Autophagolysosomal exocytosis inverts Src kinase onto the cell surface in cancer.

Overexpression of the proto-oncogene Src is common to a wide variety of cancers. In this work, we found that Src is noncanonically translocated and inverted onto the cell surface in cancer, both in vitro and in vivo. We identified autophagolysosomal exocytosis (ALE) as a secretory mechanism prominent in cancer cell lines. Src represents the prototypical example of a family of membrane-anchored proteins that are transported by this process. Furthermore, this extracellular membrane–associated Src (eSrc) was found in primary tumors, and anti-Src antibody-based therapies mediated tumor cell killing in cell culture systems and in mouse xenograft models. Thus, intracellular N-myristoylated proteins, prototypically Src, can be topologically inverted onto the cell surface in cancer and targeted with antibody therapeutics. Editor's summary: Discovering cell surface proteins that are specific to cancer cells and not normal cells can create opportunities for safer delivery of toxic drugs to tumors. Delaveris et al. identified a family of proteins that are delivered to the cell surface in cancer but not in healthy tissues (see the Perspective by Pfannenstein and Meyer). One such protein is the cancer-promoting enzyme Src. The researchers developed antibodies that target the surface form of Src and showed that these anti-Src antibodies could specifically deliver toxic radioactive isotopes or recruit killer T cells to tumors in vivo. This work highlights the potential to increase the types of cell surface targets available for cancer therapies. —Stella M. Hurtley INTRODUCTION: Antibody-targeted therapeutics for cancer (e.g., T cell engagers, antibody-based radioligand therapies, and antibody-drug conjugates) require a cancer-associated protein to be displayed on the cell surface at much higher levels than in healthy tissues. Typically, targets are proteins highly up-regulated by cancer cells for survival (e.g., HER2 and EGFR). However, therapies targeting these proteins are limited by the expression of these cell surface targets in healthy tissues. There is therefore a need to find and understand new classes of tumor-associated cell surface proteins that have greater differences in tumor versus healthy tissue expression. Interest in new targets for cancer therapeutics has led to the discovery of new ways to differentiate tumor cells from healthy cells using their cell surface proteins. These differences include genetic mutations specific to cancer, the activities of enzymes yielding new posttranslational modifications, and aberrant protein localization. RATIONALE: We developed a system to identify cell surface kinase substrates as well as to define a high-resolution cell surface proteome through photo-proximity labeling proteomics. This method allowed us to both identify protein substrates that get phosphorylated on the cell surface and gain insights into the enzymes mediating phosphorylation. RESULTS: In this work, we found that Src, the master regulatory kinase typically anchored by its N-myristoyl lipid to the inner leaflet of the plasma membrane, was noncanonically translocated to the cell surface through autophagolysosomal exocytosis (ALE). We found additional evidence that other N-myristoylated intracellular proteins could be translocated in a similar fashion. Using cell lines with varying levels of cellular Src (cSrc), we observed that the transport of Src to the cell surface was regulated both by autolysosomal flux and total Src protein level. We observed that extracellular Src (eSrc) was present on the surface of cancer cell lines, whole and disaggregated xenografts, and disaggregated patient tumor samples. We propose that the prevalence of eSrc on cancer cells is likely attributable to both cSrc protein up-regulation in cancer and dysregulated autophagic flux, a hallmark of cancer, leading to translocation and inversion of cSrc by ALE. Additionally, we characterized the cell surface phosphotyrosine substrates of eSrc and showed that eSrc activity promoted cell proliferation. Finally, we demonstrated the translational potential of eSrc as a diagnostic and therapeutic cancer antigen using an anti-Src antibody formulated as a bispecific T cell engager (TCE), an antibody-drug conjugate (ADC), or a radioligand therapeutic (RLT). CONCLUSION: We describe a class of mislocalized proteins, prototypically the proto-oncogene tyrosine kinase Src. These proteins are N-terminal lipid-modified proteins, canonically anchored to the plasma membrane, that undergo topological inversion through a noncanonical secretory pathway that we elucidate in this work. Collectively, these findings uncover a class of proteins by which cancer cells can be therapeutically targeted with antibodies. Src is translocated to the cell surface.: SRC is translated, the protein is lipidated, and the lipid moiety inserts into the inner leaflet of the plasma membrane. Nucleating phagophore membranes originate from plasma membrane components, carrying Src along. After autophagosome formation, lysosomal fusion occurs, yielding autolysosomes, which can be exocytosed resulting in cell surface presentation of Src. [Figure created in part with BioRender.com] [ABSTRACT FROM AUTHOR]