Project Summary/Abstract The discovery that GRP78, traditionally regarded as a typical endoplasmic reticulum (ER) lumenal protein, can relocalize to the cell surface under pathological stress such as cancer, changes the paradigm on how this protein exerts its pro-proliferative and anti-apoptotic functions in cancer. Cell surface GRP78 (csGRP78) is emerging as a novel co-receptor controlling cell signaling, proliferation, survival, metastasis and renewal. Its mode of function does not require ATP, thus distinct from its ER foldase activity. Furthermore, since csGRP78 is preferentially expressed in cancer cells but not in normal organs, csGRP78 represents a new target as well as mediator for cancer-specific therapy. Agents including drug-conjugated peptides, toxins and antibodies have been developed targeting csGRP78 and have shown robust activities in blocking its oncogenic functions, suppressing tumor growth in both xenografts and spontaneous tumor models, and synergizing with standard chemotherapy. Of note, monoclonal antibody targeting csGRP78 has completed Phase I/IIa clinical trials with positive response. Despite these exciting advances, how ER stress induces csGRP78 expression and how csGRP78 exerts its biological function are largely unknown. During the past grant period, we discovered that inhibition of the ER stress-inducible proto-oncogene tyrosine kinase protein SRC (c-SRC) potently blocks cell surface expression of GRP78 and other ER lumenal proteins containing the KDEL ER retention motif in a wide variety of cancer cells. We have identified novel binding partners of GRP78 on the cell surface including CD44 and CD109 with established functions in cell motility, invasion, signaling, proliferation and survival. Based on these exciting findings, we propose the following two integrated aims. Aim 1 will investigate how ER stress causes relocalization of GRP78 from the ER to the cell surface. We will test a new concept that a major pathway whereby cancer cells export ER chaperones bearing the KDEL motif to the cell surface is triggered by SRC activation and its subsequent activation of ASAP1, GBF1 and Arf1 leading to blockage of Golgi to ER retrograde. We will also investigate additional SRC downstream effectors and SRC/Golgi-independent routes for csGRP78 expression in exceptional cases. Aim 2 will address how csGRP78 regulates its cell surface partner proteins and their signaling pathways. We will study their physical and functional interactions through biochemical, mutational, as well as confocal and single molecule super-resolution imaging approaches. We will focus on CD44 with established roles in cancer invasion and stemness and perform in vitro and in vivo tests utilizing established human breast cancer cell lines with differential metastatic potential, as well as novel ex vivo culture of patient-derived circulating tumor cells exhibiting metastatic and cancer stem cell properties. We will also study novel mechanisms of csGRP78, through interaction with CD109, in regulating TGF-?-mediated tumor suppressing as well as tumor promoting functions. Collectively, these studies will reveal novel mechanisms for SRC and csGRP78 in tumorigenesis with wide clinical implications.