated with overall survival (p=0.035). The integrin β1 knock-down or integrin β1low/− HNSCC cells showed a significant reduction in lymph node and lung metastases in vivo (p<0.001 and p<0.05, respectively). Significantly reduced matrigel invasion capability was also found in integrin β1 knock-down or integrin β1low/− HNSCC cells (p< 0.01). Finally, zymography results showed integrin β1 affected HNSCC invasion by regulating MMP-2 activation.
Conclusion
These findings indicate that integrin β1 has a major impact on HNSCC prognosis through its regulation of metastasis.
This study explored whether expression of aldehyde dehydrogenase 1 (ALDH1A1) in the primary tumor correlated with lymph node metastasis (LNM) of squamous cell carcinoma of the head and neck (HNSCC). We used both quantum dot (QD)-based immunohistofluorescence (IHF) and conventional immunohistochemistry (IHC) to quantify ALDH1A1 expression in primary tumor samples taken from 96 HNSCC patients, 50 with disease in the lymph nodes and 46 without. The correlation between the quantified level of ALDH1A1 expression and LNM in HNSCC patients was evaluated with univariate and multivariate analysis. The prognostic value of ALDH1A1 was examined by Kaplan-Meier analysis and Wald test. ALDH1A1 was highly correlated with LNM in HNSCC patients (p < 0.0001 by QD-based IHF and 0.039 by IHC). The two methods (QD-based IHF and conventional IHC) for quantification of ALDH1A1 were found to be comparable (R = 0.75, p < 0.0001), but QD-IHF was more sensitive and objective than IHC. The HNSCC patients with low ALDH1A1 expression had a higher 5-year survival rate than those with high ALDH1A1 level (p = 0.025). Our study suggests that ALDH1A1 is a potential biomarker for predicting LNM in HNSCC patients, though it is not an independent prognostic factor for survival of HNSCC patients. Furthermore, QD-IHF has advantages over IHC in quantification of ALDH1A1 expression in HNSCC tissues.
Caveolin-1 (Cav-1) plays an important role in modulating cellular signalling, but its role in metastasis is not well defined. A significant reduction in Cav-1 levels was detected in lymph node metastases as compared with primary tumour of head and neck squamous cell carcinoma (HNSCC) specimens (P<0.0001), confirming the downregulation of Cav-1 observed in a highly metastatic M4 cell lines derived from our orthotopic xenograft model. To investigate the function of Cav-1 in metastasis of HNSCC, we compared stable clones of M4 cells carrying human cav-1 cDNA (CavS) with cells expressing an empty vector (EV) in vitro and in the orthotopic xenograft model. Overexpression of Cav-1 suppressed growth of the CavS tumours compared with the EV tumours. The incidence of lung metastases was significantly lower in animals carrying CavS tumours than those with EV tumours (P=0.03). In vitro, CavS cells displayed reduced cell growth, invasion, and increased anoikis compared with EV cells. In CavS cells, Cav-1 formed complex with integrin β1 and Src. Further application of integrin β1 neutralising antibody or Src inhibitor PP2 to EV cells illustrated similar phenotypes as CavS cells, suggesting that Cav-1 may play an inhibitory role in tumorigenesis and lung metastasis through regulating integrin β1- and Src-mediated cell–cell and cell–matrix interactions.
Hyperspectral imaging (HSI) is an emerging imaging modality that can provide a noninvasive tool for cancer detection and image-guided surgery. HSI acquires high-resolution images at hundreds of spectral bands, providing big data to differentiating different types of tissue. We proposed a deep learning based method for the detection of head and neck cancer with hyperspectral images. Since the deep learning algorithm can learn the feature hierarchically, the learned features are more discriminative and concise than the handcrafted features. In this study, we adopt convolutional neural networks (CNN) to learn the deep feature of pixels for classifying each pixel into tumor or normal tissue. We evaluated our proposed classification method on the dataset containing hyperspectral images from 12 tumor-bearing mice. Experimental results show that our method achieved an average accuracy of 91.36%. The preliminary study demonstrated that our deep learning method can be applied to hyperspectral images for detecting head and neck tumors in animal models.