Regulation of squamous cell differentiation in human head and neck carcinoma cells by retinoids

Retinoids have been found to be effective in the prevention of premalignant lesions and second primary cancers in the upper aerodigestive tract. Further development of retinoids for prevention and therapy of head and neck squamous cell carcinoma (HNSCC) requires a better understanding of their mechanism of action on the growth and differentiation of such cells. I have chosen to employ cultured HNSCC cell lines as a model system for investigating the mechanism underlying the effects of retinoids. These cells are useful because all-trans retinoic acid (ATRA) inhibits their proliferation. Furthermore, two HNSCC cell lines were found to express three squamous differentiation (SqD) markers characteristic of normal keratinocytes and ATRA suppressed the expression of these markers as reported for normal keratinocytes. It is thought that nuclear retinoic acid receptors (RARs and RXRs), which act as DNA-binding transcription modulating factors, mediate the effects of retinoids on the growth and differentiation of normal and tumor cells. I found that all four cell lines examined expressed RAR-$\alpha ,$ RAR-$\tau ,$ and RXR-$\alpha$ and three of four expressed RAR-$\beta .$ ATRA treatment increased the level of RAR-$\alpha ,$ -$\beta ,$ and -$\tau$ in four cell lines. Two HNSCC cell lines that exhibited a progressive increase in the expression of SqD markers during growth in culture also showed a concurrent decrease in RAR-$\beta$ level. Moreover, increasing concentrations of RA suppressed the SqD marker while inducing RAR-$\beta$ mRNA. Several synthetic retinoids which exhibit a preference for binding to specific nuclear RARs showed a differential ability to inhibit cell proliferation, transactivate transcription of the reporter genes (CAT and luciferase) from the RA response element (RARE) of the RAR-$\beta$ gene, and induce RAR-$\beta$ expression. Those retinoids that were effective inducers of RAR-$\beta$ also suppressed SqD effectively, indicating an inverse relationship exists between the expression of RAR-$\beta$ and SqD. This inverse relationship suggests a role for RAR-$\beta$ in the suppression of SqD. ^

Molecular epidemiological studies on genetic predispositions to squamous cell carcinoma of the head and neck

Two molecular epidemiological studies were conducted to examine associations between genetic variation and risk of squamous cell carcinoma of the head and neck (SCCHN). In the first study, we hypothesized that genetic variation in p53 response elements (REs) may play roles in the etiology of SCCHN. We selected and genotyped five polymorphic p53 REs as well as a most frequently studied p53 codon 72 (Arg72Pro, rs1042522) polymorphism in 1,100 non-Hispanic White SCCHN patients and 1,122 age-and sex-matched cancer-free controls recruited at The University of Texas M. D. Anderson Cancer Center. In multivariate logistic regression analysis with adjustment for age, sex, smoking and drinking status, marital status and education level, we observed that the EOMES rs3806624 CC genotype had a significant effect of protection against SCCHN risk (adjusted odds ratio= 0.79, 95% confidence interval =0.64–0.98), compared with the -838TT+CT genotypes. Moreover, a significantly increased risk associated with the combined genotypes of p53 codon 72CC and EOMES -838TT+CT was observed, especially in the subgroup of non-oropharyneal cancer patients. The values of false-positive report probability were also calculated for significant findings. In the second study, we assessed the association between SCCHN risk and four potential regulatory single nucleotide polymorphisms (SNPs) of DEC1 (deleted in esophageal cancer 1) gene, a candidate tumor suppressor gene for esophageal cancer. After adjustment for age, sex, and smoking and drinking status, the variant -606CC (i.e., -249CC) homozygotes had a significantly reduced SCCHN risk (adjusted odds ratio = 0.71, 95% confidence interval = 0.52–0.99), compared with the -606TT homozygotes. Stratification analyses showed that a reduced risk associated with the -606CC genotype was more pronounced in subgroups of non-smokers, non-drinkers, younger subjects (defined as ≤ 57 years), carriers of TP53 Arg/Arg (rs1042522) genotype, patients with oropharyngeal cancer or late-stage SCCHN. Further in silico analysis revealed that the -249 T-to-C change led to a gain of a transcription factor binding site. Additional functional analysis showed that the -249T-to-C change significantly enhanced transcriptional activity of the DEC1 promoter and the DNA-protein binding activity. We conclude that the DEC1 promoter -249 T>C (rs2012775) polymorphism is functional, modulating susceptibility to SCCHN among non-Hispanic Whites. Additional large-scale, preferably population-based studies are needed to validate our findings.^

Association of p21/p27 polymorphisms with risk of HPV16-associated oral squamous cell carcinoma

