Expression of Truncated Products at the 5'-Terminal Region of RIPK2 and Evolutive Aspects that Support Their Biological Importance.

Alternative splicing is the process of generating different mRNAs from the same primary transcript, which contributes to increase the transcriptome and proteome diversity. Abnormal splicing has been associated with the development of several diseases including cancer. Given that mutations and abnormal levels of the RIPK2 transcript and RIP-2 protein are frequent in tumors, and that RIP-2 modulates immune and inflammatory responses, we investigated alternative splicing events that result in partial deletions of the kinase domain at the N-terminus of RIP-2. We also investigated the structure and expression of the RIPK2 truncated variants and isoforms in different environments. In addition, we searched data throughout Supraprimates evolution that could support the biological importance of RIPK2 alternatively spliced products. We observed that human variants and isoforms were differentially regulated following temperature stress, and that the truncated transcript was more expressed than the long transcript in tumor samples. The inverse was found for the longer protein isoform. The truncated variant was also detected in chimpanzee, gorilla, hare, pika, mouse, rat, and tree shrew. The fact that the same variant has been preserved in mammals with divergence times up to 70 million years raises the hypothesis that it may have a functional significance.

Cetuximab chemotherapy resistance: Insight into the homeostatic evolution of head and neck cancer (Review).

The complex evolution of genetic alterations in cancer that occurs in vivo is a selective process involving numerous factors and mechanisms. Chemotherapeutic agents that prevent the growth and spread of cancer cells induce selective pressure, leading to rapid artificial selection of resistant subclones. This rapid evolution is possible because antineoplastic drugs promote alterations in tumor­cell metabolism, thus creating a bottleneck event. The few resistant cells that survive in this new environment obtain differential reproductive success that enables them to pass down the newly selected resistant gene pool. The present review aims to summarize key findings of tumor evolution, epithelial­mesenchymal transition and resistance to cetuximab therapy in head and neck squamous cell carcinoma.

Plasma metabolomics of oral squamous cell carcinomas based on NMR and MS approaches provides biomarker identification and survival prediction.

Metabolomics has proven to be an important omics approach to understand the molecular pathways underlying the tumour phenotype and to identify new clinically useful markers. The literature on cancer has illustrated the potential of this approach as a diagnostic and prognostic tool. The present study aimed to analyse the plasma metabolic profile of patients with oral squamous cell carcinoma (OSCC) and controls and to compare patients with metastatic and primary tumours at different stages and subsites using nuclear magnetic resonance and mass spectrometry. To our knowledge, this is the only report that compared patients at different stages and subsites and replicates collected in diverse institutions at different times using these methodologies. Our results showed a plasma metabolic OSCC profile suggestive of abnormal ketogenesis, lipogenesis and energy metabolism, which is already present in early phases but is more evident in advanced stages of the disease. Reduced levels of several metabolites were also associated with an unfavorable prognosis. The observed metabolomic alterations may contribute to inflammation, immune response inhibition and tumour growth, and may be explained by four nonexclusive views-differential synthesis, uptake, release, and degradation of metabolites. The interpretation that assimilates these views is the cross talk between neoplastic and normal cells in the tumour microenvironment or in more distant anatomical sites, connected by biofluids, signalling molecules and vesicles. Additional population samples to evaluate the details of these molecular processes may lead to the discovery of new biomarkers and novel strategies for OSCC prevention and treatment.

Investigating the effect of sexual behaviour on oropharyngeal cancer risk: a methodological assessment of Mendelian randomization.

