Rationale & Hypotheses. Past Studies Have Shown That Podxl
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RATIONALE & HYPOTHESES
Past studies have shown that Podxl overexpression identifies a highly aggressive subset of breast carcinomas. Moreover, our previous results show an important role of Podxl on in vivo primary tumor growth and metastasis of MDA-MB-231 cells. However, the mechanism of action by which Podxl plays a role in tumor progression it still unclear. I hypothesize that the structure and biochemical and signalling properties of podocalyxin promote breast cancer tumor progression and, therefore, podocalyxin may be a novel target for future therapeutics.
OBJECTIVES & AIMS
Objective #1: Identify the Podxl functional domains required for cancer metastasis.
Aim 1.1: Generation of human Podxl mutants
Aim 1.2: Identify critical…show more content… We have selected different hPodxl mutants to generate (Figure 5). 1) PodxlDTHL: Podxl mutant lacking the C-terminal PDZ docking site, the docking site for NHERF1/2. This mutant will allow us to determine whether or not the ability to bind NHERF1/2 is critical for tumor progression. 2) PodxlTail, lacking all but three aminoacids of the cytoplasmic tail and, therefore, eliminating all intracellular binding (ezrin, NHERF1/2 and other potential unknown ligands). 3) PodxlEC missing the whole extracellular domain, allowing us to account for the lack of the highly negatively charged glycosylated portion as well as the stalk domain.
We previously demonstrated that a high-affinity monoclonal antibody (mAb) targeting Podxl (PODOC1) could be used to block tumor growth and metastasis in a pre-clinical tumor model in mice(28). Administration of PODOC1 mAb to tumor-bearing mice inhibited primary tumor growth and attenuated distant metastases to the lung(28). The PODOC1 epitope is the stalk domain of Podxl’s extracellular portion (a core protein domain). Because other very similar anti-podocalyxin mAbs that we generated to Podxl’s extracellular core protein domains had no therapeutic effect, we post that this critical domain (the PODOC1 epitope) serves a functional role in Podxl mode of action in metastasis. Since PODOC1’s epitope appears to have a significant role in tumorigenesis, we will generate an additional hPodxl mutant (ECGlyco) lacking only the