DARPP-32 and t-DARPP promote non-small cell lung cancer growth through regulation of IKKα-dependent cell migration

Abstract

Lung cancer is the leading cause of cancer-related death worldwide. Here we demonstrate that elevated expression of dopamine and cyclic adenosine monophosphate-regulated phosphoprotein, Mr 32000 (DARPP-32) and its truncated splice variant t-DARPP promote lung tumor growth, while abrogation of DARPP-32 expression in human non-small cell lung cancer (NSCLC) cells reduces tumor growth in orthotopic mouse models. We observe a novel physical interaction between DARPP-32 and inhibitory kappa B kinase-α (IKKα) that promotes NSCLC cell migration through non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells 2 (NF-κB2) signaling. Bioinformatics analysis of 513 lung adenocarcinoma patients reveals elevated t-DARPP isoform expression is associated with poor overall survival. Histopathological investigation of 62 human lung adenocarcinoma tissues also shows that t-DARPP expression is elevated with increasing tumor (T) stage. Our data suggest that DARPP-32 isoforms serve as a negative prognostic marker associated with increasing stages of NSCLC and may represent a novel therapeutic target.

Keywords: Akt; DARPP-32; Erk; IKKα; NF-κB2; NSCLC; cancer; migration; survival; t-DARPP.

Fig. 1

DARPP-32 promotes cell survival and negatively regulates apoptosis. a NSCLC A549, b H1650 and c H226 cell lines were transduced with lentivirus encoding LacZ control shRNA or DARPP-32 shRNAs (clone numbers 03 and 04). DARPP-32 and α-tubulin (loading control) proteins were detected by immunoblotting of cell lysates. Immunoblots are representative of three independent experiments. d A549, e H1650 and f H226 cells transduced with control or DARPP-32 shRNAs were seeded into 60 mm culture dishes for 16 h. Flow cytometry-based apoptosis assays were performed following incubation with anti-annexin V antibodies conjugated with APC. g A549, h H1650 and i H226 cells were transduced with control or DARPP-32 shRNAs and immunoblotted with antibodies to detect cleaved and uncleaved PARP, cleaved and uncleaved (i.e., pro-) caspase-3, DARPP-32 and α-tubulin (loading control). j A549 and k H226 cells were transduced with retrovirus containing control (LacZ), DARPP-32- or t-DARPP-overexpressing clones. DARPP-32, t-DARPP and α-tubulin (loading control) proteins were detected by immunoblotting of cell lysates. Immunoblots are representative of three independent experiments. l A549 and m H226 cells transduced with control, DARPP-32- or t-DARPP-overexpressing clones were seeded into 96-well cell culture plates for 72 h. Colorimeter-based cell survival assay was conducted using MTS reagents. Each open circle on a graph represents an independent experiment. Uncropped images of depicted immunoblots are shown in Supplementary Figs. 13-15. Error bars indicate SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, one-way ANOVA followed by Dunnett’s test for multiple comparison

Fig. 2

DARPP-32 regulates cell survival through Akt and Erk1/2. a A549, H1650 and H226 cells were transduced with control or DARPP-32 shRNAs. Cell lysates were collected and immunoblotted with antibodies against phosphorylated Akt (p-Akt; S473), total Akt (T-Akt), b phosphorylated Erk1/2 (p-Erk1/2), total Erk1/2 (T-Erk1/2), DARPP-32 and α-tubulin (loading control). c A549 and H226 cells were transduced with control, DARPP-32-, t-DARPP- or T34A DARPP-32-overexpressing clones. Phosphorylated Akt (p-Akt; S473), total Akt (T-Akt), d phosphorylated Erk1/2 (p-Erk1/2), total Erk1/2 (T-Erk1/2), DARPP-32 and α-tubulin (loading control) proteins were detected by immunoblotting of cell lysates. Densitometry of the indicated immunoblots was performed using ImageJ software. Each open circle on a graph represents an independent experiment. All immunoblots are representative of three independent experiments. Uncropped images of depicted immunoblots are shown in Supplementary Figs. 16, 17. All bar graphs represent mean ± SEM (n = 3). *P < 0.05, one-way ANOVA followed by Dunnett’s test for multiple comparison

Fig. 3

DARPP-32 positively regulates cell migration. a A549 and b H1650 cells transduced with lentivirus encoding control or DARPP-32 shRNAs were plated into 60 mm cell culture dishes, scratched and imaged at 0 and 14 h. c A549 and d H1650 cells infected with retrovirus encoding control, DARPP-32, t-DARPP or T34A DARPP-32 clones were scratched and imaged at 0 and 14 h. Dashed lines indicate the boundary of the edges of the wound at 0 h. Experiments were repeated at least three times in triplicate. Scale bar, 200 µm. Distance traveled by the migratory cells were calculated using ImageJ software. Each open circle on a graph represents an independent experiment. Results represent mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 and ****P < 0.0001, one-way ANOVA followed by Dunnett’s test for multiple comparison

