Metastasis formation is the life-threatening end stage of cancer. It is therefore vital to understand how this process is regulated so that therapeutic intervention becomes possible. ASAP1 (Arf-GAP with SH3-domains, Ankyrin-repeats and PH-domains) was discovered in an unbiased genetic screen for genes that are involved in metastasis formation. Subsequently it was shown that this protein promotes tumor cell motility and invasiveness. Loss and gain of function experiments in a pancreatic carcinoma model demonstrated a functional role for ASAP1 in regulating metastasis. In human colorectal cancer patients ASAP1 expression strongly correlates with short metastasisfree survival and poor prognosis. At the molecular level, co-immunoprecipitation experiments have shown that ASAP1 binds to both h-prune and Nm23-H1 (Non- Metastatic protein 23-H1), proteins that are also involved in the metastatic process. Using the highly metastatic breast cell line MDA-MB-231 that endogenously expresses ASAP1, Nm-23H1 and h-prune, in my PhD thesis I aimed to characterize how ASAP1 contributes to metastasis by addressing the following issues: a) the influence of ASAP1 and its interaction partners on the internalization of EGF; b) effects of ASAP1- containing protein complexes on cellular motility; c) further possible interaction partners that may modulate ASAP1 activity. My results showed that, despite their role in regulating motility and metastasis formation, neither Nm23-H1, nor h-prune influence the effects of ASAP1 on cell migration. Moreover, these two proteins also do not affect the ability of ASAP1 to regulate the internalization of EGF. I then focused my attention on other possible proteins that might contribute to ASAP1-mediated metastasis by screening for potential SH3 domain-bearing interaction partners, because the SH3 binding domain of ASAP1 is crucial for its motility-promoting activity. I thereby identified SLK (Ste-20 Like Kinase) as a new interaction partner of ASAP1, and analyzed its effects on ASAP1 function. Although SLK co-immunoprecipitated with ASAP1, and is itself involved in the control of cell motility, this protein did not affect the motility-promoting activity of ASAP1. Together, my results demonstrate the role of ASAP1 in promoting both cell motility and receptor internalization in the context of tumor cells, important biological processes that regulate metastasis formation.
|Supervisor:||Sleeman, Prof. Dr. Jonathan|
|Place of Publication:||Heildeberg|
|Date of thesis defense:||8 May 2013|
|Date Deposited:||14 May 2013 05:57|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||500 Natural sciences and mathematics
570 Life sciences