The tumor suppressor protein Par-4, which is secreted by normal cells, selectively induces apoptosis in cancer cells. We identified a 3-arylquinoline derivative, designated Arylquin 1, as a potent Par-4 secretagogue in cell cultures and mice. Mechanistically, Arylquin 1 binds to vimentin, displaces Par-4 from vimentin for secretion and triggers the efficient paracrine apoptosis of diverse cancer cells. Thus, targeting vimentin with Par-4 secretagogues efficiently induces paracrine apoptosis of tumor cells.

Par-4 is a pro-apoptotic, tumor suppressor protein that induces apoptosis selectively in cancer cells. Endoplasmic reticulum-stress and higher levels of protein kinase A in tumor cells confer the coveted feature of cancer selective response to extracellular and intracellular Par-4, respectively. Recent studies have shown that systemic Par-4 confers resistance to tumor growth in mice, and that tumor-resistance is transferable by bone marrow transplantation. Moreover, recombinant Par-4 inhibits the growth of tumors in mice. As systemic Par-4 induces apoptosis via cell surface GRP78, strategies that promote GRP78 trafficking to the cell surface are expected sensitize cancer cells to circulating levels of Par-4. This review illustrates the domains and mechanisms by which Par-4 orchestrates the apoptotic process in both cell culture models and in physiological settings.

Tumor suppressor PAR-4 acts in part by modulating sensitivity to apoptosis but the basis for its activity is not fully understood. In this study, we describe a novel mechanism of anti-apoptosis by NF-kappaB, revealing that it can block PAR-4-mediated apoptosis by downregulating trafficking of the PAR-4 receptor GRP78 from the endoplasmic reticulum (ER) to the cell surface. Mechanistic investigations revealed that NF-kappaB mediated this anti-apoptotic mechanism by upregulating expression of UACA, a pro-inflammatory protein in certain disease settings. In clinical specimens of cancer, a strong correlation existed between NF-kappaB activity and UACA expression, relative to normal tissues. UACA bound to intracellular PAR-4 in diverse cancer cells, where it prevented translocation of GRP78 from the ER to the cell surface. This pathway of anti-apoptosis could be inhibited by suppressing levels of NF-kappaB or UACA expression, which enhanced ER stress and restored GRP78 trafficking to the cell surface, thereby sensitizing cancer cells to apoptosis by extracellular PAR-4 or GRP78 agonistic antibody. In summary, our results identify a novel intracellular pathway of apoptosis mediated by NF-kappaB through UACA elevation, which by attenuating ER stress and GRP78 translocation to the cell surface can blunt the sensitivity of cancer cells to apoptosis.

SUMMARYThe induction of tumor suppressor proteins capable of cancer cell apoptosis represents an attractive option for the re-purposing of existing drugs. We report that the anti-malarial drug, chloroquine (CQ), is a robust inducer of Par-4 secretion from normal cells in mice and cancer patients in a clinical trial. CQ-inducible Par-4 secretion triggers paracrine apoptosis of cancer cells and also inhibits metastatic tumor growth. CQ induces Par-4 secretion via the classical secretory pathway that requires the activation of p53. Mechanistically, p53 directly induces Rab8b, a GTPase essential for vesicle transport of Par-4 to the plasma membrane prior to secretion. Our findings indicate that CQ induces p53- and Rab8b-dependent Par-4 secretion from normal cells for Par-4-dependent inhibition of metastatic tumor growth.

Developing CAR T cells for acute myeloid leukemia (AML) has been hampered by a paucity of targets that are expressed on AML blasts and not on hematopoietic progenitor cells (HPCs). Here we demonstrate that GRP78 is expressed on the cell surface of primary AML blasts but not HPCs. To target GRP78, we generate T cell expressing a GRP78-specific peptide-based CAR, which show evidence of minimal fratricide post activation/transduction and antigen-dependent T cell differentiation. GRP78-CAR T cells recognize and kill GRP78-positive AML cells without toxicity to HPCs. In vivo, GRP78-CAR T cells have significant anti-AML activity. To prevent antigen-dependent T cell differentiation, we block CAR signaling and GRP78 cell surface expression post activation by using dasatinib during GRP78-CAR T cell manufacturing. This significantly improves their effector function in vitro and in vivo. Thus, targeting cell surface GRP78-positive AML with CAR T cells is feasible, and warrants further active exploration.

Summary The guardian of the genome, p53, is often mutated in cancer and may contribute to therapeutic resistance. As p53 is intact and functional in normal tissues, we harnessed its potential to inhibit the growth of p53-deficient cancer cells. Specific activation of p53 in normal fibroblasts selectively induced apoptosis in p53-deficient cancer cells. This paracrine effect was mediated by p53-dependent secretion of the tumor suppressor Par-4. Accordingly, activation of p53 in normal mice, but not in p53−/− or Par-4−/− mice, caused systemic elevation of Par-4 that induced apoptosis of p53-deficient tumor cells. Mechanistically, p53 induced Par-4 secretion by suppressing the expression of UACA, which sequesters Par-4. Thus, normal cells can be empowered by p53 activation to induce Par-4 secretion for inhibition of therapy-resistant tumors.

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Primary tumors are often heterogeneous, composed of therapy-sensitive and emerging therapy-resistant cancer cells. Interestingly, treatment of therapy-sensitive tumors in heterogeneous tumor microenvironments results in apoptosis of therapy-resistant tumors. In this study, we identify a Par-4 amino-terminal fragment (PAF) that is released by diverse therapy-sensitive cancer cells following therapy-induced caspase cleavage of the tumor suppressor Par-4 protein. PAF caused apoptosis in cancer cells resistant to therapy and inhibited tumor growth. A VASA segment of Par-4 mediated its binding and degradation by the ubiquitin ligase Fbxo45, resulting in loss of Par-4 pro-apoptotic function. Conversely, PAF, which contains this VASA segment, competitively bound to Fbxo45 and rescued Par-4-mediated induction of cancer cell-specific apoptosis. Collectively, our findings identify a molecular decoy naturally generated during apoptosis that inhibits a ubiquitin ligase to overcome therapy resistance in tumors.