ASCL1-regulated DARPP-32 and t-DARPP stimulate small cell lung cancer growth and neuroendocrine tumour cell proliferation

Abstract

Background: Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, and new molecular insights are necessary for prognostic and therapeutic advances.

Methods: Dopamine and cAMP-regulated phosphoprotein, Mr 32000 (DARPP-32) and its N-terminally truncated splice variant, t-DARPP, were stably overexpressed or ablated in human DMS-53 and H1048 SCLC cells. Functional assays and immunoblotting were used to assess how DARPP-32 isoforms regulate SCLC cell growth, proliferation, and apoptosis. DARPP-32-modulated SCLC cells were orthotopically injected into the lungs of SCID mice to evaluate how DARPP-32 and t-DARPP regulate neuroendocrine tumour growth. Immunostaining for DARPP-32 proteins was performed in SCLC patient-derived specimens. Bioinformatics analysis and subsequent transcription assays were used to determine the mechanistic basis of DARPP-32-regulated SCLC growth.

Results: We demonstrate in mice that DARPP-32 and t-DARPP promote SCLC growth through increased Akt/Erk-mediated proliferation and anti-apoptotic signalling. DARPP-32 isoforms are overexpressed in SCLC patient-derived tumour tissue, but undetectable in physiologically normal lung. Achaete-scute homologue 1 (ASCL1) transcriptionally activates DARPP-32 isoforms in human SCLC cells.

Conclusions: We reveal new regulatory mechanisms of SCLC oncogenesis that suggest DARPP-32 isoforms may represent a negative prognostic indicator for SCLC and serve as a potential target for the development of new therapies.

Fig. 1. DARPP-32 and t-DARPP decrease cell death by reducing apoptosis.

a Retrovirus containing control- (LacZ), DARPP-32- or t-DARPP-overexpressing clones and b lentivirus encoding control (LacZ) or DARPP-32 shRNAs were transduced in DMS-53 cells. Cells were incubated with anti-annexin V antibodies conjugated with FITC followed by propidium iodide incorporation. Flow cytometry-based apoptosis assays were performed to determine the total number of annexin V-positive cells. The average number of annexin V-positive cells of three independent experiments were plotted in a bar graph. Each open circle on a graph represents an independent experiment. The numerical values on quadrants of the scatter plots represent the percentage of total cells in one single representative experiment. c DMS-53 and d H1048 cells were transduced with control-, DARPP-32- or t-DARPP-overexpressing clones. Cell lysates were collected and immunoblotted with antibodies to detect cleaved and uncleaved PARP-I, cleaved and uncleaved (i.e., pro-) caspase-3, DARPP-32 and α-tubulin (loading control). e DMS-53 and f H1048 cells were transduced with lentivirus encoding control or DARPP-32 shRNAs. Cleaved and uncleaved PARP-I, cleaved and uncleaved (i.e., pro-) caspase-3, DARPP-32 and α-tubulin (loading control) proteins were detected by immunoblotting of cell lysates. Immunoblots are representative of three independent experiments. All bar graphs represent mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, one-way ANOVA followed by Dunnett’s test for multiple comparison.

Fig. 2. DARPP-32 and t-DARPP positively regulate cell survival through Akt and Erk1/2.

a Lysates of DMS-53 and b H1048 cells overexpressing control, DARPP-32 or t-DARPP clones were immunoblotted using antibodies against phosphorylated Akt (p-Akt; S473), total Akt, phosphorylated Erk1/2 (p-Erk1/2, T202/Y204), total Erk1/2, DARPP-32 and α-tubulin (loading control). c DMS-53 and d H1048 cells transduced with control or DARPP-32 shRNAs were subjected to western blotting using antibodies against phosphorylated Akt (p-Akt; S473), total Akt, phosphorylated Erk1/2 (p-Erk1/2), total Erk1/2, DARPP-32 and α-tubulin (loading control). All Immunoblots are representative of three independent experiments. Densitometry analysis was performed using Image J software and numerical values are reported below each immunoblot. e Flow cytometry-based BrdU cell proliferation assays were performed in DMS-53 cells transduced with control or DARPP-32 shRNAs following incubation with anti-BrdU antibodies conjugated with FITC. DNA intercalating fluorescent agent (propidium iodide) was used to quantify total DNA in the cells. The bar graph represents mean ± SEM of at least 3 independent experiments. ***P < 0.001, one-way ANOVA followed by Dunnett’s test for multiple comparison.

