Cytohesin-4/ARF6 facilitates the progression of acute myeloid leukemia through activating PIK3R5/PI3K/AKT pathway

Abstract

In silico analysis revealed an elevated expression of cytohesin-4 (CYTH4) in acute myeloid leukemia (AML) cells, correlating with a poorer prognosis for AML patients. However, its role in AML is not fully understood. Our study using loss-of-function assays identified CYTH4 as an oncogene promoting leukemogenesis. Silencing CYTH4 in MV4-11 and THP-1 cells reduced cell proliferation and colony formation, and induced apoptosis and cell-cycle arrest at G0/G1, whereas overexpression had no significant impact. CYTH4 silencing also increased chemosensitivity to cytarabine. In a THP-1 xenograft model, CYTH4 silencing slowed AML progression and reduced leukemic cell homing and infiltration. Mechanistically, CYTH4 silencing inhibited PI3K/AKT pathway by lowering PIK3R5 and decreased ARF6-GTP levels, as confirmed by pull-down assays. Overexpression of PIK3R5 and AKT activation via SC-79 successfully countered the cellular dysfunctions from CYTH4 silencing. Thus, CYTH4 may play a role in AML progression, and targeting its pathway could be a promising anti-leukemic treatment strategy.

Keywords: Cancer; Cell biology.

Graphical abstract

Figure 1

CYTH4 is highly expressed in AML cells and inversely correlated with the overall survival of AML patients (A) After intersecting three datasets, 25 upregulated and 8 downregulated differentially expressed genes (DEGs) were identified. (B) The significantly upregulated expression of CYTH4 in AML patients compared to healthy donors (HD) (left, GSE67936, ∗p = 0.0116; middle, GSE65409, ∗p = 0.0102; and right, GEPIA2, ∗p < 0.05). (C) CYTH4 expression is reversely associated with the overall survival of AML patients (left, GEPIA2, p = 0.0014; middle, TIMER, p = 0.0015; and right, UALCAN, p = 0.019). Kaplan-Meier curve was used to analyze the survival distribution of the two groups. (D) RT-qPCR assay for CYTH4 mRNA in bone marrow mononuclear cells (BMMCs) from AML patients (n = 35) and HD (n = 15). Data are represented as mean ± SD. Statistical significance was assessed by unpaired t test. (E) Immunoblotting assay for CYTH4 protein in BMMC from AML patients (n = 6) and HD (n = 4). (F) RT-qPCR assay for CYTH4 mRNA in human AML cell lines (n = 3). The mRNA level of CYTH4 in the mixed BMMCs from five HD was set as 1.0. Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (G) Immunoblotting assay for CYTH4 protein expression in human AML cell lines. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 2

CYTH4 silencing alleviates the proliferation of human AML cells and increases the chemosensitivity of AML cells to Ara-C (A) Validation of the downregulated expression of CYTH4 in four shCYTH4-transduced MV4-11 and THP-1 cells by RT-qPCR (n = 3). β-actin was used as an internal control. Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (B) Validation of the downregulated expression of CYTH4 in shCYTH4-transduced MV4-11 and THP-1 cells by immunoblotting. GAPDH was used as a loading control. (C) CYTH4 silencing inhibits the proliferation of MV4-11 and THP-1 cells. CCK-8 assay (n = 3) was conducted to detect the viability of MV4-11 and THP1 cells at the indicated time points (0, 24, 48, 72, and 96 h). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (D) Inhibitory effect of CYTH4 silencing on colony formation of AML cell was assessed by microscopically counting the numbers and sizes of colonies at 14 days after plating. The quantification is shown on the right (n = 3).Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (E) CYTH4 silencing in MV4-11 and THP-1 cells using shRNAs induces cell apoptosis. Following annexin V-APC and 7-AAD staining, the percentages of apoptotic cells were detected by flow cytometry. Representative flow cytometry plots (top) and quantification (bottom) are shown (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (F) CYTH4 silencing in MV4-11 and THP-1 cells using shRNAs induces cell-cycle arrest at the G0/G1 phase. Representative flow cytometry histogram plots (top) and quantification (bottom) are shown (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (G) Immunoblotting assay for c-MYC, BCL-2, BAX, CYCLIN B1, and CYCLIN D1 upon CYTH4 silencing in MV4-11 and THP-1 cells. GAPDH was used as a loading control. (H) CYTH4 silencing sensitizes MV4-11 and THP-1 cells to Ara-C. The growth inhibitory effects of AML cells were evaluated through a CCK-8 assay following treatment with varying concentrations of Ara-C for a duration of 48 h. IC50 values were calculated utilizing GraphPad Prism 8.0 software based on the dose-response curve. Data are represent as mean ± standard deviation. Statistical significance was assessed by unpaired t test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Sc, scramble control shRNA; shCYTH4, CYTH4 shRNA.

