The NHERF1 PDZ2 Domain Regulates PKA-RhoA-p38-mediated NHE1 Activation and Invasion in Breast Tumor Cells

Rosa A. Cardone,* Antonia Bellizzi,* ${dagger}$ Giovanni Busco,* Edward J. Weinman, ${ddagger}$ ${sect}$ Maria E. Dell’Aquila,|| Valeria Casavola,* Amalia Azzariti, ${dagger}$ Anita Mangia, ${dagger}$ Angelo Paradiso, ${dagger}$ ¶ and Stephan J. Reshkin*¶

Departments of ^*General and Environmental Physiology and ^||Animal Production, University of Bari, 70126 Bari, Italy; Clinical Experimental Oncology Laboratory, National Cancer Institute Giovanni Paolo II, 70126 Bari, Italy; Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201; Medical Service, Department of Veterans Affairs Medical Center, Baltimore, MD 21201
Departments of ^*General and Environmental Physiology and ^||Animal Production, University of Bari, 70126 Bari, Italy; Clinical Experimental Oncology Laboratory, National Cancer Institute Giovanni Paolo II, 70126 Bari, Italy; Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201; Medical Service, Department of Veterans Affairs Medical Center, Baltimore, MD 21201

Monitoring Editor: John Cleveland

Understanding the signal transductionsystems governing invasion is fundamental for the design oftherapeutic strategies against metastasis. NHERF1 is a PDZ domaincontaining protein that recruits membrane receptors/transportersand cytoplasmic signaling proteins into functional complexes.NHERF1 expression is altered in breast cancer but its effectiverole in mammary carcinogenesis remains undefined. We reporthere that NHERF1 overexpression in human breast tumor biopsiesis associated with metastatic progression, poor prognosis andHIF-1 ${alpha}$ expression. In cultured tumor cells, hypoxia and serumdeprivation increase NHERF1 expression, promote the formationof leading-edge pseudopodia and redistribute NHERF1 to thesepseudopodia. This pseudopodial localization of NHERF1 was verifiedin breast biopsies and in 3D matrigel culture. Further, serumdeprivation and hypoxia stimulate the Na⁺/H⁺ exchanger (NHE1),invasion and activate a PKA-gated RhoA/p38 invasion signal module.Significantly, NHERF1 overexpression was sufficient to inducethese morphological and functional changes and potentiated theirinduction by serum deprivation. Functional experiments withtruncated and binding groove mutated PDZ domain constructs demonstratedthat NHERF1 regulates these processes through its PDZ2 domain.We conclude that NHERF1 overexpression enhances the invasivephenotype in breast cancer cells, both alone and in synergywith exposure to the tumor microenvironment, via the coordinationof PKA-gated RhoA/p38 signaling.

^¶These authors contributed equally to this work.

Address correspondence to: Stephan J. Reshkin (reshkin{at}biologia.uniba.it)