BACKGROUND: Human papilloma virus infection is known to influence oropharyngeal cancer (OPC) risk, likely via sexual transmission. However, sexual behaviour has been correlated with other risk factors including smoking and alcohol, meaning independent effects are difficult to establish. We aimed to evaluate the causal effect of sexual behaviour on the risk of OPC using Mendelian randomization (MR). METHODS: Genetic variants robustly associated with age at first sex (AFS) and the number of sexual partners (NSP) were used to perform both univariable and multivariable MR analyses with summary data on 2641 OPC cases and 6585 controls, obtained from the largest available genome-wide association studies (GWAS). Given the potential for genetic pleiotropy, we performed a number of sensitivity analyses: (i) MR methods to account for horizontal pleiotropy, (ii) MR of sexual behaviours on positive (cervical cancer and seropositivity for Chlamydia trachomatis) and negative control outcomes (lung and oral cancer), (iii) Causal Analysis Using Summary Effect estimates (CAUSE), to account for correlated and uncorrelated horizontal pleiotropic effects, (iv) multivariable MR analysis to account for the effects of smoking, alcohol, risk tolerance and educational attainment. RESULTS: In univariable MR, we found evidence supportive of an effect of both later AFS (IVW OR = 0.4, 95%CI (0.3, 0.7), per standard deviation (SD), p = < 0.001) and increasing NSP (IVW OR = 2.2, 95%CI (1.3, 3.8) per SD, p = < 0.001) on OPC risk. These effects were largely robust to sensitivity analyses accounting for horizontal pleiotropy. However, negative control analysis suggested potential violation of the core MR assumptions and subsequent CAUSE analysis implicated pleiotropy of the genetic instruments used to proxy sexual behaviours. Finally, there was some attenuation of the univariable MR results in the multivariable models (AFS IVW OR = 0.7, 95%CI (0.4, 1.2), p = 0.21; NSP IVW OR = 0.9, 95%CI (0.5 1.7), p = 0.76). CONCLUSIONS: Despite using genetic variants strongly related sexual behaviour traits in large-scale GWAS, we found evidence for correlated pleiotropy. This emphasizes a need for multivariable approaches and the triangulation of evidence when performing MR of complex behavioural traits.

Using genetic variants to evaluate the causal effect of cholesterol lowering on head and neck cancer risk: A Mendelian randomization study.

Head and neck squamous cell carcinoma (HNSCC), which includes cancers of the oral cavity and oropharynx, is a cause of substantial global morbidity and mortality. Strategies to reduce disease burden include discovery of novel therapies and repurposing of existing drugs. Statins are commonly prescribed for lowering circulating cholesterol by inhibiting HMG-CoA reductase (HMGCR). Results from some observational studies suggest that statin use may reduce HNSCC risk. We appraised the relationship of genetically-proxied cholesterol-lowering drug targets and other circulating lipid traits with oral (OC) and oropharyngeal (OPC) cancer risk using two-sample Mendelian randomization (MR). For the primary analysis, germline genetic variants in HMGCR, NPC1L1, CETP, PCSK9 and LDLR were used to proxy the effect of low-density lipoprotein cholesterol (LDL-C) lowering therapies. In secondary analyses, variants were used to proxy circulating levels of other lipid traits in a genome-wide association study (GWAS) meta-analysis of 188,578 individuals. Both primary and secondary analyses aimed to estimate the downstream causal effect of cholesterol lowering therapies on OC and OPC risk. The second sample for MR was taken from a GWAS of 6,034 OC and OPC cases and 6,585 controls (GAME-ON). Analyses were replicated in UK Biobank, using 839 OC and OPC cases and 372,016 controls and the results of the GAME-ON and UK Biobank analyses combined in a fixed-effects meta-analysis. We found limited evidence of a causal effect of genetically-proxied LDL-C lowering using HMGCR, NPC1L1, CETP or other circulating lipid traits on either OC or OPC risk. Genetically-proxied PCSK9 inhibition equivalent to a 1 mmol/L (38.7 mg/dL) reduction in LDL-C was associated with an increased risk of OC and OPC combined (OR 1.8 95%CI 1.2, 2.8, p = 9.31 x10-05), with good concordance between GAME-ON and UK Biobank (I2 = 22%). Effects for PCSK9 appeared stronger in relation to OPC (OR 2.6 95%CI 1.4, 4.9) than OC (OR 1.4 95%CI 0.8, 2.4). LDLR variants, resulting in genetically-proxied reduction in LDL-C equivalent to a 1 mmol/L (38.7 mg/dL), reduced the risk of OC and OPC combined (OR 0.7, 95%CI 0.5, 1.0, p = 0.006). A series of pleiotropy-robust and outlier detection methods showed that pleiotropy did not bias our findings. We found limited evidence for a role of cholesterol-lowering in OC and OPC risk, suggesting previous observational results may have been confounded. There was some evidence that genetically-proxied inhibition of PCSK9 increased risk, while lipid-lowering variants in LDLR, reduced risk of combined OC and OPC. This result suggests that the mechanisms of action of PCSK9 on OC and OPC risk may be independent of its cholesterol lowering effects; however, this was not supported uniformly across all sensitivity analyses and further replication of this finding is required.

