In order to address whether the increased activation of Arf6 and Rac1 is specific for HCT116 cells or whether it is a general mechanism important for tumorigenesis and metastasis of colorectal cancer cells, we generated another colorectal cancer cell line, SW48, with a knockout of Pals1 (Supplementary Fig.
3a). Indeed, Pals1-deficient SW48 cells display a similar increase in cell migration as HCT116 cells (Supplementary Fig.
3b). Furthermore, active Rac1 as well as Arf6 levels are strongly increased in SW48∆Pals1 cells (Supplementary Fig.
3c-d). However, in contrast to the situation in HCT116 cells, depletion of Pals1 in SW48 results only in a slight increase in total Arf6 expression (Supplementary Fig.
3e). In HCT116 cells, it is still unclear, how deletion of Pals1 results in an increase in Arf6 transcription. Enhanced Arf6 gene expression has been reported to be induced by activation of PI3K/Akt and ERK1/2 signaling [
13]. In turn, Arf6 activates the MEK/ERK signaling pathway [
14]. HCT116 cells display a gain of function mutation in KRas, resulting in a constitutively activated MEK/ERK signaling, whereas SW48 are wild type for KRas. Thus, we hypothesized, that in Pals1-depleted cells, Arf6 activity is increased, resulting in an enhanced transcription by activation of MEK/ERK in a KRas-mutant-sensitized background (HCT116 cells), whereas in KRas wild type cells, increased Arf6 activation is not sufficient to trigger a strong increase in its own transcription. Indeed, we found that inhibition of Arf6 in HCT116∆Pals1 cells results in a significant decrease of Arf6 expression, supporting our hypothesis (Supplementary Fig.
3f). However, although inhibition of ERK led to a decrease in Arf6 mRNA expression in Pals1-depleted HCT116 cells, the effect was not as strong as for Arf6 inhibition, indicating that differences in KRas-signaling might not the only differences between HCT116∆Pals1 and SW48∆Pals1.