Syndecan-1 Is Critical in ARF6-Dependent Macropinocytosis Driven By KRAS Mutation in the Pathophysiology of Multiple Myeloma

Syndecan-1 (SDC1), also known as CD138, is a member of integral membrane heparin sulfate proteoglycans constantly expressed in plasma cells (PCs) and a primary diagnostic marker for human multiple myeloma (MM). We here further define new functions of SDC1 in the MM pathobiology. Firstly, flow cytometry and qRT-PCR analysis showed that SDC1 is expressed at relatively higher levels in AMO-1, U266, OPM2, H929, MM1S, and MM1R MM cells when compared with JJN3, RPMI 8226, and ANBL6 MM cells. SDC1 levels are comparable in paired MM cell lines sensitive or resistant to current anti-MM therapies including lenalidomide, pomalidomide, and bortezomib. Significantly increased SDC1 mRNA levels in advanced MM stages (p<0.05) were further correlated with elevated soluble SDC1 protein levels in patient serum by ELISA. As expected, higher soluble SDC1 was also detected in culture media (CM) from MM cell lines with higher mRNA levels. Next, the effects of SDC1 were studied by SDC1 knockout (KO) in OPM2, JJN3 and H929 cells via CRISPR/Ca9 gene modification, followed by RNA-Seq analysis. Neglectable shed SDC1 in CM of all SDC1 KO MM cells confirm null SDC1 expression. Expression of anti-apoptosis gene BCL2L1, cell cycle genes (CCND1, CCND2), and transcription factor RELA gene were decreased in SDC1 KO vs control MM cells. Permanent SDC1 KO cells were eventually derived, indicating additional SDC1 function besides its role in MM cell growth and survival. KEGG pathway analysis associated with genes downregulated following SDC1 KO showed biological processes (BPs) enrichment in ECM-receptor interaction (hsa04512; p< 0.001), cell adhesion molecules (hsa04514; p<0.001), focal adhesion (hsa04510; p<0.001), cytokine-cytokine receptor interaction (hsa04060; p=0.005), chemokine signaling pathway ( hsa04062; p=0.006), gap junction (hsa04540; p=0.002), axon guidance (hsa04360; p=0.016), JAK-STAT signaling pathway (hsa04630; p=0.026), lysosome (hsa04142; p=0.047).Specifically, IL-21R, related to JAK-STAT signaling pathway and cytokine-cytokine receptor interaction, was significantly decreased in SDC1 KO MM cells, as validated by qRT-PCR and human receptor array analysis. IL-21R contains the common cytokine-receptor gamma-chain shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, indicating potential cross-talks between MM cells and surrounding immune cells via SDC1. Since its natural ligand IL-21 is mainly secreted by non-myeloma bone marrow (BM) accessory cells, SDC1 could also modulate interactions between myeloid lineages and MM cells via IL-21/IL-21R circuit in the BM microenvironment. Of note, other key MM antigens, i.e., CD38, BCMA, SLAMF7 were affected at mRNA levels in SDC1 KO vs control MM cells. Moreover, human receptor array data showed decreased expression in Flt-3L, DR6, Endoglin, GITR, HVEM, IL-2RG, IL-17RA, IL-21R, PECAM-1, PDGFRB, RAGE, Trappin-2 and µPAR in SDC1 KO MM cells. BPs through GO analysis in these downregulated receptors were cell activation (GO:0001775), cell surface receptor signaling pathway (GO:0007166), and immune system process (GO:0002370). KEGG analysis showed that those receptors molecular were enriched in cytokine-cytokine receptor interaction pathway (KEGG:04060).Consistent with RNA seq data, µPAR, an important factor of ARF6-dependent trafficking, was also found significantly downregulated in SDC1 KO MM cells. Since ARF6 activation regulates macropinocytosis, an essential metabolic pathway fueling Ras-driven cancer cells, these data suggest that SDC1 may involve in ARF6-dependent macropinocytosis in MM cells. ARF6 is induced by KRAS mutation, we thus checked macropinocytic index in KRAS-mutated MM cell lines. Increased macropinocytosis occur in KRAS-mutated MM cells (KMS28-BM, MM1S, MM1R) compared with KRAS WT OPM2 and KMS12-BM. Importantly, macropinocytosis was inhibited following SDC1 depletion in KRAS-mutated MM cells, indicating that SDC1 critically mediates KRAS-driven macropinocytosis in MM cells. These data highlight the requirements for SDC1 to mediate nutrient-scavenging macropinocytosis in MM cells, most prominently harboring KRAS-mutation. Taken together, our results identify new functions of SDC1 which are crucial to enhance myeloma cell fitness and adaptation to various conditions in the BM milieu, thereby further supporting SDC1 targeted immunotherapy in MM.