CAPITULO IV: DISCUSION DE LOS DATOS
4.4. SÍNTESIS DEL ANÁLISIS
How mutant cells are identified by normal neighbours and how they trigger competitive interactions is an area of ongoing research. A growing body of work suggests a positive role for cell repulsion signals that classically have a role in axon guidance, have been identified in cell competition. For example, Slit-Robo2 signalling drives extrusion of polarity deficient cells (Vaughen and Igaki, 2016), and we have identified EphA2 signalling as a mechanism of Ras-transformed cell repulsion and extrusion (Porazinski et al., 2016). Eph receptor tyrosine kinases regulate cell position by imposing repulsive signals and modulating adhesion between cells (Marston et al., 2003; Shamah et al., 2001). In pancreatic cancer, EphA2 is associated with early PanIN lesions and pre-mestastatic tumours (Mudali et al., 2006). In this thesis, I found that EphA2 is required for the detection and repulsion of KrasG12D cells from the pancreas. As KrasG12D cells were not triggered to become contractile by other Kras-transformed cells this suggests a requirement for normal neighbours to initiate repulsion via heterotypic interactions possibly via EphrinA1 ligand on non-transformed neighbours (Porazinski et al., 2016).
Using primary co-culture experiments an EphA2 dependent increase in cell contractility occurred in KrasG12D pancreatic cells when surrounded by normal neighbours. This work also indicated the possible upregulation of active Src as observed previously in MDCK cell cultures (Porazinski et al., 2016). Activation of Src can promote tumour invasion and metastasis by destabilisation of epithelial cell-cell junctions. This occurs via direct phosphorylation of E- cadherin which promotes internalisation (Behrens et al., 1993; Lilien et al., 2005; Yap et al., 1998). Importantly, I found that E-cadherin was internalised in an EphA2 dependent manner specifically in KrasG12D cells surrounded by normal neighbours. This suggests activation of EphA2 is the initial event which triggers internalisation of E-cadherin possibly via Src activation. Src activation can also lead to RhoA GTPase activation which can disrupt adherens junctions via ROCK (Fang et al., 2008; Wójciak-Stothard et al., 2001). Finally, Eph receptor activation can lead to Rac1-dependent endocytosis of E-cadherin (Akhtar and Hotchin,
2001; Marston et al., 2003). Therefore, several possible mechanisms downstream of EphA2 could promote the loss of E-cadherin-based junctions. Figure 6.2 provides a summary of the proposed mechanisms by which EphA2 could be regulating the internalisation of E-cadherin. The co-culture technique developed in this thesis in combination with specific inhibitors could be used in future studies to further elucidate the mechanism of KrasG12D cell extrusion and loss of E-cadherin via EphA2. It is important to understand the underlying mechanism as it could improve our understanding of how extrusion is overcome
in vivo to promote pancreatic tumourigenesis.
In order for cells to be extruded they must lose contact with neighbouring cells. Using co-culture assays in vitro and mosaic expression of KrasG12D in vivo models I found evidence for the loss of E-cadherin in KrasG12D cells upon interaction with normal neighbours. Intriguingly, loss of E-cadherin can promote metastasis and is a key hallmark of epithelial-mesenchymal transition (Cano et al., 2000; Onder et al., 2008). Thus, it could be hypothesised that repulsion of KrasG12D cells by normal neighbours is promoting a metastatic phenotype in transformed cells. Future studies could test if extruded KrasG12D cells are more invasive than KrasG12D cells cultured next to KrasG12D cells. Although extrusion was not directly observed in vivo, analysis of further intermediate time points could capture pancreatic cells extruding. Another important implication in the loss of E-cadherin-based cell-cell junctions is the loss of cell polarity. Epithelial tissues display apical-basal polarity which is organised by polarity complexes physically associated with cell junction architecture. Consequently, loss of epithelial junctions confers a loss of polarity. This could be important for the direction of extrusion with loss of polarity potentially able to alter where oncogenic cells are extruded (Slattum & Rosenblatt, 2014). Apically extruded cells would be extruded in the lumen and could cleared through the ductal system (Kon et al., 2017), whereas basally extruded cells could invade and enter the circulation. Intriguingly, circulating tumour cells have been found in genetically engineered mouse models of pancreatic cancer circulating before primary tumours (Rhim et al., 2012). Moreover, pancreatic cancer is rarely diagnosed in humans without metastasis suggesting these tumours spread early (Das & Batra, 2015). Although I screened livers for RFP, robust RFP fluorescence was not detected in KrasG12D mice. This could be due to detection of RFP in the liver and future
experiments would need to stain for the pancreatic specific marker Pdx1. Another hypothesis is that cells require additional oncogenic transformations such as loss of p53 to promote survival after extrusion (Rhim et al., 2012). Recently, genetic analysis has demonstrated that spatially distal lesions share a common ancestral cell of origin suggesting precancerous cells spread and colonise the pancreas via the ductal network (Makohon-Moore et al., 2018). In 4/8 patients, a precursor cell had obtained one or two mutations to driver oncogenes such as Kras and spread through the pancreas to produce additional neoplastic lesions. Therefore, extrusion of KrasG12D cells by normal neighbours could drive the invasion and metastasis of pancreatic cancer from the earliest stages of tumourigenesis.
Figure 6.2: Proposed molecular mechanism of KrasG12D cell elimination from the pancreas. In KrasG12D mutant cells, EphA2 interacts with EphrinA1 on normal neighbouring cells which leads to activation of Src. Once active, Src can directly promote E-cadherin internalisation by phosphorylation and also activates RhoA GTPase. RhoA can activate ROCK which promotes E-cadherin internalisation. It has also been demonstrated that ROCK leads to Myosin-II activation and
cytoskeletal rearrangements driving increased contractility. EphA2 can also activate another GTPase, Rac1, which can disrupt cell-cell contacts through endocytosis of E-cadherin. In summary, activation of EphA2 in KrasG12D cells by normal
neighbours could trigger repulsion and loss of cell-cell contacts via activation of Rho GTPases and Src. Together, this promotes extrusion of mutant cells from normal pancreatic monolayers. Blue arrows indicate published studies and red arrows the findings of this thesis.