Fig. 4

DARPP-32 knockdown inhibits non-canonical NF-ĸB2 signaling. a Nuclear fractions of A549 and b H1650 cells expressing control or DARPP-32 (DP32) shRNAs were immunoblotted with antibodies against total p52 (T-p52) and histone H3 (loading control). Cytosolic fractions were also collected and subjected to western blotting using antibodies against phosphorylated p100 (p-p100), total p100 (T-p100), phosphorylated IKKα/β (p-IKKα/β), total IKKα (T-IKKα), DARPP-32 and α-tubulin (loading control). c Immunofluorescence studies were performed using a monoclonal NF-ĸB2 antibody (that detects both p100 and p52 proteins) on A549 and d H1650 cell lysates expressing control or DARPP-32 shRNAs. Nuclei were labeled with DAPI. Average red fluorescence intensity of all nuclei was calculated using ImageJ software. Experiments were repeated at least three times. Each open circle on a graph represents an independent experiment. Scale bar, 20 µm. Error bars indicate SEM (n = 3). **P < 0.01, ***P < 0.001 and ****P < 0.0001, one-way ANOVA followed by Dunnett’s test for multiple comparison. e A549 and f H1650 cells transduced with lentivirus encoding control or DARPP-32 shRNAs (DP32 shRNAs) were immunoprecipitated (IP) with anti-DARPP-32 antibody and immunoblotted (IB) with antibody against IKKα. Total cell lysates (Input) were subjected to western blotting using antibodies against IKKα, DARPP-32 (DP32) and α-tubulin (loading control). All immunoblots are representative of three independent experiments. Uncropped images of depicted immunoblots are shown in Supplementary Figs. 18, 19

Fig. 5

Knockdown of IKKα and NF-ĸB2 reduces lung cancer cell migration. a A549 and H1650 cells transduced with lentivirus encoding control or IKKα shRNAs (clone numbers 04 and 05) were plated into 60 mm cell culture dishes, scratched and imaged at 0 and 14 h. b A549 and H1650 cells transduced with lentivirus encoding control or NF-ĸB2 shRNAs (clone numbers 04 and 05) were scratched and imaged at 0 and 14 h. c A549 and d H1650 cells expressing control, IKKα or NF-κB2 shRNAs were transfected with control (pMMP-LacZ) or DARPP-32 (pMMP-DARPP-32) overexpressing plasmids using Polyfect reagent. The cells were then scratched and imaged at 0 and 14 h. Dashed lines indicate the boundary of the edges of the wound at 0 h. Experiments were repeated at least three times in triplicate. Each open circle on a graph represents an independent experiment. Scale bar, 200 µm. Distance traveled by the migratory cells were calculated using ImageJ software. Results represent mean ± SEM (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001, one-way ANOVA followed by Dunnett’s test for multiple comparison

Fig. 6

DARPP-32 knockdown decreases tumor progression in a human lung tumor xenograft model. a Luciferase-labeled human A549, b H1650 and c H226 cells transduced with lentivirus encoding control or DARPP-32 shRNAs were injected into the left thorax of SCID mice and imaged for luminescence on indicative days. d Luciferase-labeled human A549 cells overexpressing control, DARPP-32 or t-DARPP clones were orthotopically injected into the left thorax of SCID mice and imaged for luminescence on indicative days. Representative in vivo images of SCID mice are depicted. Total luminescence intensity (photon count) was calculated using molecular imaging software. The colored bar represents the numerical value of luminescence. Error bars indicate SEM. *P < 0.05, ***P < 0.001 and ****P < 0.0001, one-way ANOVA followed by Dunnett’s test for multiple comparison

Fig. 7

Elevated t-DARPP protein expression positively correlates with tumor (T) staging score in lung adenocarcinoma patients. a Immunohistochemistry was performed on human NSCLC serially sectioned specimens using an N-terminal DARPP-32 antibody to exclusively detect DARPP-32 and a C-terminal DARPP-32 antibody to detect both DARPP-32 and t-DARPP. Scale bar, 50 µm. b Differential immunohistochemistry was used to quantify t-DARPP protein expression in 62 human lung cancer tissue samples. Each tissue was scored based on the percentage of tumor cells stained positive multiplied by the staining intensity (i.e., 0 = none, 1 = weak, 2 = moderate, 3 = strong expression) to yield an immune reactive (IR) score. The IR score for t-DARPP protein expression was calculated by subtracting the IR score of DARPP-32 (detected with N-terminal antibody) from the IR score of both DARPP-32 isoforms (detected with C-terminal antibody). Each point on the graph represents an individual tissue. Error bars indicate SEM. The P value has been calculated using one-way ANOVA followed by Dunnett’s test for multiple comparison

Fig. 8

Elevated t-DARPP, NF-ĸB2 and IKKα transcripts correlate with decreased lung adenocarcinoma patient survival. a Quantification of t-DARPP mRNA expression in 513 human lung cancer tissue samples. Blue dots indicate outliers. T1–T4 represents tumor (T) staging scores from TNM system. b Kaplan–Meier plot showing overall survival within the total cohort of 513 NSCLC patients based on the expression of t-DARPP and DARPP-32 mRNAs. c Kaplan–Meier curve depicting overall survival within the total cohort of 201 NSCLC patients based on the expression of NF-ĸB2 or d IKKα mRNAs. The normalized read count for DARPP-32, t-DARPP, NF-ĸB2 and IKKα mRNAs were obtained from The Cancer Genome Atlas dataset (TCGA). The difference between the two groups was calculated using log-rank (Mantel–Cox) test. HR hazard ratio