# Shedding of GPP130 by PC7 and Furin: Potential Implication in Lung Cancer Progression
## 2 Results
### GPP130 Is Cleaved in Post-ER Acidic Comparments
Indeed, Furin activity is enriched >3-fold in endosomes compared to the TGN, and yet it is 10-fold less inhibited by dec-RVKR-cmk [29]. The reduced inhibition of Furin by dec-RVKR-cmk in endosomes may also be related to the higher acidity of late endosomes whereupon the Furin active site His* would be more protonated and hence resistant to irreversible alkylation by dec-RVKR-cmk. Taken together, these data suggest that GPP130 is cleaved at S1 by both Furin and PC7 in post-ER acidic compartments, e.g., TGN and/or endosomes, and at S2 by Furin in a distinct acidic compartment. GPP130 Influences Cell Proliferation in Lung Cancer-, Ovarian Cancer- and Cervical Cancer-Derived Cell Lines  
The GOLIM4/GPP130 gene enhances cell proliferation via the modulation of key regulators of the cell cycle in head-and-neck cancer cells [25]. We therefore wanted to investigate whether GPP130 shedding, as seen in the current study, could also influence cell proliferation. Hence, we first interrogated a genomics database (cBioPortal for Cancer Genomics) and noted that the GOLIM4/GPP130 gene is amplified in up to ~35% of lung cancer patients, ~20% of ovarian cancer patients, ~15% of cervical cancer and in >10% of head-and-neck cancer patients (Figure 1B). Accordingly, we selected lung cancer-derived A549, ovarian cancer-derived SKOV3, and cervical cancer-derived HeLa cells to perform cell proliferation assays. Although the overexpression of GPP130 cDNA in these cell lines resulted in an increase in cell proliferation (Figure 5A-C), we chose to continue with A549 cells owing to the high alteration frequency of GPP130 in lung cancers (Figure 1B) and the fact that these cells endogenously express PC7, Furin, and GPP130 (Figure 5D). Our results showed that upon treatment of A549 cells with siRNAs to GPP130 that effectively reduced its protein expression by >80%, the cells exhibited a significant decrease in cellular proliferation (~40%; Figure 5E). In contrast, upon overexpression of GPP130 cDNA, we observed a significant increase in cell proliferation (~20%; Figure 5C). Identification of Critical Domains and Palmitoylation of GPP130 Implicated in Enhanced Proliferation  
We next wanted to assess if GPP130 shedding by PCs could affect cell proliferation and identify the critical domains in GPP130 that may enhance the proliferation of A549 cells. Thus, according to data obtained in Figure 3, we engineered three constructs to mimic the products generated upon cleavage at the S1 and S2 sites. This included the N-terminal membrane-bound fragment consisting of amino acids aa 1-67 generated upon S1 cleavage and the corresponding aa 71-696 (soluble sol-GPP130), and the soluble secreted Furin-generated S2 product aa 278-696 (Figure 6A). Figure 5: GPP130 enhances the proliferation of HeLa, SKOV3, and A549 cells. (**A–C**) Inucocyte analysis of the cellular proliferation of human HeLa (**A**), SKOV3 (**B**), and A549 (**C**) cells overexpressing GPP130. The data are normalized to day 0, and proliferation is followed by 96 h [\(n=3\)]. (**D**) qPCR analysis of the mRNA levels of PCSK7, Furin, and GPP130 in naive A549 cells, normalized to the housekeeping gene human TATA-binding protein (hTBP) [\(n=3\)]. (**E**) Inucocyte analysis of the cellular proliferation of A549 cells lacking GPP130 [\(n=3\)]. The efficiency of the siRNA is shown in (**E**), and the overexpressed levels of GPP130 in A549 cells are shown in (**C**) [\(n=3\)]. The statistically significant results (\(p<0.05\)) at the 96 h timepoint are depicted. The Western blot images shown are representative of three independent experiments. Asterisk mean it is statistically significant \(p=0.01\) or 0.02. Figure 6: (**A,B**) GPP130 domains implicated in enhancing the proliferation of A549 cells and the paracrine effect of GPP130 shedding. (**A**) Schematic diagram of the various constructs used.