Chai Phei Gan

Valproic acid: growth inhibition of head and neck cancer by induction of terminal differentiation and senescence.

There are limited studies on the effects of drugs that modulate epigenetic regulation for head and neck squamous cell carcinoma (HNSCC). This study determined the effect of valproic acid (VPA) on HNSCC.

Genetically-defined novel oral squamous cell carcinoma cell lines for the development of molecular therapies

Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations (MLL4, USP9X, ARID2) in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups (cell cycle, xenobiotic metabolism, others). The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC.

Over-expression of MAGED4B increases cell migration and growth in oral squamous cell carcinoma and is associated with poor disease outcome.

MAGE proteins have been shown to be good targets for cancer immunotherapy. We demonstrate that MAGED4B is over-expressed in more than 50% of Oral Squamous Cell Carcinoma (OSCC) tissues and the expression of MAGED4B is associated with lymph node metastasis and poor disease specific survival. OSCC cell lines that over-express MAGED4B promote migration in vitro, exhibit an increase in cell growth both in vitro and in vivo, and are more resistant to apoptosis compared to control cells. Our data suggest that MAGED4B over-expression is a driver in oral carcinogenesis and argues strongly that this protein may represent a potential therapeutic target in OSCC.

Novel MDM2 splice variants identified from oral squamous cell carcinoma

INTRODUCTION The presence of a variety of MDM2 splice variants has been reported in a range of different tumor types and is associated with poor patient prognosis. Furthermore, several MDM2 variants have been shown to have oncogenic properties. Despite this, MDM2 splice variants have not been comprehensively characterized in oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS MDM2 splice variants were identified by polymerase chain reaction (PCR), using cDNA from 55 OSCC and 20 normal oral mucosa (NOM) tissues. MDM2 amplicons from the polymerase chain reactions were cloned and sequenced. The associations between the presence of MDM2 splice variants as well as the types of MDM2 splice variants with OSCC and patient clinico-pathological data was examined using Fisher Exact and Chi-square tests. RESULTS Thirty-eight MDM2 splice variants were identified from both OSCC and NOM tissues, where the majority (30/38) were exclusively detected in OSCC. Some of these variants were similar to those reported in other cancers whilst 14 novel MDM2 splice variants predicted to code for proteins were also identified. The majority of these variants retained their RING binding domain but had lost the p53 binding site. The presence of MDM2 splice variants was significantly associated with OSCC and increased the risk of OSCC development (OR=9.98; 95% CI=2.94-33.90). CONCLUSION MDM2 splice variants were identified in OSCC at a high frequency and were significantly associated with OSCC development. This suggests that MDM2 splice variants may play an important role in oral carcinogenesis and the functional role of these variants in OSCC should be examined further.

Identification of immunogenic MAGED4B peptides for vaccine development in oral cancer immunotherapy

The ever-increasing number of tumor-associated antigens has provided a major stimulus for the development of therapeutic peptides vaccines. Tumor-associated peptides can induce high immune response rates and have been developed as vaccines for several types of solid tumors, and many are at various stages of clinical testing. MAGED4B, a melanoma antigen, is overexpressed in oral squamous cell carcinoma (OSCC) and this expression promotes proliferation and cell migration. In this study, we have identified 9 short peptides derived from MAGED4B protein that are restricted in binding to the HLA subtypes common in the Asian population (HLA-A2, A11, and A24). The peptides had good binding affinity with the MHC-Class I molecules and stimulated ex-vivo IFN-gamma and Granzyme-B production in blood samples from OSCC patients, suggesting that they are immunogenic. Further, T cells stimulated with peptide-pulsed dendritic cells showed enhanced T-cell cytotoxic activity against MAGED4B-overexpressing OSCC cell lines. In summary, we have identified MAGED4B peptides that induce anti-tumor immune responses advocating that they could be further developed as vaccine candidates for the treatment of OSCC.

GENIPAC: A Genomic Information Portal for Head and Neck Cancer Cell Systems

Head and neck cancer (HNC)–derived cell lines represent fundamental models for studying the biological mechanisms underlying cancer development and precision therapies. However, mining the genomic information of HNC cells from available databases requires knowledge on bioinformatics and computational skill sets. Here, we developed a user-friendly web resource for exploring, visualizing, and analyzing genomics information of commonly used HNC cell lines. We populated the current version of GENIPAC with 44 HNC cell lines from 3 studies: ORL Series, OPC-22, and H Series. Specifically, the mRNA expressions for all the 3 studies were derived with RNA-seq. The copy number alterations analysis of ORL Series was performed on the Genome Wide Human Cytoscan HD array, while copy number alterations for OPC-22 were derived from whole exome sequencing. Mutations from ORL Series and H Series were derived from RNA-seq information, while OPC-22 was based on whole exome sequencing. All genomic information was preprocessed with customized scripts and underwent data validation and correction through data set validator tools provided by cBioPortal. The clinical and genomic information of 44 HNC cell lines are easily assessable in GENIPAC. The functional utility of GENIPAC was demonstrated with some of the genomic alterations that are commonly reported in HNC, such as TP53, EGFR, CCND1, and PIK3CA. We showed that these genomic alterations as reported in The Cancer Genome Atlas database were recapitulated in the HNC cell lines in GENIPAC. Importantly, genomic alterations within pathways could be simultaneously visualized. We developed GENIPAC to create access to genomic information on HNC cell lines. This cancer omics initiative will help the research community to accelerate better understanding of HNC and the development of new precision therapeutic options for HNC treatment. GENIPAC is freely available at http://genipac.cancerresearch.my/.

