An in vivo RNAi mini-screen in Drosophila cancer models reveals novel potential Wnt targets in liver cancer

Abstract

Background/aims: Aberrant activation of the Wnt/β-catenin signaling, which arises from the accumulation of mutant β-catenin in the cell, is one of the most common driving forces in hepatocellular carcinoma (HCC). We previously identified several genes that are regulated on the overexpression of β-catenin in the HCC cell line that are suggested to be novel Wnt/β-catenin targets playing effective roles in cancer. The aim of the present study was to elucidate the roles of these putative target genes in tumorigenesis with an in vivo analysis in Drosophila.

Materials and methods: We selected 15 genes downregulated in two Drosophila cancer models.

Results: The results from the RNAi mini-screen revealed novel roles for the analyzed putative Wnt/β-catenin target genes in tumorigenesis. The downregulation of the analyzed nine genes led to tumor formation as well as metastasis in Drosophila, suggesting a tumor suppressor function. On the other hand, the knockdown of the other two genes suppressed tumor and metastasis formations and disturbed the development of the analyzed eye tissues, indicating an oncogenic or developmental role for these genes.

Conclusion: These findings could serve to identify novel subjects for cancer research in order to provide insight into the diagnostic and therapeutic processes of several cancer types.

Figure 1. a, b

Experimental outline of the mini-screen in the eyeful (a) and sensitized (b) cancer models

Figure 2. a–f

Several examples of tumorigenic eye tissue formations in eyeful flies. Eyeful flies present a regular red-eye phenotype (a), whereas the white gene knockdown leads to lighter eye color development (b), as expected. Tumor formations in flies are detected as excessive outgrowth of the eye tissue in the form of foldings (c and d) or additional eye tissue generation on the head (e and f). Arrows indicate the related examples in each picture

Figure 3. a, b

(a) Normalized tumor formation prevalences in the eyeful cancer model. Decreased levels of Mgat1, YL-1, HINT1, RhoGAP68f, DEF8, Liprin-alpha, Ide, Mp20, Roughened, Cam, and Tctp significantly enhance tumorigenesis. In contrast, the knockdown of Hsc70-5 and Ip259 significantly decreases the tumor formation in the eyeful background. All results are normalized to the tumor formation rates of the downregulated negative control (NC) white gene. *p<0.05, **p<0.01, ***p<0.001; ns, not significant as analyzed by Student’s t-test. (b) Normalized metastasis formation prevalences in the eyeful cancer model. The knockdowns of Mgat1, YL-1, HINT1, RhoGAP68f, Liprin-alpha, Ide, Mp20, Cam, and Tctp significantly increase metastasis formation frequencies in the eyeful background

Figure 4. a, b

Normalized tumor (a) and metastasis (b) formation prevalences on RNAi downregulations in the sensitized cancer model