Fig. 3. DARPP-32 isoforms promote growth of human SCLC cells in mouse xenograft models.

a Luciferase-labelled human DMS-53 cells transduced with lentivirus encoding control (LacZ) or DARPP-32 shRNAs were orthotopically injected into the left thorax of SCID mice and imaged for luminescence on indicated days. b Luciferase-labelled human DMS-53 and c H1048 cells overexpressing control (LacZ), DARPP-32 or t-DARPP clones were injected into the left thorax of SCID mice and imaged for luminescence on the indicated days. In vivo images of SCID mice bearing luciferase-labelled tumours showing luminescence on the indicated days are depicted. Total luminescence intensity (photons count) was calculated using molecular imaging software. The numerical value of luminescence was represented by the colour bar. The average luminescence intensity of each time point was plotted. Error bars indicate SEM. *P < 0.05 and ****P < 0.0001, one-way ANOVA followed by Dunnett’s test for multiple comparison.

Fig. 4. Expression of DARPP-32 isoforms is elevated in human SCLC patients.

a–f Normal lung tissues (n = 6) and g–l tumour tissues from SCLC patients (n = 6) were subjected to immunohistochemistry using an antibody that recognises both DARPP-32 and t-DARPP isoforms. Nuclei were counterstained using haematoxylin stain. N and T denotes normal and tumour, respectively. Scale bar indicates 20 µm.

Fig. 5. Transcripts associated with Notch signalling are upregulated in a subset of SCLC tumours expressing high levels of t-DARPP.

a Expression of t-DARPP transcripts was quantified using a previously published RNA-Seq database. Blue points indicate individual SCLC patients (n = 6) with an elevated t-DARPP expression (t-DARPP: both DARPP-32 isoforms) in tumour tissue compared to mRNA derived from corresponding adjacent normal tissue. b Quantification of mRNA transcripts in a subset of SCLC patients (n = 6; blue points) expressing high level of t-DARPP isoforms in tumour relative to normal tissue. c Gene set enrichment analysis (GSEA) was performed to identify cellular pathways regulated in a subset of SCLC patients with elevated t-DARPP levels in tumour tissue. Notch signalling was enriched by GSEA. d Heat map showing the expression of Notch family member genes identified through GSEA. N and T denotes normal and tumour tissue, respectively. Red colour represents highest fold change in expression in tumour tissue relative to normal. Colour bar represents normalised read counts in a log scale.

Fig. 6. ASCL1 positively regulates DARPP-32 expression in SCLC cells.

a DMS-53 cells stably transduced with lentivirus encoding either LacZ shRNAs (control) or ASCL1 shRNAs (clone #3 and #4) were lysed using RIPA cell lysis buffer. Immunoblotting was performed with antibodies against ASCL1, DARPP-32 and α-Tubulin (loading control). Immunoblot is representative of three independent experiments. b Dual-luciferase assays (right) were performed in DMS-53 cells transduced with either control or ASCL1 shRNAs following transient transfection of pGL3-DARPP-32-Luc (Firefly) and pRL-SV40 (Renilla) plasmids. Firefly and renilla luminescence were measured and plotted as a ratio. The bar graph represents mean ± SEM of three independent experiments, two-tailed unpaired t-test. Immunoblotting (left) was performed in parallel to confirm shRNA-mediated knockdown of ASCL1 in DMS-53 cells. c Three potential ASCL1 binding sites are present within the DARPP-32 promoter (orange boxes). ASCL1 binding sites on the DARPP-32 promoter have been mutated by site-directed mutagenesis. The crossed boxes refer to the mutated sites. d DMS-53 cells transfected with either unmodified pGL3-DARPP-32-Luc plasmids (A) or site-directed mutagenesis pGL3-DARPP-32-Luc constructs (indicated as B to H) were subjected to dual-luciferase assay. e Immunoblotting was performed in H1048 cells transfected with ASCL1 expressing plasmids (cDNA). Immunoblotting results represent three independent experiments. f Transient transfection of either unmodified pGL3-DARPP-32-Luc plasmids (A) or site-directed mutagenesis pGL3-DARPP-32-Luc constructs (indicated as B to H) was carried out in H1048 cells exogenously overexpressing ASCL1. Dual-luciferase assays were performed after 48 h post-transfection. Each open circle on a graph represents an independent experiment. Bar graph represents an average of three independent experiments. Error bars indicate SEM. *P < 0.0001, one-way ANOVA followed by Dunnett’s test for multiple comparison.