Figure 3

CYTH4 silencing results in the deactivation of PI3K/AKT pathway via downregulating PIK3R5 (A) Volcano plots for the DEGs between Sc shRNA-transduced and shCYTH4-transduced MV4-11 and THP-1 cells. (B) The KEGG pathway analysis utilizing mRNA-sequencing data from Sc shRNA-transduced and shCYTH4-transduced MV4-11 and THP-1 cells. (C) Immunoblot analysis confirmed the inhibition of the PI3K/AKT signaling pathway upon CYTH4 silencing in MV4-11 and THP-1 cells, with GAPDH serving as a loading control. (D) The intersection analysis of differential transcripts within the PI3K-AKT signaling pathway between MV4-11 and THP-1 cells. Five upregulated and 10 downregulated transcripts were identified at last. (E) Clustering heatmap of differential transcripts in PI3K-AKT signaling pathway. (F) Potential candidates involving in “PI3K-AKT signaling pathway” were examined in Sc shRNA-transduced and shCYTH4-transduced MV4-11 and THP-1 cells by RT-qPCR (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. (G) The gene expression correlation analyses between CYTH4 and MAP2K1, HRAS, TSC2, or PIK3R5 in AML cells were conducted using GEPIA2 data. (H) Immunoblot confirmation for the downregulation of PIK3R5 following CYTH4 silencing in MV4-11 and THP-1 cells. (I) The activities of ARF1 and ARF6 between Sc shRNA- and shCYTH4-transduced MV4-11/THP-1 cells were compared using a pull-down assay. (J) The gene expression correlation analysis between ARF6 and PIK3R5 in AML cells was conducted using GEPIA2 data (R = 0.51). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Sc, scramble control shRNA; shCYTH4, CYTH4 shRNA.

Figure 4

Overexpression of CYTH4 has no effect on AML cell proliferation (A) The expression of CYTH4 was validated to be upregulated in CYTH4-transduced MV4-11 and THP-1 cells by RT-qPCR, with β-actin serving as the internal control (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. (B) Immunoblotting confirmed the upregulated expression of CYTH4 in MV4-11 and THP-1 cells transduced with CYTH4, with GAPDH as the loading control. (C) CYTH4 overexpression did not influence the proliferation of MV4-11 and THP-1 cells. A CCK-8 assay was carried out to assess the viability of these cells at the indicated time points (0, 24, 48, and 72 h). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. (D) The impact of CYTH4 overexpression on the colony formation of MV4-11 and THP-1 cells was evaluated by counting the number and size of colonies under a microscope at 14th day post-plating. The quantification is depicted on the right (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. (E) Overexpression of CYTH4 in MV4-11 and THP-1 cells did not significantly impact cell apoptosis. Apoptotic cell percentages were measured using flow cytometry after annexin V-APC and 7-AAD staining. Representative plots from flow cytometry are on the left, and their quantifications are on the right (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. OE, overexpression.

Figure 5

CYTH4 silencing delays the progression and extramedullary invasion of AML in a xenografts model (A) Representative images of the livers and spleens of the recipients following transplantation for five weeks. Quantifications of liver weight and spleen weight are shown on the right (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. (B) Human CD45 chimerism in the peripheral blood, bone marrow, liver, and spleen of recipients was assessed using flow cytometry following transplantation for five weeks (n = 3). THP-1 cells (4.0 × 10⁵ cells per mouse) were transplanted into NCG mice via tail vein injection. Following euthanizing, peripheral blood, femurs, livers, and spleens of the recipients were harvested for human CD45 chimerism analysis, with quantifications presented at the bottom. Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. (C) Representative hematoxylin/eosin staining images for the femurs, livers, and spleens. The infiltrated leukemic cells are indicated by black arrows. (D) Kaplan-Meier survival curve for the mice transplanted with Sc shRNA/shCYTH4-transduced THP-1 cells (n = 10 per group). (E) Flow cytometry analysis was conducted on the bone marrow and spleen of the xenograft recipient mice 16 h post tail vein injection for homing evaluation (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by unpaired t test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Sc, scramble control shRNA; shCYTH4, CYTH4 shRNA.

Figure 6

PIK3R5 overexpression effectively counteracts the AML cell growth defect resulting from CYTH4 silencing (A) PIK3R5 overexpression in CYTH4-silenced MV4-11 and THP1 cells were confirmed at the mRNA levels by RT-qPCR (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (B) PIK3R5 overexpression in CYTH4-silenced MV4-11 and THP1 cells were confirmed at the protein levels by immunoblotting. (C) CCK-8 assays showed that the impaired cell proliferations caused by CYTH4 silencing were reversed by PIK3R5 overexpression in MV4-11 and THP-1 cells (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (D) PIK3R5 overexpression could effectively reverse the decrease in clone formation caused by CYTH4 silencing (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (E) The apoptosis of cells in MV4-11 and THP1 lines transduced with shCYTH4 was examined by flow cytometry, and this could be alleviated by overexpressing PIK3R5 with a lentivirus packaging system (n = 3). Apoptosis was determined with flow cytometry following annexin V-APC and DAPI staining (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (F) Immunoblotting showing that overexpression of PIK3R5 in CYTH4-silenced MV4-11 and THP-1 cells counteracted the reduction of p-AKT, c-Myc, and Bcl-2. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Sc, scramble control shRNA; shCYTH4, CYTH4 shRNA; pCDH, pCDH-CMV-MCS-EF1-mCherry-P2A-Puro vector.

Figure 7

Activation of AKT by SC-79 rescues AML cell growth defects induced by CYTH4 silencing (A) Immunoblot confirmation for restoration of AKT activity and the downregulated expression of c-Myc and BCL-2 induced by CYTH4 silencing following SC-79 treatment. (B) SC-79 treatment rescued the growth defect phenotype induced by CYTH4 silencing in MV4-11 and THP-1 cells. The cells were treated with 10 μM SC-79 for 0, 24, 48, 72, and 96 h. CCK-8 assay was performed to evaluate cell viability (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (C) The colony formation defect caused by CYTH4 silencing was rescued by SC-79 in MV4-11 and THP-1 cells (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. (D) CYTH4 silencing-induced apoptosis were rescued by SC-79 in MV4-11 and THP-1 cells. Apoptosis was determined with flow cytometry following annexin V-APC and 7-AAD staining (n = 3). Data are represented as mean ± SEM. Statistical significance was assessed by ANOVA with Tukey’s multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Sc: scramble control shRNA; shCYTH4, CYTH4 shRNA.