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CD44 has been described as a marker of cancer stem cells in oral squamous cell carcinoma (OSCC). The main objective of this study was to characterise expression of CD44 in both fresh samples of human OSCC and in cell lines generated from them, and to examine its correlation with selected clinicopathological parameters of the tumours of origin. The epithelial fraction in 20 fresh OSCC samples was identified by the standard method using the negative selection technique with antibodies against non-tumour cells. A novel method of identifying the epithelial fraction, termed positive selection, was also developed and used for analysis of 14 additional OSCC samples. This new method, using epithelial-specific antibodies, led to a considerable improvement in the efficiency and the accuracy of the procedure. The frequency of CD44+ cells in the epithelial fraction of the tumour specimens was assessed by FACS and varied widely (3-97%). High frequency of CD44+ cells in tumour samples was found to be associated with high tumour grade, discohesive invasion front and presence of lymph node metastases (p<0.01, as calculated with Spearman’s ranked test and Fisher’s exact test). It was also observed, that the percentage of CD44+ cells changes when cells isolated from tumour samples are propagated in culture. Nearly all cells in cell lines generated from OSCC samples showed CD44 expression when analysed by FACS. However, a markedly higher level of CD44 expression (as assessed by median fluorescence intensity for cell surface CD44) was found for early passage cell lines generated from metastatic OSCC and lymph node metastases as compared to cell lines generated from nonmetastatic OSCC. These findings show that a high frequency of CD44+ cells in fresh OSCC tissue and a high level of CD44 expression in cultured OSCC cells correlate 11 with more aggressive tumour behaviour. These results might provide important information of prognostic and therapeutic value.

Orientador: Christiane Pienna Soares / Banca: Sophie Françoise Mauricette Derchain / Banca: Maria Regina Sposto / Resumo: O mdm2 parece controlar o tráfego da proteína p53 do núcleo-citoplasma enquanto a proteína p27 é um inibidor de CDK que controla a fase G1 para S do ciclo celular. Em contraste, a catepsina B parece induzir um sinal de apoptose independente de caspase na presença do HPV. Entretanto, a influência da infecção pelo HPV na carcinogênese oral e o envolvimento na desregulação das proteínas do ciclo celular não são claras. Assim, o objetivo deste estudo foi avaliar a associação entre a expressão de proteínas do ciclo celular, p27, mdm2 e catepsina B e câncer oral na presença do HPV. Cinqüenta e cinco biópsias orais representando lesões benignas (LB), lesões potencialmente malignas (LPM) e lesões malignas (LM). A detecção e tipagem do HPV (6/11,16,18, 31 e 33) foi realizada pela reação da polimerase em cadeia (PCR). A expressão quantitativa de p27, mdm2 e catepsina B foi analisada pela reação imunohistoquímica. Vinte e uma (38%) de 55 lesões orais foram positivas para HPV. O DNA do HPV 6/11 foi encontrado em 6 (33%), HPV 16 em 1(5%) e o HPV 18 em 14 (72%). Não foi encontrado HPV 31 e 33. De 55 biópsias, foi encontrada alta expressão de todas as proteínas: o p27 foi encontrado em 37 (67,2%) lesões, mdm2 em 17 (30,9%) e a catepsina B em 37 (67,2%). Entre lesões orais de HPV positivo, uma superexpressão de p27, mdm2 e catepsina B foi encontrada, respectivamente, em 6 (33%) de 18 lesões benignas, 4 (22%) de 18 lesões potencialmente malignas e 11 (57,9%) de 19 lesões malignas. Adicionalmente, o HPV16/18 (11/58%) foi detectado em lesões malignas com alta expressão de todas as proteínas. Nós concluímos que os tipos de HPVs de alto risco devem estar associados com carcinoma oral, bem como, com a superexpressão de p27, mdm2 e catepsina B. Estes resultados sugerem que os tipos de HPVs de alto risco podem desregular o controle do ciclo celular, contribuindo para a carcinogênese oral. / Abstract: Mdm2 seems to control p53 nucleus-cytoplasm traffic while p27 is a CDK inhibitor which control G1 to S phase of the cell-cycle. In contrast, cathepsin B is reported in HPVinduced apoptotic signalling caspase-independent. However, the influence of HPV infection in oral carcinogenesis and its involvement to this cell-cycle proteins dysfunction remain still unclear. The purpose of this study was to clarify the association between the expression of cell-cycle proteins, p27, mdm2 and cathepsin B and oral cancer HPV-related. Fifty-five oral biopsies presenting benign oral lesions (BL), potentially malignant lesions (PML) and malignant oral lesions (ML). HPV detection and typing (6/11, 16, 18, 31 and 33) was performed using polymerase chain reaction. p27, mdm2 and cathepsin B quantitative expression were performed by immunohistochemistry. Twenty one (38%) of the 55 oral lesions were positive for HPV, of which HPV6/11 DNA was found in 6 (33%), HPV 16 in 1(5%) e o HPV 18 in 14 (72%). No HPV positivity was found to HPV31 and 33. Of the 55 biopsies, a high expression of all proteins was found: to p27 in 37 (67,2%), mdm2 was observed in 17 (30,9%) slides, and cathepsin B in 37 (67,2%). Among HPV-positive oral lesions, an overexpression for mdm2, p27 and cathepsin B was found, respectively, in 6 (33%) out of 18 BL, 4 (22%) out of 18 PML and 11 (57,9%) out of 19 ML. In addition, HPV16/18 (11/58%) was detected in OSSC with high expression of all proteins. We conclude that high-risk HPV types might be associated with oral carcinoma, as well as, with the overexpression of mdm2, p27 and cathepsin B. These results suggest that high-risk HPV types might deregulate cell-cycle control, contribuiting to oral carcinogenesis. / Mestre

