Unlocking the tumor suppressor-like function of SORT1 expression: Inhibiting cell proliferation and tumor progression
In light of our previous studies, where we observed in LUAD cell lines a significant down-regulation of the SORT1 gene, encoding sortilin, we employed a Tet-On induction system to enable the reversible control of SORT1 expression. This strategy allowed us to further compare how the loss of sortilin expression unlocks oncogenic pathways in LUAD. For these experiments, we used the NCI-H3255 and NCI-H1975 LUAD cell lines, which exhibit sustained-EGFR proliferative signaling due to acquired mutations (L858R and L858R/T790M, respectively). As shown with western blot (Figs. 1a to 1c) and qPCR analysis (Figs. 1d to 1e), sortilin expression remained weak in the control group (Tet-EV) while, induction with doxycycline (dox) resulted in a progressive and significant (p < 0.001) increase in sortilin expression. Because the extent of sortilin induction depends on the dox concentration (ranging from 0 to 100 nM, indicated by gray to black bars in the histogram), and that control (Tet-EV) did not exhibit sortilin expression despite dox induction, these results underscore the robustness of our cell models to control sortilin expression. Then, to investigate the phenotypic and functional effects of restoring sortilin expression, we employed a dox concentration of 100nM for subsequent analyses. Subsequently, we assessed two critical cancer hallmarks, cell proliferation and tumorigenesis, in the presence or absence of sortilin. In both Tet-SORT1 models using the NCI-H1975 and NCI-H3255 cell lines, we observed a significant (p < 0.001) reduction of approximately 20–25% in cell proliferation upon SORT1 induction compared to control Tet-EV cells (blue bars vs black and white bars, Figs. 2a and 2b). To rule out cytotoxic effects triggered by dox exposure or sortilin expression, we performed viability tests. No significant effects attributable to the administration of dox itself or the induced-expression of sortilin were observed on cell viability. Altogether, we assert that the decrease of cell proliferation rates remains independent of cell toxicity (blue bars vs black and white bars, Figures. 2c and 2d) but governed by sortilin expression induction. Remarkably, beyond its impact on cell proliferation, SORT1 induction also had a negative effect on tumorigenesis, as evidenced by monitoring spheroid growth (Figs. 2e and 2f). When we triggered sortilin expression in Tet-SORT1 spheroids derived from NCI-H1975 and NCI-H3255 cell lines, a significant reduction in tumor growth and progression was observed between days 5 and 10 compared to control Tet-EV cells (p < 0.001 at day 5 and 10 for NCI-H1975; p = 0.06 at day 5 and p < 0.001 at day 10 for NCI-H3255). In contrast, Tet-EV cell-derived spheroids showed no significant change in tumor progression (full line). Therefore, the reduction in spheroid volume in the Tet-On system can be attributed to sortilin expression (Figs. 2e to 2h).
In summary, these findings establish that when sortilin remains expressed both cell proliferation and tumorigenesis are significantly limited in LUAD cell lines.
The loss of sortilin increases cell proliferation in HEK KO SORT1 models
To deepen the pivotal role of sortilin in the previous observed cancer hallmarks, we employed CRISPR/Cas9 genome-editing technology to shut down its expression. We selected the HEK293T transformed cell line as our experimental model due to its capacity for precise genetic modifications. This cell model provides an ideal platform for in-depth research into the molecular mechanisms underlying tumor development.
We achieved SORT1 depletion through CRISPR/Cas9, followed by the selection of clonal populations from individual cells. This approach allowed us to isolate three SORT1 knockout (KO) clones (CRISPR SORT1 clones; #1, #2 and #3), as demonstrated through western blot and immunofluorescence analyses (rectangle with dotted line, Figs. 3a to 3c). Strikingly, the depletion of sortilin led to a significant increase (p < 0.001) in cell proliferation across all three SORT1 KO clones (Fig. 3d, dark blue bars) when compared to the parental cells (light blue bars, Fig. 3d). Inversely, when we transiently restore sortilin expression (pcDNA-SORT1) (Fig. 3e and 3f), we observe a significant (p < 0.001) and overall decrease in cell proliferation in all three CRISPR SORT1 clones. In parental cells (P1), where we performed no gene manipulation, these cells exhibit a similar decrease of cell proliferation.
