Investigation of developmental LIN28/let-7/SOX2/SOX9 feedback loop during cancer stem cell formation and cancer cell migration
Introduction
The RNA binding protein, LIN28A and LIN28B, and the microRNA (miRNA) let-7 family represent an evolutionarily conserved developmental feedback loop that acts as a heterochronic regulator of developmental timing in metazoans from worms to mammals. LIN28 paralogs regulate the pluripotency of embryonic stem cells and are hypothesized to drive tumor cell plasticity. From our previous work, we have demonstrated that in the developing lung and brain, Sox2 and Sox9 transcriptionally regulate Lin28a/b. Moreover, we found that LIN28A binds Sox2 and Sox9 mRNAs and regulates their stability and translational capacity. Some studies have shown that transitions of expression from SOX2 to SOX9 cause tumor conversion from a highly proliferative to a highly metastatic state. Our hypothesis is that reactivation of an early developmental pathway in which the LIN28/let-7 axis is transcriptionally and post-transcriptionally regulated by SOX2 and SOX9 occurs in tumors. Our goal is to understand feedback regulatory loops within the LIN28/let-7/SOX2/SOX9 axis during cancer stem cell (CSC) formation and cancer cell migration.
Methods
We will knockdown (KD)/knockout (KO) SOX2/SOX9 using siRNA and CRISPR in normal and cancer cell lines of lung, pancreas, and breast to examine downstream targets at confluency (NS) and during pre-migratory (T=0) and migratory stages (T=4/12/24). We will also conduct assays to evaluate CSC formation and migratory potential in vitro. For CSC formation we will investigate colony formation as well analysis of stem cell markers during migratory and pre-migratory stages. We will assess migratory potential using the Incucyte SX5 live-cell imager via high throughput migration assays.
Results
We have found that SOX9 expression becomes nuclear in some cell lines indicating a potential transcriptional activation during cell migration. We noticed that SOX9 expression is increased at different migratory stages via immunofluorescence and immunoblots. SOX9 expression is higher in cancer cell line compared to normal cell line during all migratory and pre-migratory stages. SOX2 is expressed in lung cancer cell line (HCC4017) not in the pancreatic cancer cell line (PANC1-PA). We also observed Lin28A/B expression is higher in cancer cell line compared to normal cell line, specifically Lin28B expression is very dramatic in pancreatic cancer cell line (PANC1-PA).
Conclusion
Overall, our data indicates that developmental LIN28/let-7/SOX2/SOX9 feedback loop may play a role in tumorigenesis. The connection and actions of the LIN28/let-7/SOX2/SOX9 circuit in developmental biology provide a roadmap to systematically study how these feedback loops function during CSC formation and cell migration. They also incentivize the identification and characterization of downstream targets of SOX2 and SOX9 to reveal potential new targets for cancer treatment.