X-ray repair cross complementing Group 1 (XRCC1) protein plays an important role in base excision repair. Single nucleotide polymorphisms (SNPs) in XRCC1 gene may affect DNA repairing ability, genetic susceptibility, and prognosis to oral and pharyngeal squamous cell cancer (OPSCC). This study was carried out to evaluate the association of three XRCC1 SNPs with the risk and prognosis of OPSCC. A total of 509 OPSCC cases and 678 cancer-free controls were recruited to detect the genotypes of XRCC1 by PCR-RFLP. Then, 447 case patients with surgical treatment and safety margins were included in the survival analysis. No association was observed for XRCC1 194 and the risk of OPSCC. As compared with the wild Arg/Arg genotype, the combined genotypes of 280 Arg/His and His/His were with decreased risk (AOR=0.73, 95% CI, 0.52-1.03, p = 0.069) of OPSCC and with a significantly decreased risk (AOR=0.67, 95% CI, 0.47-0.97, p = 0.035) of oral cavity. As compared with the Arg/Arg genotype of XRCC1 399, the Gln/Gln genotype was with the increased risk of OPSCC (AOR=2.06, 95%CI: 1.21-3.51, p = 0.008) and oral cavity cancer (AOR=1.89, 95%CI: 1.08-3.33, p = 0.026). We defined the ¡§putative high risk haplotypes¡¨ as ¡§Arg-Arg-Gln and Trp-Arg-Gln¡¨. The AOR were 1.29 (95% CI, 1.04-1.60, p = 0.020) for the ¡§putative high risk haplotypes¡¨ as compared with other haplotypes for OPSCC. Then, two putative high risk haplotypes were combined into ¡§putative high risk diplotypes¡¨. The AOR for the ¡§high risk diplotypes¡¨ were 1.98 (95% CI, 1.18-3.33, p = 0.010) as compared with other diplotypes for OPSCC. No association between XRCC1 polymorphisms and clinicopathological outcomes, except XRCC1 280. Those carriers of XRCC1 280His allele (combined Arg/His and His/His genotypes) were associated with late onset (≥50 yrs) of oral cavity cancers. No association between genetic variants in XRCC1 and disease-specific survival except XRCC1 399. Patients with 399 Arg/Gln and Gln/Gln genotypes showed a significant better survival as compared to Arg/Arg genotype carriers (AHR 0.41 95% CI: 0.18-0.93), especially for those patients younger than 50 years (p = 0.012), in pathological stage III or IV (p = 0.044), and without postoperative radiotherapy (p = 0.012). In summary, XRCC1 280 Arg/His and His/His genotypes were associated with decreased risk of oral cavity cancer. 399 Gln/Gln genotype was associated with increased risk of OPSCC and oral cavity cancer. The putative ¡§high risk haplotypes and diplotypes¡¨ were with increased risk of OPSCC. However, 399 Arg/Gln and Gln/Gln genotypes were prognostic factors, especially for those with young age, aggressive tumor stage, and without postoperative radiotherapy for oro and hypopharynx patients. These findings suggest that XRCC1 polymorphisms may play a role in the development and prognosis of OPSCC.