Genome-wide association meta-analysis identifies pleiotropic risk loci for aerodigestive squamous cell cancers.

Squamous cell carcinomas (SqCC) of the aerodigestive tract have similar etiological risk factors. Although genetic risk variants for individual cancers have been identified, an agnostic, genome-wide search for shared genetic susceptibility has not been performed. To identify novel and pleotropic SqCC risk variants, we performed a meta-analysis of GWAS data on lung SqCC (LuSqCC), oro/pharyngeal SqCC (OSqCC), laryngeal SqCC (LaSqCC) and esophageal SqCC (ESqCC) cancers, totaling 13,887 cases and 61,961 controls of European ancestry. We identified one novel genome-wide significant (Pmeta<5x10-8) aerodigestive SqCC susceptibility loci in the 2q33.1 region (rs56321285, TMEM273). Additionally, three previously unknown loci reached suggestive significance (Pmeta<5x10-7): 1q32.1 (rs12133735, near MDM4), 5q31.2 (rs13181561, TMEM173) and 19p13.11 (rs61494113, ABHD8). Multiple previously identified loci for aerodigestive SqCC also showed evidence of pleiotropy in at least another SqCC site, these include: 4q23 (ADH1B), 6p21.33 (STK19), 6p21.32 (HLA-DQB1), 9p21.33 (CDKN2B-AS1) and 13q13.1(BRCA2). Gene-based association and gene set enrichment identified a set of 48 SqCC-related genes rel to DNA damage and epigenetic regulation pathways. Our study highlights the importance of cross-cancer analyses to identify pleiotropic risk loci of histology-related cancers arising at distinct anatomical sites.

Biological and physical approaches on the role of piplartine (piperlongumine) in cancer.

Chronic inflammation provides a favorable microenvironment for tumorigenesis, which opens opportunities for targeting cancer development and progression. Piplartine (PL) is a biologically active alkaloid from long peppers that exhibits anti-inflammatory and antitumor activity. In the present study, we investigated the physical and chemical interactions of PL with anti-inflammatory compounds and their effects on cell proliferation and migration and on the gene expression of inflammatory mediators. Molecular docking data and physicochemical analysis suggested that PL shows potential interactions with a peptide of annexin A1 (ANXA1), an endogenous anti-inflammatory mediator with therapeutic potential in cancer. Treatment of neoplastic cells with PL alone or with annexin A1 mimic peptide reduced cell proliferation and viability and modulated the expression of MCP-1 chemokine, IL-8 cytokine and genes involved in inflammatory processes. The results also suggested an inhibitory effect of PL on tubulin expression. In addition, PL apparently had no influence on cell migration and invasion at the concentration tested. Considering the role of inflammation in the context of promoting tumor initiation, the present study shows the potential of piplartine as a therapeutic immunomodulator for cancer prevention and progression.

Elemental characterization of oral cavity squamous cell carcinoma and its relationship with smoking, prognosis and survival.

Oral cancer squamous cell carcinoma (OCSCC) mainly affects individuals aged between 50 and 70 years who consume tobacco and alcohol. Tobacco smoke contains hundreds of known toxic and carcinogenic molecules, and a few studies have sought to verify the relationship of such trace elements as risk or prognostic factors for head and neck cancer. We obtained 78 samples of tumor tissues from patients with OCSCC, and performed a qualitative elemental characterization using the micro X-Ray Fluorescence technique based on synchrotron radiation. We found the presence of magnesium, phosphorus, sulfur, chlorine, potassium, calcium, chromium, manganese, iron, zinc, cobalt, nickel, copper, arsenic and bromine in OCSCC samples. Magnesium, chlorine, chromium, manganese, nickel, arsenic and bromine are associated with smoking. We observed a significant association between relapse and chlorine and chromium. The presence of chlorine in the samples was an independent protective factor against relapse (OR = 0.105, CI = 0.01-0.63) and for best disease-free survival (HR = 0.194, CI = 0.04-0.87). Reporting for the first time in oral cancer, these results suggest a key relationship between smoking and the presence of certain elements. In addition, chlorine proved to be important in the context of patient prognosis and survival.

Mendelian Randomization and mediation analysis of leukocyte telomere length and risk of lung and head and neck cancers.

