Investigation of developmental LIN28/ let-7 /SOX2/SOX9 feedback loop during cell migration and metastasis

The RNA binding protein LIN28, and the microRNA (miRNA) let-7 family represent an evolutionarily conserved developmental feedback loop that acts as a regulator of developmental timing in metazoans. We have recently published data demonstrating that in the developing lung and brain, Sox2 and Sox9 transcriptionally regulate Lin28a/b . Moreover, previous work from our lab show that Lin28a binds Sox2 and Sox9 mRNAs and regulates their stability and translation. Our hypothesis is that reactivation of an early developmental pathway governed by SOX9 and SOX2 lead to transcriptional upregulation of LIN28A/B at various times during tumorigenesis. Our goal is to understand why developmental pathways are re-expressed during tumorigenesis as well as find ways to specifically target these pathways to inhibit metastasis. We established transient SOX9 knockdown (KD) using siRNA and stable SOX9 KD using lenti-viral shRNA construct in normal and cancer cell lines of lung and pancreas. We performed migration assays in control and SOX9 KD cells to test protein expression of SOX9, SOX2, LIN28A, and LIN28B using immunoblot and immunofluorescence experiments at different migratory stages. We performed migration/invasion assays using the Incucyte SX5 live-cell imager via high throughput migration assays to test migratory potential of control and SOX9 KD cells. We have found that SOX9 expression becomes nuclear during migratory stages in normal and cancer cells indicating a potential transcriptional activation during cell migration. We noticed that SOX9 expression is increased at different migratory stages but decreased after wound closure. Interestingly, SOX2 expression is higher in cancer cell lines after wound closure when SOX9 expression is lower. LIN28A and LIN28B expression is increased at different migratory stages in lung and pancreatic cancer cell lines, but not in the normal cell lines. Importantly, we observed that in lung and pancreatic cancer cells migration, invasion and proliferation are slow after SOX9 KD. We will now investigate whether LIN28A/B are functional downstream targets of the SOX9/SOX2 axis during the pathophysiology of non-small cell lung cancer (NSCLC) and pancreatic cancer with specific focus on migration/invasion and metastasis.