Oral cancer is now the fourth leading cause of male cancer mortality in Taiwan. Betel quid chewing is one of the main causes of oral cancer in Taiwan. CDK2AP1 is a growth suppressor gene that negatively regulates cyclin-dependent kinase 2 (CDK2) activities. Expression of p12CDK2AP1 protein is reduced and/or lost in oral cancers. Mutations in microsatellite-like sequence of CDK2AP1 gene in microsatellite instability colorectal cancer are associated with down-regulated CDK2AP1 transcription. This mutation was due to down-regulation of one DNA repair protein, MLH1. In order to understand whether CDK2AP1 mRNA and protein expression levels associate with betel-chewing oral cancers, we firstly analyzed 44 oral cancer specimens (normal and tumor, in pairs) by quantitative reverse transcription polymerase chain reaction and Western blotting. Immunohistochemistry was used to examine p12CDK2AP1 protein expression in another 167 buccal mucosa squamous cell carcinoma tissues. We have demonstrated that the expression levels of CDK2AP1 mRNAs were slightly higher in normal oral tissues than those in tumor tissues (P>0.05). Similarly, p12CDK2AP1 and CDK2 protein expression levels were up-regulated in oral cancer tissues than in normal tissues by Western blot analysis (P<0.05). Among Ca9-22, CAL27, SAS and in betel-chewing oral cancer cells TW2.6 and human normal skin keratinolial cells (HaCaT) that we examined, p12CDK2AP1 and CDK2 proteins were detected to be highest expressed in Ca9-22 and TW2.6 cells, respectively, when compared to HaCaT cells. Immunocytochemistry indicated p12CDK2AP1 expressed in nucleus and cytoplasm in Ca9-22, CAL27, SAS and HaCaT cells, however predominant present in nucleus in TW2.6 cells. On the other hand, immunohistochemistry demonstrated that nuclear (P=0.157) and cytoplasmic p12CDK2AP1 (P=0.350) in 167 patients with buccal mucosa squamous cell carcinoma were slightly down-regulated. Reduction of nuclear p12CDK2AP1 was not significantly correlated with any clinicopathologic characteristics or prognosis. Direct sequencing indicated that lack of microsatellite-like instability of CDK2AP1 3¡¦-UTR in four oral cancer cell lines, HaCaT and six patients with down-regulated MLH1 protein. In conclusion, we demonstrated that: (1) p12CDK2AP1 was located in both the nucleus and cytoplasm in most oral cancer cell lines and HaCaT cells but predominate present in the nucleus in betel-chewing oral cancer cells, TW2.6; (2) Reduction of nuclear p12CDK2AP1 in buccal mucosa squamous cell carcinoma tissues were identified, however, not significantly correlated with any clinicopathologic characteristics, prognosis or betel chewing; (3) In six patients with down-regulated MLH1, lack of micorsatellite-like instability in the CDK2AP1 3¡¦-UTR region has been found.

Phage display is a molecular technique accomplished by incorporation of the nucleotide sequence encoding the protein to be displayed into a phage or phagemid genome as a fusion to a gene encoding a phage coat protein. After several rounds of selection and amplification, high affinity phage clones, and thus high affinity ¡§homing peptides¡¨ can be obtained. Cell-binding homing peptides selected in this manner could be linked by physical or genetic manipulation to gene therapy vectors that mediate their own entry (viral or non-viral vectors) to facilitate targeting. Homing peptides that target specific cellular receptors can also be used as a treatment modality to induce various signal transduction pathways or even apoptotic signals of cancer cells. Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world. It has become the fourth cancer death reason of males in Taiwan. Radical surgery combined with postoperative chemotherapy and/or radiotherapy is still the major modality for treatment of OSCC. The 5-year survival rate of OSCC is still discouraged in recent years. Patients with OSCC present numerous challenges to treating physicians. In this study, we aimed to isolate and identify homing phage clones specific to oral cancer cells by panning with a random phage peptide library. The homing phage clones will be used as a basis to improve targeting specificity of gene therapy vectors. A NCBI BLAST search was performed and close similarities were found to several important molecules biologically with the homing peptides carried by phage clones. Characterization of the selected phage-29 was then studied by immunohistochemical methods. Internalization of this phage-29 is sequence-specific and mediated by integrin £\v£]6 in HSC-3 cells rapidly. We also confirmed that the integrin £\v£]6-targeting homing peptide is universally useful in all major kinds of head and neck cancer. We will further study the possible biological functions of the other homing peptides to see whether these peptides could have potential applications for oral cancer treatment.