In vitro evaluation of dual-antigenic PV1 peptide vaccine in head and neck cancer patients

ABSTRACT Peptide vaccines derived from tumour-associated antigens have been used as an immunotherapeutic approach to induce specific cytotoxic immune response against tumour. We previously identified that MAGED4B and FJX1 proteins are overexpressed in HNSCC patients; and further demonstrated that two HLA-A2-restricted 9–11 amino acid peptides derived from these proteins were able to induce anti-tumour immune responses in vitro independently using PBMCs isolated from these patients. In this study, we evaluated the immunogenicity and efficacy of a dual-antigenic peptide vaccine (PV1), comprised of MAGED4B and FJX1 peptides in HNSCC patients. We first demonstrated that 94.8% of HNSCC patients expressed MAGED4B and/or FJX1 by immunohistochemistry, suggesting that PV1 could benefit the majority of HNSCC patients. The presence of pre-existing MAGED4B and FJX1-specific T-cells was detected using a HLA-A2 dimer assay and efficacy of PV1 to induce T-cell to secrete cytotoxic cytokine was evaluated using ELISPOT assay. Pre-existing PV1-specific T-cells were detected in all patients. Notably, we demonstrated that patients’ T-cells were able to secrete cytotoxic cytokines upon exposure to target cells expressing the respective antigen post PV1 stimulation. Furthermore, patients with high expression of MAGED4B and FJX1 in their tumours were more responsive to PV1 stimulation, demonstrating the specificity of the PV1 peptide vaccine. Additionally, we also demonstrated the expression of MAGED4B and FJX1 in breast, lung, colon, prostate and rectal cancer suggesting the potential use of PV1 in these cancers. In summary, PV1 could be a good vaccine candidate for the treatment of HNSCC patients and other cancers expressing these antigens.

Resistance to dasatinib is associated with the activation of Akt in oral squamous cell carcinoma

Objectives: Overexpression and aberrant activation of Src promote the development of oral squamous cell carcinoma (OSCC), thus therapies targeting Src-related kinases may afford an improvement in patient survival. However, limited clinical activity of the Src-targeted drug, dasatinib, in cancer patients warrants further investigation to better understand the underlying basis of resistance to dasatinib in OSCC. Methods: Response to dasatinib was evaluated in a panel of oral cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA synthesis, cell cycle and apoptosis analysis. The underlying mechanism of drug response was investigated using immunoblotting. Xenograft models were used to test efficacy. Results: All cell lines were sensitive to dasatinib (IC50 < 250 nM), but this was not associated with CDKN2a/p14ARF mutations. Dasatinib-induced cell cycle arrest and apoptosis, while inhibiting Src, Akt and FAK activity in all lines tested. However, dasatinib failed to inhibit tumour growth in xenograft models and treated tissues showed Akt activity despite the loss of Src activity. Conclusions: Our data revealed that reactivation of Akt (Ser473) could be a compensatory mechanism that bypasses Src inhibition by dasatinib, providing important clues that could improve treatment strategies to overcome dasatinib resistance.

Abstract 1573: MAGED4B drives oral carcinogenesis and is a promising peptide vaccine target for the treatment of oral squamous cell carcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL MAGED4B is a member of the Melanoma Antigen Gene (MAGE) family. We demonstrated that MAGED4B is over-expressed in more than 50% of oral squamous cell carcinoma (OSCC) tissues and that the expression of MAGED4B is associated with lymph node metastasis and poor disease specific survival. OSCC cell lines over-expressing MAGED4B exhibited an increase in cell growth both in vitro and in vivo, and were significantly more resistant to UV-induced apoptosis compared to control cells, suggesting a role for MAGED4B in modulating cell death. Further, MAGED4B also promoted cell migration. MAGE proteins are good targets for cancer immunotherapy as they are highly immunogenic. We identified and synthesized MAGED4B peptides that were HLA-A2- specific and tested the ability of these peptides to generate an anti-tumour response using PBMCs from OSCC patients and healthy individuals. Firstly, the PBMC samples obtained from patients and healthy individuals were tested for HLA-A2 expression. Positive samples were analyzed for peptide-specific stimulation of IFN-γ and granzyme secretion ex-vivo via ELISPOT assay. Dendritic cells pulsed with these peptides were used to stimulate PBMC cultures, and were studied for IFN-γ and granzyme secretion. In parallel, the binding of the peptides to the MHC-Class I molecule was determined by the binding assay. Further, the ability of these bound peptides to interact with T cells was measured by the dimer assay. We demonstrated that all the peptides have good binding capacity with the MHC-Class I molecules. Moreover, the binding of these peptides on MHC-Class I molecules could attract and further interact with T cells. Notably, the PBMC stimulated once with peptide-pulsed dendritic cells or twice with peptide and peptide-pulsed dendritic cells showed peptide-specific cytotoxic activity against MAGED4B-expressing OSCC cell lines. In summary, we first demonstrated that MAGED4B drives oral carcinogenesis and second, showed that MAGED4B-specific peptides are apable of inducing anti-tumour specific immune responses. Taken together, this indicates that MAGED4B could be a promising peptide vaccine target for the treatment of OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1573. doi:1538-7445.AM2012-1573