BACKGROUND: Evidence from observational studies of telomere length (TL) has been conflicting regarding its direction of association with cancer risk. We investigated the causal relevance of TL for lung and head and neck cancers using Mendelian Randomization (MR) and mediation analyses. METHODS: We developed a novel genetic instrument for TL in chromosome 5p15.33, using variants identified through deep-sequencing, that were genotyped in 2051 cancer-free subjects. Next, we conducted an MR analysis of lung (16 396 cases, 13 013 controls) and head and neck cancer (4415 cases, 5013 controls) using eight genetic instruments for TL. Lastly, the 5p15.33 instrument and distinct 5p15.33 lung cancer risk loci were evaluated using two-sample mediation analysis, to quantify their direct and indirect, telomere-mediated, effects. RESULTS: The multi-allelic 5p15.33 instrument explained 1.49-2.00% of TL variation in our data (p = 2.6 × 10-9). The MR analysis estimated that a 1000 base-pair increase in TL increases risk of lung cancer [odds ratio (OR) = 1.41, 95% confidence interval (CI): 1.20-1.65] and lung adenocarcinoma (OR = 1.92, 95% CI: 1.51-2.22), but not squamous lung carcinoma (OR = 1.04, 95% CI: 0.83-1.29) or head and neck cancers (OR = 0.90, 95% CI: 0.70-1.05). Mediation analysis of the 5p15.33 instrument indicated an absence of direct effects on lung cancer risk (OR = 1.00, 95% CI: 0.95-1.04). Analysis of distinct 5p15.33 susceptibility variants estimated that TL mediates up to 40% of the observed associations with lung cancer risk. CONCLUSIONS: Our findings support a causal role for long telomeres in lung cancer aetiology, particularly for adenocarcinoma, and demonstrate that telomere maintenance partially mediates the lung cancer susceptibility conferred by 5p15.33 loci.

Differentially expressed proteins in positive versus negative HNSCC lymph nodes.

BACKGROUND: Lymph node metastasis is one of the most important prognostic factors in head and neck squamous cell carcinomas (HNSCCs) and critical for delineating their treatment. However, clinical and histological criteria for the diagnosis of nodal status remain limited. In the present study, we aimed to characterize the proteomic profile of lymph node metastasis from HNSCC patients. METHODS: In the present study, we used one- and two-dimensional electrophoresis and mass spectrometry analysis to characterize the proteomic profile of lymph node metastasis from HNSCC. RESULTS: Comparison of metastatic and non-metastatic lymph nodes showed 52 differentially expressed proteins associated with neoplastic development and progression. The results reinforced the idea that tumors from different anatomical subsites have dissimilar behaviors, which may be influenced by micro-environmental factor including the lymphatic network. The expression pattern of heat shock proteins and glycolytic enzymes also suggested an effect of the lymph node environment in controlling tumor growth or in metabolic reprogramming of the metastatic cell. Our study, for the first time, provided direct evidence of annexin A1 overexpression in lymph node metastasis of head and neck cancer, adding information that may be useful for diagnosing aggressive disease. CONCLUSIONS: In brief, this study contributed to our understanding of the metastatic phenotype of HNSCC and provided potential targets for diagnostic in this group of carcinomas.

Genomic analysis of head and neck cancer cases from two high incidence regions.

We investigated how somatic changes in HNSCC interact with environmental and host risk factors and whether they influence the risk of HNSCC occurrence and outcome. 180-paired samples diagnosed as HNSCC in two high incidence regions of Europe and South America underwent targeted sequencing (14 genes) and evaluation of copy number alterations (SCNAs). TP53, PIK3CA, NOTCH1, TP63 and CDKN2A were the most frequently mutated genes. Cases were characterized by a low copy number burden with recurrent focal amplification in 11q13.3 and deletion in 15q22. Cases with low SCNAs showed an improved overall survival. We found significant correlations with decreased overall survival between focal amplified regions 4p16, 10q22 and 22q11, and losses in 12p12, 15q14 and 15q22. The mutational landscape in our cases showed an association to both environmental exposures and clinical characteristics. We confirmed that somatic copy number alterations are an important predictor of HNSCC overall survival.

ATM, BCL2, and TGFß Gene Polymorphisms as Radiotherapy Outcome Biomarkers in Head and Neck Squamous Cell Carcinoma Patients.