In summary, our results highly suggest that loss of SORT1 induces significant phenotypic changes in HEK293T cells, by triggering cell proliferation. These results highlight the substantial impact of SORT1 on cell behavior and its potential relevance in limiting proliferative signaling beyond oncogene-related pathways in LUAD.
Restoring sortilin expression attenuates EGFR mutant dominance in HEK293T cells
Since HEK293T cells exhibit weak endogenous expression of wild-type EGFR, we aimed to create a model mimicking LUAD cells upon the oncogenic driving force of EGFR mutations. Consequently, we arbitrary restored sortilin expression in one of the three SORT1 KO clones, clone #3 in the presence of either wild-type EGFR or various EGFR mutations; including L858R, G719X, L861Q, or the L858R/T790M double mutation.
Our results unveiled that in the genetic SORT1-depleted cells background (dark blue bars) we observe a significant increase (p < 0.01) of cell proliferation rate in cells carrying EGFR mutations (L858R, G719X, L861Q, or L858R/T790M) in comparison to control cells (#3 Ctrl - dark blue bars) (Fig. 3g). Strikingly, when we restored sortilin, a significant decrease (p < 0.001) in proliferation was observed (light blue bars, Fig. 3g). Because cell toxicity induced by successive transfections would comprise results, we performed in parallel viability tests. No critical cell toxicity between cells co-transfected either with empty vector (control, pcDNA-EV), pcDNA-SORT1 or various EGFR mutations (pBabe-EGFR) was observed attesting thereby that the decrease in cell proliferation was not linked to cell death (Fig. 3h).
These results suggest that sortilin overcame the dominance of EGFR mutants, and further underline the essential role of sortilin as a key regulator of cell proliferation, independent of EGFR status. The knowledge gained from these results reinforces sortilin's role as a tumor suppressor.
Sortilin plays a key role in transcriptional regulation of several oncogenic genes
Our previous research revealed the nuclear role of sortilin in the regulation of EGFR nuclear target genes. This first discovery led us to further investigate its potential involvement in genome-wide transcriptional regulation. To support this hypothesis, we investigated the nuclear translocation of sortilin after dox induction in the NCI-H1975 and NCI-H3255 cell lines, using cell fractionation and western blotting techniques (red rectangle, Fig. 4a). Surprisingly, our results showed that nuclear translocation of sortilin was directly proportional to its total concentration, whatever the cell line studied.
To further investigate the transcriptional role of sortilin in this doxycycline-induced model, we decided to study the expression profiles of various genes involved in other major oncogenic pathways in LUAD, such as MTOR, AKT, SOX2, and UBC in addition to the EGFR and CCND1 previously studied (see Figs. 4b to 4h). Our observations revealed a significant decrease (p < 0.001) of EGFR, MTOR and AKT gene expression upon sortilin overexpression in both cell lines (as shown in Figs. 4c to 4f). In contrast, CCND1 and SOX2 expression was significantly reduced only in the NCI-H1975 cell line (as shown in Fig. 4d and 4g), which could be explained by the higher decrease in EGFR mRNA levels in this cell line. Notably, among these genes, UBC mRNA is the only one to show a significant increase in expression levels in both cell lines (as shown in Fig. 4h). Importantly, no significant impact of dox on the expression of selected genes was observed in Tet-EV cells.
Overall, these results highlight the distinctive role of sortilin in transcriptional regulation, revealing its diverse influence on key genes involved in cancer-related pathways, a phenomenon not observed in control cells (Tet-EV) with low sortilin expression. These results also support the hypothesis that sortilin may control cell proliferation and tumor growth upstream of the signaling pathway by directly regulating oncogene expression.
