GCC2 regulates cell proliferation, invasion, and migration in NSCLC cell lines
First, we analyzed GCC2 protein expression levels in various NSCLC cell lines and a pulmonary alveolar epithelial cell line, and we found H460 and H1299 cell lines had the highest GCC2 expression levels (Supplementary Figure S1A). To investigate the role of GCC2 in lung cancer cell lines, we inhibited the expression of GCC2 in two NSCLC cell lines with the highest levels of GCC2 protein, H460 and H1299. First, we confirmed the effects of shRNA-mediated GCC2 knockdown, and GCC2 mRNA and protein expression in both NSCLC cell lines decreased by more than 70% relative to control (Fig. 1A and B, Supplementary Fig. S1B and C). There were noticeable differences in cell counts following GCC2 knockdown, so we analyzed the cell viability of H460 cells after shGCC2 treatment in a time-dependent manner (Fig. 1C). GCC2 knockdown H460 cells had a 50% reduction in cell viability 8 hours post-transfection, and this effect was maintained for another 16 hours. H460 cells with decreased GCC2 expression demonstrated a 10-fold increase of TUNEL-positive cells and similar results were seen in H1299 cells, suggesting apoptosis induction in both NSCLC cell lines following GCC2 knockdown (Fig. 1D, Supplementary Fig. S1D). Since GCC2 silencing reduced cell viabilities and induced apoptosis in H460 cells, we further looked into the potential additive or synergistic effects with additional treatment of gefitinib, an EGFR TKI. Combination therapies involving existing EGFR-TKI are of clinical significance, and GCC2 knockdown may serve as a combination therapeutic option in NSCLC [28]. When comparing control and GCC2 knockdown H460 cells, the latter were more sensitive to gefitinib treatment, as IC50 values were 1.323 µM and 0.8602 µM, respectively (Supplementary Fig. S1E). These results suggest shRNA-mediated GCC2 knockdown impairs proliferation and survival of H460 and H1299 cells.
Next, we explored the changes in gene expression patterns of NSCLC cell lines following GCC2 knockdown. Cancer stem cells (CSCs) are a subpopulation of cancer cells with self-renewal and differentiation capacities similar to those of stem cells, and they play a significant role in tumor initiation, relapse and resistance to treatments [29]. qPCR analysis demonstrated GCC2 knockdown cells had expression of CSC-related genes, such as OCT4 and SOX2, decrease by more than 50% relative to control cells (Fig 1E, Supplementary Fig. S1F). The process of adherent epithelial cells acquiring mesenchymal characteristics and migratory properties is known as epithelial-mesenchymal transition (EMT). EMT is a critical process linked with stem-cell characteristics and metastatic progression in cancers and has specific, identifiable changes in gene expression [30]. Silencing of GCC2 expression in NSCLC cell lines decreased mRNA expression of mesenchymal-associated genes, such as Snail, Slug, Zeb, while expression of E-cadherin, a marker of epithelial state, in H460 cells was unchanged (Fig. 1F, Supplementary Fig. S1G). Having observed changes in CSC- and EMT-related genes at the mRNA level, we investigated whether there were concomitant changes in cancer cell phenotypes. shGCC2-treated NSCLC cell lines showed decreased colony formation abilities (Fig. 1G, Supplementary Fig. S1H). GCC2 knockdown inhibited the migratory potential of cancer cells, as demonstrated by a lowered capacity for gap recovery in the scratch assay in H460 and H1299 cells (Fig. 1H, Supplementary Fig. S1I). In addition, GCC2 knockdown H460 and H1299 cells exhibited approximately 85% and 60% decreases, respectively, in cell movement for both transwell migration and invasion assays (Fig. 1I, Supplementary Fig. S1J). Therefore, shRNA-mediated silencing of GCC2 in lung cancer cell lines decreased CSC- and EMT-related gene expression profiles, which were further supported by inhibition of cancer cell growth and metastatic potential in vitro.
GCC2 regulates the secretion and pro-tumor effects of cancer cell-derived exosomes
Formation and cargo sorting of early sorting endosomes, which become MVBs that secrete exosomes, are regulated by the trans-Golgi network [31]. Therefore, we investigated the effects of inhibiting GCC2 expression on exosome secretion. Nanoparticle tracking analysis (NTA) results demonstrated a decrease in number of total exosomes released from GCC2 knockdown H460 cells relative to control (Fig. 2A). Additionally, immunostaining for CD63, a tetraspanin protein and an exosome marker, revealed a significant decrease in CD63+ green fluorescence puncta upon GCC2 knockdown (Fig. 2B). Here, GCC2 gene silencing inhibited exosome secretion in H460 cells.
