Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive carcinomas, with very high early metastasis rates. Increased Lamellipodin (Lpd) expression is correlated with poor prognosis in breast cancer patients, while also promoting breast cancer metastasis. We found that high Lpd mRNA abundance also correlates with poor prognosis in PDAC. In 90-95% of PDAC cases it is driven by mutated KRas, a small GTPase which controls cellular proliferation. Lpd functions downstream of growth factor receptors as an adapter protein and interacts with KRas and Rac, a related GTPase, which controls cell migration. Previous data showed that Lpd promotes cell proliferation of fibroblasts, HeLa endometrium carcinoma cells, and glioblastoma cells. In breast cancer cells, Lpd promotes cell migration via interaction with the F-actin nucleating Scar/WAVE-Arp2/3 complexes and with the F-actin elongating Ena/VASP proteins. We therefore hypothesised that in PDAC cells Lpd may promote cell proliferation downstream of KRas and cell migration downstream of KRas and/or Rac. Furthermore, we postulated that Lpd may be a biomarker for PDAC prognosis. We generated Lpd cDNA mutated in specific binding and regulatory sites and used these to investigate whether the interaction between Lpd and KRas or the Scar/WAVE complex is required for mediating cell proliferation. We found that downregulation of Lpd expression significantly reduced proliferation in two PDAC cell lines. We also observed that re-expressing an Lpd mutant, which constitutively binds KRas, significantly increased proliferation. In addition to this, Lpd that cannot bind the Scar/WAVE complex also increased proliferation. From these findings we conclude that Lpd has two proliferaCve mediaCng funcCons in PDAC cells, both promoCng proliferaCon downstream of KRas and inhibiCng it via the Scar/WAVE complex. Similarly, to test whether Lpd is activated by KRas to promote migration or whether Lpd’s function in cell migration is mediated by the Scar/WAVE complex, the previous mutants were re-expressed in Lpd-depleted cells in random cell migration assays. The results showed that cell speed and persistence were significantly reduced when Lpd that cannot bind the Scar/WAVE complex was re-expressed. In addition to this our results showed that rescuing Lpd with a dominant active Lpd-KRas interacting mutant also rescued the persistence defect observed when Lpd was knocked down. Taken together, this suggests that Lpd promotes proliferation and cell persistence as a downstream KRas effector, while Lpd inhibits proliferation and promotes the speed and persistence of cells via the Scar/WAVE complex. To explore whether Lpd is a biomarker for PDAC prognosis, we also analysed Lpd’s protein expression levels in PDAC samples and correlated this with patient outcome data. Taken together, Lpd may be an important new target for future PDAC studies and treatments.
The Role of Lamellipodin in the Proliferation and Migration of Pancreatic Cancer Cells
Schlatter, J. (Author). 1 Jun 2024
Student thesis: Doctoral Thesis › Doctor of Philosophy