Scribble knockdown induced metastasis tracking, identification of its associated novel molecular candidates through Proteome and in silico studies

Metastasis is the primary cause of cancer associated death globally. Loss of function of Scribble , a cell polarity regulator/tumor suppressor gene, is associated with many forms of human cancer but its role in cell proliferation and metastasis remains unknown. We generated metastatic cancer in Drosophila using UAS-GAL4 system, through knockdown of Scribble in the wing imaginal discs and tracked metastasis events from 0hr early pupae to 84hrs late pupae using fluorescence microscope. Here, we report, for the first time, that knockdown of Scribble alone could lead to the development of primary tumor in the wing imaginal discs, which is capable of establishing metastasis, leading to secondary tumor formation, eventually resulting in absolute pupal lethality. Further, we checked a metastasis biomarker, MMP1 levels during pre-and post-metastatic phases in Drosophila pupae using qRT-PCR and Western blot analysis. In addition, we analyzed the proteome of Scribble knockdown induced tumor-bearing pupae by 2-D gel electrophoresis followed by MALDI-TOF MS to identify novel proteins involved in the process of tumorigenesis and metastasis. We identified six differentially expressed proteins, Obp 99b, Fer2LCH,CG13492, Hsp23, Ubiquitin and Colt in Scrib knockdown pupae compared to wild-type and validated their expression at the transcriptional level using qRT-PCR. In-silico studies show these novel protein interaction with Scrib . Thus, our results suggested that loss of Scrib alone causes metastasis, without the need for cooperative interaction with oncogenic Ras. The newly identified Fer2LCH (ferritin) and colt proteins could be important candidates for therapeutic target against Scrib associated cell proliferation and metastasis.