Cancer cell-derived exosomes not only confer pro-tumorigenic traits to surrounding cancer cells, but also are diagnostic, predictive, and prognostic biomarkers [26, 27]. Jeong et al. identified GCC2-containing exosomes as a biomarker for NSCLC progression, and exosomal GCC2 protein levels increased with pathological stage, suggesting GCC2 enrichment in NSCLC-derived exosomes [13]. Therefore, we investigated the role of NSCLC cell-derived, GCC2 enriched exosomes, which will hereafter be denoted as “GCC2+ exosomes”, and explored the effects of cancer cell GCC2 expression on exosome functions. Briefly, exosomes were isolated from H460 cells with either normal or silenced GCC2 expression, analyzed for proper isolation, calculated for dosage determination, and treated to cancer cells in an autocrine and dose-dependent manner. Upon treatment of GCC2+ exosomes on H460 cancer cells in increasing doses, we found that there were parallel increases in expression of CSC- and EMT-related genes, such as OCT4, NANOG, CD133, Snail, and Twist (Fig. 2C). Interestingly, once treatment doses exceeded 10x, there were no statistically significant differences in gene expression between 10x, 20x, and 30x treatment conditions, suggesting a saturation effect of GCC2+ exosomes. At the level of cell phenotype, transwell migration and invasion assays demonstrated a similar dose-dependent, migratory potential promoting effect of GCC2+ exosomes in H460 cells (Fig. 2D). Like the saturation effect observed with gene expression, the migration promoting effects of GCC2+ exosomes appeared maximal at, but not beyond, the 10x dose. Colony formation abilities demonstrated increasing trends upon treatment with lower concentrations of GCC2+ exosome, and 10x or more GCC2+ exosomes significantly upregulated colony formation ability in H460 cells (Fig. 2E). Together, these results demonstrated GCC2+ exosomes promote cancer cell proliferation and migration.
To investigate the relationship between GCC2 expression and functions of cancer cell-derived exosomes, we first inhibited GCC2 expression in cancer cell lines, isolated GCC2 knockdown cancer cell-derived exosomes, henceforth referred to as “GCC2 KD exosomes”, and treated GCC2 KD exosomes to H460 cells in a dose-dependent manner. Since GCC2 silencing reduced exosome secretion in H460 cells (Fig. 2A and B), we standardized the number of particles treated at each dose to ensure equal treatment conditions for GCC2+ and GCC2 KD exosomes; NTA results were used to set 2.5x108 particles as 1x. In addition, dose-dependent treatment of GCC2 KD exosomes on H460 cells was performed until 10x, as evidenced by the saturation effect observed with GCC2+ exosomes. Surprisingly, despite treating the same number of particles as GCC2+ exosomes, GCC2 KD exosomes had a less prominent effect on promoting the expression of CSC- and EMT-related genes in H460 cells (Fig. 2F). Relative to untreated, control conditions, the expression of most genes in H460 cells did not exhibit an increasing response with dose-dependent GCC2 KD exosome treatment. At lower doses, the treatment GCC2 KD exosomes decreased the expression EMT-related genes N-cadherin, Snail, Slug, Zeb, and Twist relative to control in H460 cells. Transwell migration and invasion assays did not reveal a difference in migratory potential of cancer cells treated with GCC2 KD exosomes, no matter the dose (Fig. 2G). Colony formation ability of H460 cells increased only after treatment of 10x GCC2 KD exosomes, and lower treatment doses did not enhance colony formation abilities (Fig. 2H). The effects of GCC2+ and GCC2 KD exosomes on migration, invasion, and colony formation are summarized in Fig. 2I. Here, we found that silencing GCC2 expression in H460 cells inhibited migration, but not proliferation, promoting effects of GCC2+ exosomes in H460 cells. This suggests GCC2 in cancer cells may regulate cargo sorting of exosomes to contain key factors that alter CSC- and EMT- gene expression and migratory potential in recipient cells.
GCC2 regulates RTK expression and MAPK signaling pathways
Dysregulated growth receptor activity is associated with cancer progression, and binding of growth factors to their cognate receptors have been shown to induce EMT and CSC-like states in cancer cells through MAPK signaling cascades [32, 33]. Previous results demonstrated GCC2 knockdown decreased CSC- and EMT-related gene expression in lung cancer cell lines, so we investigated whether GCC2 expression was related to the expression of growth receptors and/or downstream signaling pathways. qPCR analysis of various receptor tyrosine kinase (RTK) genes demonstrated decreased expression in H460 and H1299 GCC2 knockdown cells (Fig. 3A, Supplementary Fig. S2A). Immunoblot analysis for MEK1/2 and ERK1/2, both of which are key downstream components of growth receptor signaling cascades, revealed decreased expression and phosphorylation-mediated activation in GCC2 knockdown NSCLC cell lines (Fig. 3B, Supplementary Fig. S2B). In addition to MAPK/ERK signaling factors, the mRNA expression of other MAPK pathway-related genes, such as KRAS and cyclin D1, were decreased upon GCC2 knockdown H460 and H1299 cell lines (Fig. 3C, Supplementary Fig. S2C). Here, GCC2 silencing in NSCLC cell lines decreased expression of multiple RTKs and components of the downstream MAPK/ERK signaling pathway.