AIMS: Polymorphisms in cell cycle genes are considered prognostic as radiosensitivity markers in patients with head and neck squamous cell carcinoma. Therefore, we aimed to investigate the relationship of ATM 5557G>A, ATM IVS62 + 60G>A, TP53 215G>C, BCL2-938C>A, TGFß-509C>T, and TGFß 29C>T with radiotherapy response. MATERIALS AND METHODS: Genotyping was performed by polymerase chain reaction followed by restriction fragment length polymorphism in 210 patients with oral cavity/oropharyngeal carcinoma and 101 patients with laryngeal tumors. RESULTS: In irradiated oral cavity/oropharyngeal tumors, the ATM IVS62 + 60G>A AA genotype significantly increased local recurrence risk (odds ratio [OR] = 4.43; confidence interval [CI] = 1.22-16.13) and the BCL2-938C>A C allele and the TGFß-509C>T T allele were associated with worse disease-specific survival (hazard ratio [HR] = 0.46; CI = 0.24-0.90 and HR = 2.20; CI = 1.12-4.29, respectively). In irradiated laryngeal carcinoma, the TGFß 29C>T C allele was associated with increased local recurrence risk (OR = 0.09; CI = 0.02-0.53), death rate (OR = 0.18; CI = 0.04-0.86), and worse local disease-free and disease-specific survival rates (HR = 0.13; CI = 0.03-0.59 and HR = 0.21; CI = 0.07-0.60, respectively), while the BCL2-938C>A C allele was related to a worse disease-specific survival (HR = 0.32; CI = 0.12-0.83). DISCUSSION: These results can help individualize treatment according to a patient's genetic markers. We demonstrated that ATM IVS62 + 60G>A, TGFß 29C>T, TGFß-509C>T, and BCL2-938C>A can function as biomarkers of tumor radiosensitivity, being candidates for a predictive genetic profile of radiotherapy response.

ANXA1Ac2-26 peptide, a possible therapeutic approach in inflammatory ocular diseases.

The eye is immunologically privileged when inflammatory responses are suppressed. One component responsible for the suppression of inflammatory responses is the blood retinal barrier, which comprises the retinal pigment epithelium. The destruction of this barrier initiates inflammation, which can affect any part of the eye. Therefore, inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1) protein acts as a modulator of inflammation. In this study we aimed to improve the knowledge of this area by investigating how a peptide of the ANXA1 protein (ANXA1Ac2-26) modulates the morphology, proliferation, migration and expression of genes and proteins in human retinal pigment epithelium cells (ARPE-19). Determining how signaling pathways (NF-κB and UBC) are modulated by the ANXA1Ac2-26 peptide could be important for understanding the inflammatory process. ARPE-19 cells were activated by bacterial lipopolysaccharide endotoxin (LPS) and treated with ANXA1Ac2-26 peptide, in a concentration of 1.7µM and 33.8µM. We observed that the LPS activation diminished the levels of endogenous ANXA1 after 2h and 24h and ANXA1Ac2-26 peptide decreased the proliferation and re-establishes the migration of ARPE-19 cells. After using a hybridization approach, 80 differentially expressed genes were found. Five of these genes were selected (LRAT, CTGF, MAP1B, ALDH1A3 and SETD7) and all were down-regulated after treatment with the peptide. The genes CTGF and LRAT would be considered as potential molecular markers of ophthalmologic inflammation. The expression of pro-inflammatory cytokines was also decreased after the treatment, indicating the efficiency of the anti-inflammatory peptide at high concentrations, since the reduction in the levels of these mediators were observed after the treatment with ANXA1Ac2-26 peptide at 33.8µM. Our results suggest that the retinal pigment epithelial cells are a potential target of the ANXA1 protein and point to possible applications of the ANXA1Ac2-26 peptide as an innovative therapy for the treatment of ocular inflammation.

Geographic heterogeneity in the prevalence of human papillomavirus in head and neck cancer.

Human papillomavirus (HPV) causes oropharyngeal squamous cell carcinoma (OPSCC), although strongly divergent results have been reported regarding the prevalence of HPV16 in different countries, whether this represents important differences in etiology remains unclear. Applying rigorous protocols for sample processing, we centrally evaluated 1,420 head and neck tumors (533 oropharynx, 395 oral cavity and 482 larynx) from studies conducted in the US, Europe and Brazil for mucosal HPV DNA and p16INK4a expression to evaluate regional heterogeneity in the proportion of HPV16-associated OPSCC and other head and neck cancer, and to assess covariates associated with the risk of HPV16-positive OPSCC. While majority of OPSCC in the US (60%) were HPV16-positive, this proportion was 31% in Europe and only 4% in Brazil (p < 0.01). Similar differences were observed for other head and neck tumors, ranging from 7% in the US and 5% in Europe, to 0% in South America. The odds of HPV16-positive OPSCC declined with increasing pack years of smoking (OR: 0.75; 95% CI: 0.64-0.87) and drink years of alcohol use (OR: 0.64; 95% CI: 0.54-0.76). These results suggest that while the contribution of HPV16 is substantial for the oropharynx, it remains limited for oral cavity and laryngeal cancers.

