# Shedding of GPP130 by PC7 and Furin: Potential Implication in Lung Cancer Progression
## 3 Discussion
It is possible that endogenous GPP130 shedding may be readily observed in cells derived from such cancers that overexpress Furin and/or PC7 endogenously, as observed in our previous report in breast cancer tumors, e.g., MDA-MB-231 [4], which is what this study is trying to mimic using PC7 or Furin overexpression in model cell lines. We next tested the effect of silencing endogenous GPP130 (siRNA) or its overexpression on the cellular proliferation of A549 cells (Figure 5). As shown for head and neck cancer, it is plausible that the knockdown of GPP130 also leads to the perturbation of Stromal Interaction Molecule 1 (STIM1)-GOLIM4 signal axis in A549 cells, thereby reducing the cellular growth [25]. Our data extends this GPP130-induced pro-proliferative activity to lung cancer A549 cells and reveals that it is the GPP130 shedding at S1 by PC7 and/or Furin that enhances such proliferative activity (Figure 6). In contrast, the GPP130 R70A-R277 Avariant that is completely resistant to shedding showed no proliferative activity at all (Figure 7). Structure-function studies demonstrated that two domains in GPP130 regulate its pro-proliferative activity: (1) The N-terminal domain aa(1-67), in which the prevention of the likely reversible Cys\({}_{6}\)-palmitoylation aa(1-67)C6A [36] of GPP130 [37] significantly enhances cellular growth (Figure 6C); and (2) the N-terminal domain of the secreted shed GPP130 at S1 [aa(71-696)] (Figure 6B) that encompasses domains responsible for endosome to Golgi recycling and TGN localization (Figure 6A). Since this fragment seems to enhance cellular proliferation upon its incubation with naive A549 cells (Figure 6B), future studies may identify a putative cell-surface binding partner/receptor and its subsequent signaling pathway. Interestingly, sortilin has been shown to interact with the luminal domain of GPP130 [38], it is plausible that shed GPP130 may bind sortilin at the cell surface and that triggers a cascade of events resulting in higher cellular proliferation [39]. By comparison, CASC4 was also shown to be shed at a single site by Furin and PC7, and the generated N-terminal transmembrane fragment (aa 1-66) also enhances cellular migration and invasion [4]. Thus, both GPP130 and CASC4 have N-terminal membrane-bound domains that enhance cellular proliferation and/or migration, which may be implicated in cancer progression. Indeed, the overexpression of CASC4 or the efficiently shed CASC4-5 REL variant in A549 cells led to increased proliferation compared to EV (Figure 7). The presence of multiple basic residues in the cytosolic tails of both GPP130 and CASC4 suggests that they may act as nuclear localization signals that could sort them to the nucleus [40] if released into the cytosol following a putative g-secretase cleavage within their transmembrane domain, as observed with various transmembrane proteins [41]. Previous studies have shown that the dysregulation of palmitoylation is indeed critically involved in many cancers [42]. This dysregulation, combined with GPP130 shedding via PC7/Furin, may explain the proliferative effects of GPP130. In conclusion, we showed for the first time that GPP130 is cleaved by PC7 and Furin and that this shedding could lead to the release of an ectodomain fragment, which may enhance cell proliferation in a similar fashion to that of another family member protein, GP73 that is highly oncogenic [43; 44]. The present study sheds light on a poorly characterized Golgi-resident substrate of PC7 and Furin, and future work will help to elucidate its biological roles, binding partners, and how PCs modulate its functions. Our findings highlight the functional significance of the shedding of GPP130 by PC7 and Furin and its effect on the proliferation of human lung carcinoma A549 cells. Our data suggest that PC7 and/or Furin, which are implicated in many cancers via their enzymatic activity [3; 4; 45], may also enhance the shedding of novel cancer growth factors such as GPP130 and CASC4.