The increasing incidence of oral squamous cell carcinoma (OSCC) among young adults has been associated with sexually transmitted infection of human papillomavirus (HPV), particularly HPV16. Given the roles of p21 (WAF1/Cip1/CDKN1A) and p27 (Kip1/CDKNIB) in cell-cycle regulation and of HPV16 E6 and E7 oncoproteins in p53 degradation and pRb inactivation, the effect of HPV16 L1 seropositivity and three putatively functional single-nucleotide polymorphisms (SNPs) of p21 (p21 C70T and p21 C98A) and p27 (p27 T109G), individually and in combination, on the risk of OSCC was evaluated in a hospital-based case-control study of 327 cases and 401 cancer-free controls who were frequency-matched on age, gender and smoking status. Individuals with HPV16 L1 seropositivity had an overall 3-fold increased risk of having OSCC than those with HPV16 seronegativity. The increased risk of HPV16-associated OSCC was particularly found among younger people (aged ≤ 50 years), males, never smokers, never drinkers and oropharynx cancer patients. None of three p21 and p27 polymorphisms alone was significantly associated with risk of OSCC. Individuals with variant genotypes for both p21 polymorphisms were more likely to have OSCC than individuals with wild-type genotypes or variant genotypes for either one of the p21 polymorphisms (adjusted OR, 1.4; 95% CI, 0.9-2.1). There was a borderline significant or significant interaction between the p21 C70T, combined p21 and combined p21/p27 genotypes and HPV16 L1 seropositivity on risk of OSCC. The three studied p21 and p27 polymorphisms, individually or in combination, did not appear to have an effect on HPV16-related clinical outcomes (overall and disease-free survival and tumor recurrence). Despite the fact that the exact biological mechanism remains to be explored, these findings suggest possible involvement of p21variants, particularly the p21 C70T variant genotypes (CT/TT), in the etiology of HPV16-associated OPSCC. Further large and functional studies are required to validate the findings.^

Factors associated with HPV16 status and survival among patients with squamous cell carcinoma of the oropharynx

Background: Squamous cell carcinoma of the oropharynx (SCCOP) is characterized by local tumor aggressiveness, high recurrence rates, a high incidence of second primary tumors, and medical comorbidities. Significant trends in demographic and clinical characteristics as well as survival among SCCOP patients have been observed over time, likely owing to the changing etiology of the disease. Human papillomavirus type 16 (HPV16) infection is associated with a significant proportion of these cancers. Biomarkers that may aid in identifying patients that are at higher risk of recurrence and death are important so that these patients may be followed more closely to improve their quality of life. ^ Study population and methods: The retrospective review (Specific Aim 2) included 3891 newly diagnosed, previously untreated patients presenting to our institution between 1955 and 2004. A total of 2299 patients treated at our institution were included in survival and recursive partitioning analysis. The prospective cohort study (Specific Aim 3) included 266 patients presenting to our institution between January 2006 and September 2009. ^ Results: The results from the retrospective review showed that over time, patients presented at younger ages and were more likely to have base of tongue/tonsil tumors and to be never/former smokers and moreover survival improved significantly over time. In survival and recursive partitioning analyses, the TNM staging system was efficient in prognosticating patients prior to 1995. However, in the recent decade, the TNM staging system was completely inadequate. The factors having the greatest positive effect on overall survival since 1995 were those common to HPV-associated SCCOP. The results from the prospective cohort study indicate that patients with high nodal stage and those with late stage disease have increased levels of pretreatment serum HPV DNA. ^ Conclusions: We saw a distinct improvement in survival among SCCOP patients over the past 50 years at our institution. The main factors contributing to this were changes in clinical characteristics, in particular surrogates for HPV status. The current TNM staging system for SCCOP is inadequate and incorporation of HPV status (and perhaps smoking status) is encouraged. Furthermore, although pretreatment circulating levels of HPV DNA was associated with higher N category and overall disease stage, it has limited utility as a marker for recurrence among SCCOP patients.^

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.^

AIS is an oncogene amplified in squamous cell carcinoma

We and others recently isolated a human p53 homologue (p40/p51/p63/p73L) and localized the gene to the distal long arm of chromosome 3. Here we sought to examine the role of p40/p73L, two variants lacking the N-terminal transactivation domain, in cancer. Fluorescent in situ hybridization (FISH) analysis revealed frequent amplification of this gene locus in primary squamous cell carcinoma of the lung and head and neck cancer cell lines. (We named this locus AIS for amplified in squamous cell carcinoma.) Furthermore, amplification of the AIS locus was accompanied by RNA and protein overexpression of a variant p68AIS lacking the terminal transactivation domain. Protein overexpression in primary lung tumors was limited to squamous cell carcinoma and tumors known to harbor a high frequency of p53 mutations. Overexpression of p40AIS in Rat 1a cells led to an increase in soft agar growth and tumor size in mice. Our results support the idea that AIS plays an oncogenic role in human cancer.

