CDKL3 promotes osteosarcoma progression by activating Akt/PKB

(A) Genomic DNA sequencing of CDKL3-KO clones. (B) CDKL3 expression in CDKL3-KO and parental cells by Western blot analysis. (C) Microscopy images of wound closure of parental, CDKL3-KO (U19), and CDKL3-rescued (Res) U2OS cells at 0, 24, and 48 h after scratching. Scale bar = 75 μm. (D) Quantification of the wounded area invaded during 48 h of U2OS cells (n = 3). (E) Quantification of wound healing speed (μm/h) of U2OS cells (n = 3). (F) Microscopy images of wound closure of parental, CDKL3-KO (S4), and CDKL3-rescued (Res) Saos-2 cells at 0, 24, and 48 h after scratching. Scale bar = 75 μm. (G) Quantification of the wounded area invaded during 48 h of Saos-2 cells (n = 3). (H) Quantification of wound healing speed (μm/h) of Saos-2 cells (n = 3). (I) Representative images of Transwell-invaded U2OS cells (purple stained, red arrows). Scale bar = 100 μm. (J) Quantification of stained/invaded cells in (I) (n = 3). (K) Representative images of Transwell-migrated U2OS cells (purple stained, red arrows). Scale bar = 100 μm. (L) Quantification of stained/migrated cells in (K) (n = 3). (M) Representative images of Transwell-invaded Saos-2 cells (purple stained, red arrows). Scale bar = 100 μm. (N) Quantification of stained/invaded cells in (M) (n = 3). (O) Representative images of Transwell-migrated Saos-2 cells (purple stained, red arrows). Scale bar = 100 μm. (P) Quantification of stained/migrated cells in (O) (n = 3). (Q) Representative images of senescent cells stained with CellEvent senescence green probe. Scale bar = 75 μm. (R) FACS analysis of green-stained senescent cells in (Q). (S) Quantification of senescent U2OS cells in (R) (n = 3). (T) Quantification of senescent Saos-2 cells (n = 3). Error bars indicate SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance of the differences was estimated by unpaired two-tailed t test. Linear regression was run on the wound width data using the GraphPad Prism software.

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(A) Representative images of tumor xenografts obtained from the indicated groups. Scale bar = 1 cm. (B) Left: tumor volumes over treatment. Right: tumor weight at harvest. Error bars represent SD (n = 8). *P < 0.05, ***P < 0.001, two-tailed t test.

(A) An outline of OS clinical dataset analysis (GEO accession: GSE21257). 33 out of 53 patient samples were selectively analyzed based on the CDKL3 expression level for the differential gene expression patterns. (B) Signaling pathway was mapped by the R package clusterProfiler. Upper and bottom figures represent down-regulated and up-regulated pathways (CDKL3 top 0.3 versus bottom 0.3), respectively. KEGG enrichment analysis using the clusterProfiler R package was performed on differential expressed genes with a strict cutoff of P < 0.01 and false discovery rate of less than 0.05. The size of the dot indicates the number of differentially expressed genes in the pathway. Color of the dot represents the value of Benjamini and Hochberg false discovery rate–adjusted P-value. (C) Molecular connection of PI3K-Akt and AMPK pathway in regulation of cell growth, autophagy, etc. This summarized mechanism was extracted from the GEO dataset GSE21257. mTORC1 plays key roles in eukaryotic cell metabolism by promoting cell growth and inhibiting autophagy and apoptosis. (D) Representative images of parental cells and CDKL3-KO U2OS and Saos-2 cells transfected with mRFP-eGFP-LC3, at normal conditions (DMEM with 10% FBS, control) or 6 h EBSS treatment (starvation). Colocalized mRFP and eGFP (yellow dots) indicate autophagosomes, only mRFP (red dots) indicates autolysosomes. Nuclei (blue) were labeled with DAPI. Scale bar = 5 μm. (E) Quantification of autophagosomes (eGFP⁺mRFP⁺; yellow) and autolysosomes (eGFP⁻mRFP⁺; red) per cell from (D) (n = 3). (F) Western blot analysis of LC3B, Beclin-1, and Atg5-Atg12 in indicated cells. The number below each blotting strip is intensity quantification derived from ImageJ. Error bars indicate SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

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(A) Akt and mTOR activation under regular or starvation conditions in parental and CDKL3-KO U2OS cells. Upon CDKL3 KO, Akt and mTORC1 activation were significantly alleviated. The numbers below each blotting strip is intensity quantification by ImageJ. (B) The expression patterns of mTORC1 and FoxO downstream target genes confirm that CDKL3 regulates both pathways at the transcription level. Cells were cultured under the regular growth condition. (C) Overexpression of CDKL3 in U2OS cells causes hyper-activation of Akt in the presence and absence of FBS. (D) General working mechanism of rapamycin, AZD5363, and MK-2206. (E) U2OS cell growth under different conditions (n = 3). (F) Co-immunoprecipitation (Co-IP) reveals the physical interaction between HA-CDKL3 and Myc-Akt1. (G) Endogenous CDKL3 coexists with endogenous Akt shown by co-IP. (H) Schematic diagrams of Akt1 constructs for mapping. (I) Co-IP of HA-CDKL3 with different Akt1 constructs shows that the Akt kinase domain is indispensable for CDKL3 interaction. (I, J) Reverse IP of CDKL3 and Akt1 confirms the findings in (I). Error bars indicate SD (n = 3). *P < 0.05 parental DMSO versus CDKL3^−/− DMSO, or parental rapamycin, or parental AZD5363, or parental MK-2206; **P < 0.01 CDKL3^−/− DMSO versus CDKL3^−/− rapamycin, or CDKL3^−/− AZD5363, or CDKL3^−/− MK-2206; ^##P < 0.01 parental MK-2206 versus CDKL3^−/− MK-2206. Statistical significance of the differences was estimated by unpaired two-tailed t test.

Source data are available for this figure.