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No description available.

INTRODUCTION: The lysyl oxidase family consists of 5 members and oxidizes specific lysine residues in biosynthetic collagen and elastin maturation. Lysyl oxidase like-2 (LOXL2) is elevated in oral cancer and promotes metastasis and correlates with poor prognosis. The objective of this study was to determine the mechanism by which LOXL2 promotes the progression and invasiveness of oral squamous cell carcinoma. METHODS: In vitro: The effects of LOXL2 inhibitor (PXS-S1C) on human gingival fibroblasts treated with tumor cell conditioned medium (CM) were investigated. Cell proliferation assays, signaling arrays, gene knock down and western blots were used to evaluate the effect of PXS-S1C on CM-treated fibroblasts. The effects of PXS-S1C on cancer cell expression of LOXL2 and proliferation were determined. To find potential LOXL2 substrates, carbonyl-containing proteins of gingival fibroblasts treated with CM +/- PXS-S1C were affinity-labeled and then purified by affinity chromatography and identified by western blot. In vivo: The effects of PXS-S1C on cancer cell growth and metastasis in vivo were investigated using orthotopic oral tongue cancer mouse models in both immunodeficient and immunocompetent mice. PXS-S1C at 10 mg/kg and 30 mg/kg was injected immediately following tumor cell injections. Tumor growth was monitored by both caliper measurement and in vivo imaging (IVIS). The mice were sacrificed and their organs were subjected to immunohistochemical staining with proliferation markers. RESULTS: PXS-S1C significantly inhibited gingival fibroblast proliferation stimulated by tumor cell CM and attenuated phosphorylation of PDGFRβ at the Y771 and Y857, but not Y751 residues in response to CM treatment. PXS-S1C inhibited ERK1/2-signaling in fibroblasts but not AKT pathway in response to CM treatment. PDGFR activation by oral tumor cells was mimicked by PDGF-AB but not PDGF-BB. PXS-S1C decreased the expression of LOXL2 in HSC3 oral cancer cells in vitro, suggesting the existence of a positive autoregulatory loop. Assessing for direct LOXL2 substrates in fibroblasts with functional consequences identified PDGFR. In vivo studies: Caliper measurements, IVIS, and immunohistochemistry demonstrated that inhibition of LOXL2 by injections of PXS-S1C significantly decreased both progression and metastasis of oral cancers in vivo in both mouse models. Mice without PXS-S1C treatment developed larger tongue volumes (p<0.05), and in immunocompetent mice larger lymph nodes (9 out 12) were observed compared to the PXS-S1C-treated mice (4 out of 12). IVIS imaging of immunodeficient mice revealed inhibition of metastasis by PXS-S1C treatment. The expression of proliferation marker (Ki-67 or PCNA) and LOXL2 was lower in tongue tumors treated with PXS-S1C in both in vivo models (p<0.05). CONCLUSIONS: LOXL2 secreted by cancer cells stimulates fibroblast proliferation by oxidizing PDGFR and thereby enhancing PDGF-mediated signaling. Inhibition of LOXL2 can be used as a therapeutic strategy to suppress the growth and metastasis of oral cancers by modulating tumor microenvironment. / 2019-10-24T00:00:00Z

Objective: With the changes in healthcare, patients with cancer now have to assume greater responsibility for their own care. Oral cancer medications with complex regimens are now a part of cancer treatment. Patients have to manage these along with the management of medications for their other chronic illnesses. This results in medication burden as patients assume the self-management. Methods: This paper describes the treatment burdens that patients endured in a randomized, clinical trial examining adherence for patients on oral cancer medications. There were four categories of oral agents reported. Most of the diagnoses of the patients were solid tumors with breast, colorectal, renal, and gastrointestinal. Results: Patients had 1u4 pills/day for oral cancer medications as well as a number for comorbidity conditions (3), for which they also took medications (10u11). In addition, patients had 3.7u5.9 symptoms and side effects. Patients on all categories except those on sex hormones had 49%u57% drug interruptions necessitating further medication burden. Conclusions: This study points out that patients taking oral agents have multiple medications for cancer and other comorbid conditions. The number of pills, times per day, and interruptions adds to the medication burden that patients' experience. Further study is needed to determine strategies to assist the patients on oral cancer medications to reduce their medication burden.