Cyclin D1, a downstream effector of the EGFR-MAPK signaling cascade, is a key regulator of cell cycle progression, and cyclin D1 is pivotal in the malignant transformation of NSCLC [34]. Therefore, we analyzed the expression of cyclin D1 in GCC2 control and knockdown NSCLC cell lines and found decreased cyclin D1 protein levels in GCC2 knockdown cancer cells (Fig. 3D, Supplementary Fig. S2D). Immunostaining for cyclin D1 in H460 cells revealed a more than 50% decrease in CCND1-positive cell counts after GCC2 knockdown, and Ki67-positive cells decreased by approximately 60% in shGCC2 treated H460 cells (Fig. 3E and F). Similar immunostaining results were seen for both cyclin D1 and Ki67 in H1299 cells following GCC2 knockdown (Supplementary Fig. S2E and F). Here, we demonstrated GCC2 expression affects RTK expression in cancer cell lines, and GCC2 knockdown reduced MAPK signaling activity, including the downstream effector cyclin D1, suggesting GCC2 may regulate multiple growth signaling pathways.
GCC2 regulates NSCLC tumor growth in vivo
To investigate whether growth-inhibiting effects of GCC2 silencing in vitro could be replicated in vivo, we established a tumor xenograft model in BALB/c nude mice. Immunodeficient mice were inoculated with either control H460 cells expressing GCC2 or shGCC2-treated H460 cells. Mice were observed for 3 weeks after tumor inoculation. Tumor growth was significantly inhibited in mice bearing GCC2 knockdown H460 cells compared to those inoculated with control H460 cells (Fig. 4A). Both tumor volumes and tumor weights had statistically significant decreases in the GCC2 knockdown group at endpoint, and mice bodyweights remained unchanged between the two groups throughout the experiment (Fig. 4B-D). In some mice, GCC2 knockdown H460 cells formed tumors soon after inoculation but were no longer detected at endpoint, suggesting impaired survival and growth abilities of GCC2 knockdown cancer cells (Fig. 4B)
Morphological analysis of tumors with H&E staining not only confirmed the smaller absolute size of GCC2 knockdown cancer cell-derived tumors, but also revealed a less dense population of cells within these tumors (Fig. 4E). Here, we demonstrated the tumor growth inhibiting effects of GCC2 knockdown in a NSCLC cell line in-vivo.
GCC2 regulates the integrity of Golgi structure and intracellular trafficking of EGFR
GCC2 is involved in the maintenance of Golgi apparatus structure and Golgi-associated processes, and disrupted Golgi structures inhibits the transport and aberrant activity of EGFR [3, 35]. To investigate the role of GCC2 in Golgi apparatus structure and changes in cellular EGFR activity, we first demonstrated the co-localization of GCC2 and Golgi marker GM130 through immunostaining in H460 and H1299 cell lines (Fig. 5A). There was a clear co-localization of GCC2 and GM130 in a compact structure that is presumed to be the Golgi apparatus. Next, we investigated the changes in Golgi structure after GCC2 silencing in NSCLC cell lines. Immunostaining for GM130 in Fig. 5B demonstrated GCC2 knockdown resulted in a dispersed, less compact Golgi apparatus morphology in both H460 and H1299 cell lines. This suggests, GCC2 expression is associated with maintenance of Golgi structure in NSCLC cell lines.
In our previous results, we observed decreased expression of various RTKs in GCC2 knockdown H460 and H1299 cells, so we explored the relationship between GCC2 and EGFR, a common oncogenic driver mutation in cancers that drives CSC and EMT induction. In Fig. 5C and Supplementary Fig. S2G, we confirmed the effects of GCC2 knockdown and decreased EGFR expression at the protein level. qPCR analyses for EGFR in H460 cells treated with GCC2+ or GCC2 KD exosomes revealed EGFR expression increased in a dose-dependent manner upon treatment of GCC2+ exosomes but not GCC2 KD exosomes (Supplementary Fig. S2H). Nuclear translocation of EGFR allows EGFR to act as a co-transcription factor to promote cell proliferation and cancer progression [32]. Control H460 cells expressing GCC2 have significant amounts of EGFR residing in the nucleus region, as demonstrated by red puncta in immunostaining results (Fig. 5D). On the contrary, GCC2 knockdown H460 cells had a drastic decrease of EGFR localized in the nucleus, suggesting aberrant GCC2 overexpression in cancer cells may promote the transport of EGFR into the nucleus for cancer progression.