CTDSPL2, a potential novel target of the tumor suppressor miR-193a-3p, is upregulated in NSCLC and associated with poor patient survival.
The inhibitory effect of miR-193a-3p on tumor progression has been reported in many reports (16–19). However, the specific downstream target acts as an oncogenic gene in NSCLC is yet to be identified. In this experiment, miR-193a-3p-overexpressing stable cells were generated in H1299 cells, and miR-193a-3p mimics were transiently transfected into A549 cells (Supplementary Fig. 1A, B). Our results confirmed that the overexpression of miR-193a-3p inhibited the proliferation, migration, and invasion of NSCLC cells (Supplementary Fig. 1C-H).
To search for the potential targets implicated in promoting NSCLC progression, we first predicted the target genes of miR-193a-3p using four databases: TargetScan, miRDB, PicTar, and miRanda. A Venn diagram was generated with an online tool (http://bioinformatics.psb.ugent.be/webtools/Venn/) and a total of 56 shared target genes were obtained (Supplementary figfigfigfig. 2A). We subsequently conducted transcriptome sequencing of H1299 cells stably expressing miR-193a-3p or miR-NC. Using a combination of database prediction and sequencing results, we analyzed the changes in the expression of these 56 common genes following miR-193a-3p overexpression. Among these, 12 were upregulated and 10 were downregulated, as shown in the heatmap (Supplementary Fig. 2B). Subsequently, TCGA database was used to compare the expression levels of the 10 downregulated genes between tumor and normal tissues in NSCLC patients. The findings indicated that the mRNA levels of AP2M1, BRWD3, and CTDSPL2 were significantly elevated in cancerous tissues (Fig. 1A and Supplementary Fig. 2C). Then overall survival (OS) analysis was performed using GEPIA2 (http://gepia2.cancer-pku.cn/#index). Among the three candidates, only higher expression of CTDSPL2 was correlated with a poorer survival rate in patients with NSCLC (Fig. 1B). The association between CTDSPL2 expression and survival outcome was confirmed using Kaplan-Meier plotter (https://kmplot.com/analysis/) (Fig. 1B). In addition, CTDSPL2 expression at different stages was investigated in LUAD and LUSC by GEPIA2. The results revealed that CTDSPL2 was differentially expressed in LUAD [F value = 2.78, Pr (> F) = 0.0409] but not in LUSC (Supplementary Fig. 2D). To further evaluate CTDSPL2 protein expression in NSCLC tissues, we performed immunohistochemical staining of paraffin-embedded tissue samples from 14 patients with NSCLC. The protein expression level of CTDSPL2 was higher in tumor tissues than that in non-tumor tissues (Fig. 1C, D). This finding was consistent with the results obtained from TCGA data analysis. Therefore, CTDSPL2 is highly expressed in NSCLC and may have potential oncogenic functions.
CTDSPL2 is directly targeted by miR-193a-3p in NSCLC cells.
TargetScan prediction revealed a putative binding site between miR-193a-3p and the 3’-UTR of CTDSPL2 (Fig. 1E). To validate the direct targeting of the CTDSPL2 3’-UTR by miR-193a-3p, a dual luciferase reporter assay was performed. As shown in Fig. 1E, the wild-type or mutant 3’-UTR of CTDSPL2 was cloned into the luciferase reporter plasmid. Overexpression of miR-193a-3p significantly reduced luciferase activity of the wild-type CTDSPL2 3’-UTR, but not the mutant 3’-UTR, suggesting that CTDSPL2 was a direct target of miR-193a-3p (Fig. 1E). Next, we evaluated the effect of miR-193a-3p overexpression on CTDSPL2 expression in NSCLC cells (H1299 and A549). Our data revealed that ectopic miR-193a-3p expression inhibited CTDSPL2 expression at both the mRNA and protein levels (Fig. 1F, G). Together, CTDSPL2 is negatively regulated by miR-193a-3p through direct targeting, indicating that CTDSPL2 elevation in NSCLC could be attributed to decreased miR-193a-3p expression.
CTDSPL2 promotes malignant progression of NSCLC cells.
To investigate the function of CTDSPL2 in NSCLC, lentiviral shRNA transduction was used to stably knockdown CTDSPL2 in H1299 and A549 cells (Fig. 2A, B). The CCK8 assay demonstrated a significant decrease in NSCLC cell proliferation upon silencing of CTDSPL2 (Fig. 2C, D), which was further confirmed by colony formation assay (Fig. 2E). To explore the mechanism by which CTDSPL2 influences cell proliferation, we examined the cell cycle distribution and cell apoptosis using flow cytometry. CTDSPL2 depletion resulted in G1 phase arrest and increased apoptosis in both the cell lines (Fig. 2F-H and Supplementary Fig. 3). Additionally, transwell assays were conducted to determine the effect of CTDSPL2 on migration and invasion. Inhibition of CTDSPL2 significantly attenuated the migratory and invasive abilities of NSCLC cells (Fig. 2I). Wound-healing assay was also used to assess the migratory ability of the cells. NSCLC cells lacking CTDSPL2 exhibited reduced wound closure rates (Fig. 2L and Supplementary Fig. 4). Collectively, these findings demonstrate that CTDSPL2 plays an oncogenic role by inhibiting apoptosis and facilitating the proliferation, cell cycle progression, migration, and invasion of NSCLC cells.