A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene.

The squamous cell carcinoma antigen (SCCA) is a member of the ovalbumin family of serine proteinase inhibitors (serpins). A neutral form of the protein is found in normal and some malignant squamous cells, whereas an acidic form is detected exclusively in tumor cells and in the circulation of patients with squamous cell tumors. In this report, we describe the cloning of the SCCA gene from normal genomic DNA. Surprisingly, two genes were found. They were tandemly arrayed and flanked by two other closely related serpins, plasminogen activator inhibitor type 2 (PAI2) and maspin at 18q21.3. The genomic structure of the two genes, SCCA1 and SCCA2, was highly conserved. The predicted amino acid sequences were 92% identical and suggested that the neutral form of the protein was encoded by SCCA1 and the acidic form was encoded by SCCA2. Further characterization of the region should determine whether the differential expression of the SCCA genes plays a causal role in development of more aggressive squamous cell carcinomas.

Canine intra-nasal squamous cell carcinoma treated with chemotherapy prior to surgical resection

A 12-year-old male castrated Samoyed dog was presented with left-sided epistaxis and sneezing. Diagnostic procedures included haematology and biochemistry testing, thoracic radiography, fine needle aspiration of regional lymph nodes, CT, rhinoscopy, incisional biopsy and histopathology. Squamous cell carcinoma of the rostral nasal cavity was diagnosed, with no evidence of metastatic disease. External beam radiation was not an accessible treatment option. Complete surgical resection of the tumour would have required a larger, more disfiguring resection of nasal planum and maxilla than the owner was prepared to accept and may have been associated with an unacceptable morbidity. As an alternative, the extent of disease was reduced using a combination of carboplatin, doxorubicin and piroxicam chemotherapy. This allowed a less extensive nasal planum removal to be performed to remove residual disease with clean margins. The patient achieved a 14 month disease free interval from the time of surgery to the time of local recurrence. Survival time from diagnosis to eventual euthanasia for progressive local disease was 18 months.

E2F suppression and Sp1 overexpression are sufficient to induce the differentiation-specific marker, transglutaminase type 1, in a squamous cell carcinoma cell line

Recently, E2F function has expanded to include the regulation of differentiation in human epidermal keratinocytes (HEKs). We extend these findings to report that in HEKs, Sp1 is a differentiation-specific activator and a downstream target of E2F-mediated suppression of the differentiation-specific marker, transglutaminase type 1 (TG-1). Deletion of elements between -0.084 to -0.034 kb of the TG-1 promoter disabled E2F1-induced suppression of promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and Sp3 bound this region. Protein expression analysis suggested that squamous differentiation was accompanied by increased Sp1/Sp3 ratio. Cotransfection of proliferating HEKs or the squamous cell carcinoma (SCC) cell line, KJD-1/SV40, with an E2F inhibitor (E2Fd/n) and Sp1 expression plasmid was sufficient to activate the TG-1 promoter. The suppression of Sp1 activity by E2F in differentiated cells appeared to be indirect since we found no evidence of an Sp1/E2F coassociation on the TG-1 promoter fragment. Moreover, E2F inhibition in the presence of a differentiation stimulus induced Sp1 protein. These data demonstrate that (i) Sp1 can act as a differentiation stimulus, (ii) E2F-mediated suppression of differentiation-specific markers is indirect via Sp1 inhibition and (iii) a combination of E2F inhibition and Sp1 activation could form the basis of a differentiation therapy for SCCs.

Total pharyngolaryngectomy for squamous cell carcinoma of the hypopharynx: A review

Objectives. To evaluate our experience with total pharyngolaryngectomy in the treatment of hypopharyngeal squamous cell carcinoma. Study Design: Retrospective analysis of consecutively treated patients in an academic otolaryngology, head and neck department. Methods. One hundred eighty patients who had total pharyngolaryngectomy performed for hypopharyngeal carcinoma were included in this study. Patients with a history of previous head and neck cancer were excluded. Clinicopathologic parameters were recorded and survival calculated using the Kaplan-Meier method. Results. One hundred sixty-two (90%) of the patients were male, and the patients had a mean age of 62 years. The majority (91%) of patients had advanced overall clinical stage disease (stage 3,4). Thirty-one (17.8%) and 43 (24%) patients developed locoregional and metastatic disease recurrence, respectively. The 2- and 5-year disease-specific survival rates were 72% and 52%, respectively. Advanced nodal stage, perineural invasion, lymphovascular invasion, and positive margins were predictors of poor survival on univariate analysis, and lymphovascular invasion was an independent prognostic factor on multivariate analysis. Conclusion: Surgery and postoperative radiotherapy remains the treatment against which other modalities should be compared for advanced stage hypopharyngeal squamous cell carcinoma.