Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy

Kimberly A Snyder, Michael R Hughes, Bradley Hedberg, Jill Brandon, Diana Canals Hernaez, Peter Bergqvist, Frederic Cruz, Kelvin Po, Marcia L Graves, Michelle E Turvey, Julie S Nielsen, John A Wilkins, Shaun R McColl, John S Babcook, Calvin D Roskelley, Kelly M McNagny

Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression. We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell-lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF-7. We evaluated how podocalyxin affects tumorsphere-formation in vitro and compared the ability of podocalxyin-deficient (shPODXL) and -replete cell lines to form tumors and metastasize using xenogenic- or syngeneic-transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of anti-human podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL) cells, we found that podocalyxin-deficient cells exhibit a striking decrease in the ability to form clonal tumors in the lung, liver, and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy we screened a series of novel anti-human podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.