Proximity proteomics identifies cancer cell membrane cis‐molecular complex as a potential cancer target

Abstract

Cancer‐specific antigens expressed in the cell membrane have been used as targets for several molecular targeted strategies in the last 20 years with remarkable success. To develop more effective cancer treatments, novel targets and strategies for targeted therapies are needed. Here, we examined the cancer cell membrane‐resident “cis‐bimolecular complex” as a possible cancer target (cis‐bimolecular cancer target: BiCAT) using proximity proteomics, a technique that has attracted attention in the last 10 years. BiCAT were detected using a previously developed method termed the enzyme‐mediated activation of radical source (EMARS), to label the components proximal to a given cell membrane molecule. EMARS analysis identified some BiCAT, such as close homolog of L1 (CHL1), fibroblast growth factor 3 (FGFR3) and α2 integrin, which are commonly expressed in mouse primary lung cancer cells and human lung squamous cell carcinoma cells. Analysis of cancer specimens from 55 lung cancer patients revealed that CHL1 and α2 integrin were highly co–expressed in almost all cancer tissues compared with normal lung tissues. As an example of BiCAT application, in vitro simulation of effective drug combinations used for multiple drug treatment strategies was performed using reagents targeted to BiCAT molecules. The combination treatment based on BiCAT information moderately suppressed cancer cell proliferation compared with single administration, suggesting that the information about BiCAT in cancer cells is useful for the appropriate selection of the combination among molecular targeted reagents. Thus, BiCAT has the potential to contribute to several molecular targeted strategies in future.

Bimolecule detection for Extracellular Vesicle Screening

“…Western blot analysis of CD63 confirmed that EVs could be crudely purified from mouse serum by a polymer precipitation method (Figure 2A). CHL1, which is the molecule constituting LC BiCAT (Kotani et al, 2019), was also detected at higher levels in serum EVs from EML4-ALK transgenic mice (Supplementary Figure 1A). EMARS products from precipitated serum EVs (crude EVs) were detectable in CHL1 probe (+) samples, but not in EMARS probe (-) samples, indicating that EMARS reacts well with CHL1 probes and no serum factors induced non-specific EMARS reactions (Figure 2B).…”

“…The mouse and human anti-CHL1 antibodies (AF2147 and MAB2126; R&D systems, MN) were partially reduced and bound to HRP using a peroxidase labeling kit SH (Dojindo, Kumamoto, Japan). The binding capacity of the HRP-labeled antibody was evaluated as described previously (Kotani et al, 2019).…”

“…6 to 8, which contained EVs with HRP-conjugated CHL1 antibody, were collected depend on each experiment, and applied to Nanosep® centrifugal devices (30K; Pall Life Sciences, MI) followed by centrifugation at 8,000 g for 5 min to concentrate EV particles. The concentrated EV particles on the filters were washed with 100 µl PBS once by centrifugation, and then fluorescein-tyramide (FT) reagent as EMARS reaction reagent (Kotani et al, 2019) was added to the filter with gentle mixing. After 10 min incubation at room temperature, remaining FT reagent was removed by centrifugation at 8,000 g for 5 min followed by wash with PBS once by centrifugation.…”

“…EMARS products were purified and enriched with anti-fluorescein antibody-conjugated Sepharose as described previously (Kotani et al, 2019). Briefly, the flow-through fraction of G-50 column described above was diluted with 300 µL of NP-40 lysis buffer.…”

“…Cell membrane or membrane-attached proteins non-randomly form heterocomplexes accompanied by a fluid biological membrane known as a “lipid raft” structure (Brown and London, 1998). We reported that the membrane heterocomplex “ Bi molecular Ca ncer T arget” (BiCAT) could be a potential lung cancer (LC) antigen consisting of a hetero bimolecular complex (Kotani et al, 2019). In the BiCAT strategy, because cancer specific-antigens are defined by two molecules, the specificity is theoretically higher than that of a single-molecule antigen.…”