A functional genomics approach to identify genes and pathways that can bypass p16INK4a-mediated cellular senescence

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The p16INK4a tumour suppressor gene, commonly known as p16, is frequently mutated, silenced or deleted in melanomas and other solid tumours. It can induce a phenotype known as cellular senescence, which is defined as a state of permanent proliferative arrest despite the presence of mitogens. Senescent cells can be characterised by low Ki-67 antigen positivity, decreased DNA synthesis, enlarged morphology and increased senescence-associated b-galactosidase positivity. Although p16 is known to inhibit cyclin-dependent kinase 4/6 and thus reinforce functional retinoblastoma tumour suppressor protein, the downstream mechanisms of p16-mediated senescence are not clear. Furthermore, bypass of p16-mediated senescence may have relevance to tumour progression, for example in the case of constitutively-active mutant CDK4.In order to identify potential genes and pathways that can bypass the initiation of p16-mediated senescence in a global fashion, we have designed a high-throughput, high-content cellular senescence assay for screening the human ORFeome lentiviral expression library developed at the ARVEC facility at the Diamantina Institute. We have established multiple parameters for cellular senescence in a p53-mutant but pRb-wild-type melanoma cell line that is able to express inducible p16. Immunofluorescent imaging for Ki-67 antigen can be combined with alpha-tubuin staining and DNA staining using DAPI to simultaneously assess proliferation and morphology. We have also established positive controls for this cellular senescence assay. The identification of genes whose over-expression can prevent or diminish the senescent phenotype induced by p16 may lead to the discovery of novel diagnostic markers or drug targets for melanoma.

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