Macropinocytosis has long been known as a primary method for cellular intake of fluid-phase and membrane-bound bulk cargo. This review seeks to re-examine the latest studies to emphasize how cancers exploit macropinocytosis to further their tumorigenesis, including details in how macropinocytosis can be adapted to serve diverse functions. Furthermore, this review will also cover the latest endeavors in targeting macropinocytosis as an avenue for novel therapeutics.

The mammalian Golgi complex is a highly dynamic organelle consisting of stacks of flattened cisternae with associated coated vesicles and membrane tubules that contribute to cargo import and export, intra-cisternal trafficking, and overall Golgi architecture. At the morphological level, all of these structures are continuously remodeled to carry out these trafficking functions. Recent advances have shown that continual phospholipid remodeling by phospholipase A (PLA) and lysophospholipid acyltransferase (LPAT) enzymes, which deacylate and reacylate Golgi phospholipids, respectively, contributes to this morphological remodeling. Here we review the identification and characterization of four cytoplasmic PLA enzymes and one integral membrane LPAT that participate in the dynamic functional organization of the Golgi complex, and how some of these enzymes are integrated to determine the relative abundance of COPI vesicle and membrane tubule formation.

Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into tumor cells. Ideal antibodies for developing such therapeutics should be capable of both tumor-selective binding and efficient endocytosis. The macropinocytosis pathway is capable of both rapid and bulk endocytosis, and recent studies have demonstrated that it is selectively up-regulated by cancer cells. We hypothesize that receptor-dependent macropinocytosis can be achieved using tumor-targeting antibodies that internalize via the macropinocytosis pathway, improving potency and selectivity of the antibody-based targeted therapeutic. Although phage antibody display libraries have been utilized to find antibodies that bind and internalize to target cells, no methods have been described to screen for antibodies that internalize specifically via macropinocytosis. We hereby describe a novel screening strategy to identify phage antibodies that bind and rapidly enter tumor cells via macropinocytosis. We utilized an automated microscopic imaging-based, High Content Analysis platform to identify novel internalizing phage antibodies that colocalize with macropinocytic markers from antibody libraries that we have generated previously by laser capture microdissection-based selection, which are enriched for internalizing antibodies binding to tumor cells in situ residing in their tissue microenvironment (Ruan, W., Sassoon, A., An, F., Simko, J. P., and Liu, B. (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol. Cell. Proteomics. 5, 2364–2373). Full-length human IgG molecules derived from macropinocytosing phage antibodies retained the ability to internalize via macropinocytosis, validating our screening strategy. The target antigen for a cross-species binding antibody with a highly active macropinocytosis activity was identified as ephrin type-A receptor 2. Antibody-toxin conjugates created using this macropinocytosing IgG were capable of potent and receptor-dependent killing of a panel of EphA2-positive tumor cell lines in vitro. These studies identify novel methods to screen for and validate antibodies capable of receptor-dependent macropinocytosis, allowing further exploration of this highly efficient and tumor-selective internalization pathway for targeted therapy development.

For the first time, a cytoplasmic phospholipase A2 enzyme, platelet-activating factor acetylhydrolase (I)b, is described that is directly involved in the formation of membrane tubules from endosomes and trafficking through the endocytic recycling pathway.