Effect of HPV on head and neck cancer patient survival, by region and tumor site: A comparison of 1362 cases across three continents.

OBJECTIVES: To explore whether HPV-related biomarkers predict oropharyngeal squamous cell cancer (OPSCC) survival similarly across different global regions, and to explore their prognostic utility among non-oropharyngeal (non-OP) head and neck cancers. METHODS: Data from 1362 head and neck SCC (HNSCC) diagnosed 2002-2011 was used from epidemiologic studies in: Brazil (GENCAPO study, n=388), U.S. (CHANCE study, n=472), and Europe (ARCAGE study, n=502). Tumors were centrally tested for p16INK4a and HPV16 DNA (by PCR). Risk of mortality was examined using Cox proportional hazard models. RESULTS: There were 517 OPSCC and 845 non-OP HNSCC. Cases were primarily male (81%), ever smokers (91%), with median age of 58yearsandmedian follow-up of 3.1years (IQR=1.4-5.9). Among OPSCC, the risk of mortality was significantly lower among 184 HPV-related (i.e., p16+/HPV16+) compared to 333 HPV-unrelated (p16- and/or HPV16-) cases (HR=0.25, 95%CI=0.18-0.34). Mortality was reduced among HPV-related OPSCC cases from the U.S., Europe, and Brazil (each p⩽0.01) and after adjustment, remained significantly reduced (aHR=0.34, 95%CI=0.24-0.49). Among non-OP HNSCC, neither p16 (aHR=0.83, 95%CI=0.60-1.14), HPV16 DNA (aHR=1.20, 95%CI=0.89-1.63), or p16+/HPV16+ (aHR=0.59, 95%CI=0.32-1.08) was a significantly predictor of mortality. When interaction was tested, the effect of HPV16/p16 was significantly different in OPSCC than non-OP HNSCC (p-interaction=0.02). CONCLUSION: HPV-related OPSCCs had similar survival benefits across these three regions. Prognostic utility of HPV among non-OP HNSCC is limited so tumor HPV/p16 testing should not be routinely done among non-OP HNSCC.

Genome-wide association analyses identify new susceptibility loci for oral cavity and pharyngeal cancer.

We conducted a genome-wide association study of oral cavity and pharyngeal cancer in 6,034 cases and 6,585 controls from Europe, North America and South America. We detected eight significantly associated loci (P < 5 × 10-8), seven of which are new for these cancer sites. Oral and pharyngeal cancers combined were associated with loci at 6p21.32 (rs3828805, HLA-DQB1), 10q26.13 (rs201982221, LHPP) and 11p15.4 (rs1453414, OR52N2-TRIM5). Oral cancer was associated with two new regions, 2p23.3 (rs6547741, GPN1) and 9q34.12 (rs928674, LAMC3), and with known cancer-related loci-9p21.3 (rs8181047, CDKN2B-AS1) and 5p15.33 (rs10462706, CLPTM1L). Oropharyngeal cancer associations were limited to the human leukocyte antigen (HLA) region, and classical HLA allele imputation showed a protective association with the class II haplotype HLA-DRB1*1301-HLA-DQA1*0103-HLA-DQB1*0603 (odds ratio (OR) = 0.59, P = 2.7 × 10-9). Stratified analyses on a subgroup of oropharyngeal cases with information available on human papillomavirus (HPV) status indicated that this association was considerably stronger in HPV-positive (OR = 0.23, P = 1.6 × 10-6) than in HPV-negative (OR = 0.75, P = 0.16) cancers.

Involvement of Kallikrein-Related Peptidases in Normal and Pathologic Processes.