Unveiling transcriptional changes in NCI-H1975 and NCI-H3255 upon sortilin overexpression
To further characterize the genes influenced by the restoration of sortilin expression and elucidate its role in modulating their expression, we conducted a comparison study through RNA-seq analysis between Tet-EV and Tet-SORT1 cell lines derived from both NCI-H1975 and NCI-H3255. Subsequently, we inspected the alterations in gene expression levels induced by SORT1 restoration in each model. An initial screening was carried out to exclude genes whose differential expression (DEG) could be attributed to the impact of dox rather than a leak of sortilin expression in Tet-On models (Refer to Supplementary Data, Fig. 1).
As anticipated, the restoration of sortilin resulted in significant shifts in the DEG profiles where we observed in NCI-H1975, the repression of 2218 genes and the induction of 1783 genes (Fig. 5a & Supplementary Data, Fig. 1a & 1c). Similarly, in NCI-H3255, 1322 genes were repressed, and 1887 genes were induced upon sortilin overexpression (Fig. 5a & Supplementary Data, Figs. 1b & 1d). We further investigated the genes that were commonly regulated in both cell lines, identifying 318 under-expressed genes (Fig. 5c) and 312 over-expressed genes (Fig. 5d).
Interestingly, Reactome enrichment analysis of these gene sets revealed that sortilin-regulated genes were predominantly associated with functions related to cell sorting and retrograde transport, which is consistent with the well-known role of sortilin in these processes. This analysis also revealed that sortilin expression deregulates genes involved in essential signaling pathways, in particular the EGFR pathway (Fig. 5e), as well as the PI3K/mTOR/AKT pathway involved in processes such as epithelial-mesenchymal transition, proliferation and differentiation (Fig. 5g & supplementary data, Table 1). These results would correlate with our previous qPCR analyses (Fig. 4).
Moreover, these observations highlight sortilin's pivotal role in transcriptional regulation. It can either repress genes related to chromatin organization (e.g., PBRM1 and ATF7IP), transcription (e.g., NCOA2) (Fig. 5e & supplementary data, Tables 1), or induce the expression of genes associated with DNA repair and transcription (e.g., INO80E, PQBP1, POLR2F, POLR2L, and POLR1C encoding specific RNA polymerases) (Fig. 5f & supplementary data, Tables 2).
To deepen its putative nuclear mode of action and nuclear partners, we analyzed sortilin's interactome in the nuclear compartment.
Elucidation of the sortilin nuclear interactome: Implications for oncogenic processes
To further investigate the hypothesis concerning the role of nuclear sortilin, identified by RNA seq, we used mass spectrometry (MS) to map the sortilin nuclear interactome. To this end, we used the BirA-Biotin system in CRISPR SORT1 clone #3 cells avoiding thereby its endogenous expression, as described in the supplemental methods and in the (Fig. 6a and 6b) 8,9.
After purification and validation of the presence of biotinylated sortilin by western blot (Fig. 6c), MS analysis revealed 99 candidate proteins in the SORT1 condition at the nuclear level (Fig. 6d & Supplementary Data, Table 4). The STRING analysis tool, combined with the gene enrichment study, enabled us to deduce the protein-protein interaction networks and functions of the proteins identified by MS in the presence of sortilin (SORT1 condition). The identification of the retromer complex, associated with the protein-sorting functions of sortilin, is consistent with previous data demonstrating that sortilin is involved in the transport and internalization of proteins, including EGFR in LUAD10,6. These results validate our experimental approach. Our analyses also highlight a predominant role for the proteins detected in RNA transport from the nucleus to the cytoplasm, DNA replication, DNA repair and the cell cycle (Fig. 6e and 6f). These results are consistent with the enrichment pathways identified by RNA-seq analysis in both adenocarcinoma cell lines. Furthermore, the identification of an interaction between sortilin and 10 proteins of the nuclear pore-associated complex suggests that sortilin translocation may depend on the nuclear pore complex to reach the nucleus.
Overall, our observations point to a potential function for sortilin in the nucleus, particularly in DNA repair, gene regulation and by extension, RNA regulation.