CTDSPL2 facilitates tumor growth and metastasis in mouse models, while inhibiting the infiltration of CD4 + T cells into tumor tissues.
In our animal experiments, a murine lung cancer cell line, LLC, was used to stably deplete CTDSPL2 (Fig. 3A). CTDSPL2 knockdown or control cells were subcutaneously injected into C57/BL6 mice and tumor formation was monitored. Tumors derived from the CTDSPL2 knockdown group exhibited reduced growth rates, smaller size, and lighter weight than the control group (Fig. 3B-D). Furthermore, IHC staining revealed that expression of the proliferation marker Ki-67 was reduced upon CTDSPL2 silencing (Fig. 3H). The expression of CTDSPL2 protein in tumor tissues was examined by western blot analysis. The results confirmed a reduction in CTDSPL2 expression in tumor tissues from the shCTDSPL2 group (Fig. 3E). The above LLC-derived cells were also inoculated into the tail vein of C57/BL6 mice. The CTDSPL2 knockdown group exhibited reduced metastatic foci in lung tissues (Fig. 3F, G). Overall, these data suggested that CTDSPL2 significantly enhanced NSCLC tumor growth and metastasis in vivo.
Using the tumor tissues from immune-competent C57/BL6 mice subcutaneously injected with LLC cells, we performed IHC staining to evaluate the influence of CTDSPL2 on T cell recruitment. The shCTDSPL2 group exhibited increased infiltration of CD4+ T cells into tumor tissues. However, CD8+ T cell infiltration was not observed in tumor tissues (Fig. 3H). Hence, our findings demonstrated for the first time that CTDSPL2 suppresses CD4+ T cell infiltration in tumors. Further investigation is necessary to evaluate the effects of CTDSPL2 on different CD4+ T cell subsets.
CTDSPL2 promotes lung cancer progression by activating the PI3K/AKT pathway via the regulation of JAK1 expression.
In an attempt to elucidate the underlying molecular mechanisms by which CTDSPL2 promotes the malignant progression of NSCLC, tandem mass tagging (TMT) quantitative proteomics analysis was applied to compare H1299-shCTDSPL2 cells with control cells. Heatmap visualization revealed the top 40 downregulated proteins, among which JAK1 attracted our interest (Fig. 4A). A previous report has implicated the interaction between CTDSPL2 and JAK1 in HeLa cells by mass spectrometry (20). This interaction was also validated by immunoprecipitation of endogenous CTDSPL2 (Fig. 4B). In NSCLC, activation of the JAK1/STAT3 pathway is considered to be crucial for tumor progression (21–23). Additionally, JAK also mediates other signaling pathways involved in tumor progression, such as PI3K pathway (24, 25). Accordingly, the effect of CTDSPL2 on the expression of key components of the JAK1/STAT3 and PI3K/AKT signaling pathways was investigated by western blot assay. The results revealed that depletion of CTDSPL2 reduced the expression of JAK1, PI3K, p-AKT(Ser473), and p-ERK1/2(Thr202/Tyr204) in H1299, A549, and LLC cells (Fig. 4C). However, altering CTDSPL2 expression did not affect the levels of STAT3, p-STAT3(Tyr705), AKT, or ERK1/2 proteins (Fig. 4C). Meanwhile, overexpression of CTDSPL2 showed the opposite results (Fig. 4C). To assess the effect of JAK on PI3K/AKT signaling, we administered different doses of ruxolitinib, a selective JAK1/2 inhibitor, to three lung cancer cell lines. As expected, JAK inhibition resulted in a dose-dependent decrease in the expression of PI3K, p-AKT, and p-ERK1/2 (Fig. 4D). In contrast, JAK inhibition had minimal effects on CTDSPL2 expression, suggesting that CTDSPL2 acted as a regulator of JAK1. To investigate the potential of CTDSPL2 to promote NSCLC cell proliferation via JAK1/PI3K/AKT signaling, we administered ruxolitinib (20 µM) or LY294002 (20 µM), a PI3K inhibitor, to A549 cells stably expressing CTDSPL2. CCK8 assay demonstrated that CTDSPL2 overexpression-induced lung cancer cell proliferation was reversed by inhibiting JAK or PI3K (Fig. 5A). Furthermore, SC79, an AKT activator, was used to reverse AKT inactivation induced by CTDSPL2 knockdown in A549 cells. Cell growth inhibition resulting from CTDSPL2 knockdown was rescued by SC79 treatment (20µM) (Fig. 5B). Interestingly, this rescue effect was eliminated when ruxolitinib (20 µM) or LY294002 (20 µM) was co-administered with SC79 (Fig. 5B). In addition, decreased migration and invasion caused by CTDSPL2 knockdown was also rescued by SC79 treatment, while co-administration of ruxolitinib or LY294002 with SC79 abolished this rescue effect (Fig. 5C-E). In summary, these findings suggest that CTDSPL2 potentially promotes NSCLC progression by activating the PI3K/AKT pathway through its interaction with and upregulation of JAK1 (Fig. 6).