Human kallikrein-related peptidases (KLKs) are a subgroup of serine proteases that participate in proteolytic pathways and control protein levels in normal physiology as well as in several pathological conditions. Their complex network of stimulatory and inhibitory interactions may induce inflammatory and immune responses and contribute to the neoplastic phenotype through the regulation of several cellular processes, such as proliferation, survival, migration, and invasion. This family of proteases, which includes one of the most useful cancer biomarkers, kallikrein-related peptidase 3 or PSA, also has a protective effect against cancer promoting apoptosis or counteracting angiogenesis and cell proliferation. Therefore, they represent attractive therapeutic targets and may have important applications in clinical oncology. Despite being intensively studied, many gaps in our knowledge on several molecular aspects of KLK functions still exist. This review aims to summarize recent data on their involvement in different processes related to health and disease, in particular those directly or indirectly linked to the neoplastic process.

PROX1 gene is differentially expressed in oral cancer and reduces cellular proliferation.

Homeobox genes are a family of transcription factors that play a pivotal role in embryogenesis. Prospero homeobox 1 (PROX1) has been shown to function as a tumor suppressor gene or oncogene in various types of cancer, including oral squamous cell carcinoma (OSCC). We have previously identified PROX1 as a downregulated gene in OSCC. The aim of this study is to clarify the underlying mechanism by which PROX1 regulates tumorigenicity of OSCC cells. PROX1 mRNA and protein expression levels were first investigated in 40 samples of OSCC and in nontumor margins. Methylation and amplification analysis was also performed to assess the epigenetic and genetic mechanisms involved in controlling PROX1 expression. OSCC cell line SCC9 was also transfected to stably express the PROX1 gene. Next, SCC9-PROX1-overexpressing cells and controls were subjected to proliferation, differentiation, apoptosis, migration, and invasion assays in vitro. OSCC samples showed reduced PROX1 expression levels compared with nontumor margins. PROX1 amplification was associated with better overall survival. PROX1 overexpression reduces cell proliferation and downregulates cyclin D1. PROX1-overexpressing cells also exhibited reduced CK18 and CK19 expression and transcriptionally altered the expression of WISP3, GATA3, NOTCH1, and E2F1. Our results suggest that PROX1 functions as a tumor suppressor gene in oral carcinogenesis.

Stable SET knockdown in head and neck squamous cell carcinoma promotes cell invasion and the mesenchymal-like phenotype in vitro, as well as necrosis, cisplatin sensitivity and lymph node metastasis in xenograft tumor models.

BACKGROUND: SET/I2PP2A is a multifunctional protein that is up-regulated in head and neck squamous cell carcinoma (HNSCC). The action of SET in HNSCC tumorigenicity is unknown. METHODS: Stable SET knockdown by shRNA (shSET) was established in three HNSCC cell lines: HN12, HN13, and Cal27. Protein expression and phosphorylated protein levels were determined by Western blotting and immunofluorescence, cell migration and invasion were measured by functional analysis, and PP2A activity was determined using a serine/threonine phosphatase assay. A real-time PCR array was used to quantify 84 genes associated with cell motility. Metalloproteinase (MMP) activity was assessed by zymographic and fluorometric assays. HN12shSET xenograft tumors (flank and tongue models) were established in Balb/c nude mice; the xenograft characteristics and cisplatin sensitivity were demonstrated by macroscopic, immunohistochemical, and histological analyses, as well as lymph node metastasis by histology. RESULTS: The HN12shSET cells displayed reduced ERK1/2 and p53 phosphorylation compared with control. ShSET reduced HN12 cell proliferation and increased the sub-G1 population of HN12 and Cal27 cells. Increased PP2A activity was also associated with shSET. The PCR array indicated up-regulation of three mRNAs in HN12 cells: vimentin, matrix metalloproteinase-9 (MMP9) and non-muscle myosin heavy chain IIB. Reduced E-cadherin and pan-cytokeratin, as well as increased vimentin, were also demonstrated as the result of SET knockdown. These changes were accompanied by an increase in MMP-9 and MMP-2 activities, migration and invasion. The HN12shSET subcutaneous xenograft tumors presented a poorly differentiated phenotype, reduced cell proliferation, and cisplatin sensitivity. An orthotopic xenograft tumor model using the HN12shSET cells displayed increased metastatic potential. CONCLUSIONS: SET accumulation has important actions in HNSCC. As an oncogene, SET promotes cell proliferation, survival, and resistance to cell death by cisplatin in vivo. As a metastasis suppressor, SET regulates invasion, the epithelial mesenchymal transition, and metastasis.