(13) Developing Novel Therapeutics for Oral Cancer Leveraging Drosophila Models

Abstract: The prevalence of oral squamous cell carcinoma (OSCC) has been increasing worldwide. Surgical resection and chemoradiotherapy are main OSCC treatments currently, but their effectiveness is limited. One of their major problems is the decline in patients' quality of life due to oral dysfunction caused by the extensive resection and oral mucositis. Although targeted therapies have emerged for treating OSCC and have improved the patients’ survival rate, these therapies also cause severe side effects that hamper completion of the regimens. Hence, five-year overall survival rate of OSCC patients remains to be approximately 50% for past decades. Therefore, developing novel, effective OSCC therapeutics with decreased toxicity is highly demanded.
　Thus far, mammalian models such as cultured cells obtained from human OSCC and their xenografts in mice have provided critical insights into the molecular mechanisms in OSCC development. However, these models are difficult to execute comprehensive analysis due to their expensive breeding and time-consuming aspects. Recently, the fruit fly Drosophila melanogaster has made contributions to cancer research. Our previous works based on a novel platform combining flies and mammalian models have successfully developed therapeutic seeds and leads for various types of cancers (1,2).
　Here, we employed flies to generate the novel animal model reproducing OSCC patients’ genotype and to discover novel therapeutic targets and candidates for OSCC treatment. To generate flies modeling OSCC patients’ genotype, we employed the binary GAL4/UAS system obtained from yeast which enables targeted expression of exogenous genes in a desired tissue in flies. We first obtained GAL4 driver strains which express a transcription factor GAL4 specifically in the compound eye (for confirming the functions of each transgene) or in squamous cells in wing discs (for modeling OSCC patients’ complex genotype and for evaluating phenotypes). In parallel, we generated UAS transgenic strains which carry a mutated ‘A’, ‘B’ or ‘C’ gene that are frequently found in OSCC patients downstream of UAS, the GAL4 target DNA sequence.
　Expression of wild-type A in fly eyes using eye-specific driver Glass Multimer Reporter-GAL4 caused ‘rough eye’ phenotype by promoting apoptosis, as previously reported. Moreover, we found that additional expression of missense-mutated form of A suppressed the rough eye phenotype, confirming that this form works as a dominant negative mutant against wild-type as expected. Next, we found that the expression of missense-mutated B or overexpression of C using the same driver increased the fly eye area. These results indicated that both genes induced hyperplasia in fly eyes as previously reported.
　After confirming function of each gene, we induced all of these three genes in fly squamous cells by employing squamous cells-specific driver Ultrabithorax-GAL4 to reproduce OSCC patients’ genotype. Resulting flies showed decreased viability (60%). Additionally, expression of these three genes under patched-GAL4 that expresses GAL4 in a limited area of squamous cells induced statistically significant enlargement of squamous cell area compared with non-transgenic control (Dunnett’s test).
　In this study, we confirmed that the eye phenotypes induced by each transgene were consistent with their reported functions and our expectation. Furthermore, we identified that the flies displayed hyperplastic phenotype in the squamous tissue. These findings prove our strategy effective to validate transgene functions and to model at least a portion of OSCC phenotypes in flies. We will perform whole-body assays such as comprehensive genetic and drug screenings in these flies to obtain novel insights into the OSCC mechanisms and novel therapeutic targets and drug candidates in OSCC, finally validating them in the mouse model. We expect that the flies modeling OSCC genotypes accelerate development of novel therapeutics for OSCC.
(1) Sonoshita et al. Nat Chem Biol 2018
(2) Jiang & Kimura et al. Front Oncol 2022