Expression of DLG members with L27 domains were inversely correlated to survival and risk
The main difference between different proteins encoded by DLG gene members and their isoforms is the presence or absence of an N-terminal L27 domain (Fig. 1a). The exon structure of DLG2 showing the initiation sites of the ζ, β, α, ε, δ and γ isoforms (Fig. 1b) (16, 17). We evaluated the association of DLG family expression with survival and risk, using online microarray data using the neuroblastoma patient dataset (GSE49710) obtained from the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). The data was divided into survival outcome; alive or deceased (Fig. 1c). The L27-containing DLG members DLG1 (log2 fc = 0.30, p < 0.001), DLG2 (log2 fc = 0.72, p < 0.001) and DLG4 (log2 fc = 0.49, p < 0.001) all showed higher expression in surviving cases, whereas the non-L27-containing DLG3 (log2 fc =-0.23, p < 0.001) showed lower expression, DLG5 showed no difference in expression (Fig. 1c). The same trend was seen for risk stratification with DLG1 (log2 fc = 0.40, p < 0.001), DLG2 (log2 fc = 0.68, p < 0.001) and DLG4 (log2 fc = 0.72, p < 0.001), all showed higher expression in low risk neuroblastoma whereas DLG3 (log2 fc =-0.47, p < 0.001) showed lower expression in low risk neuroblastoma (Fig. 1d).
We evaluated the expression levels in neuroblastoma of the L27-domain containing DLG family members, DLG1, DLG2 and DLG4, by comparing the total gene expression and transcript encoding the alpha or beta protein using the using online data using the neuroblastoma patient dataset (TARGET) obtained from the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). The data was divided into INSS stage for DLG1, DLG2 and DLG4. DLG1 showed a decrease in DLG1 isoform 1 (DLG1-iso1) (ENST00000452595) expression corresponding to SAP-97α between stage 4 and the favorable stage 4 s (log2 FC = 0.44, p < 0.001) with no difference between stage 3 and 4 (Fig. 2a). DLG1 showed a decrease in DLG1 isoform 2 (DLG1-iso2) (ENST00000357674) transcript expression corresponding to L27-containing SAP-97β, between stage 4 and stage 4 s (log2 FC = 0.44, p < 0.001) and between stage 3 and stage 4 s (log2 FC = 0.76, p < 0.05) (Fig. 2a). At the total gene expression level a similar decrease in expression as the DLG1-iso2 transcript was observed between stage 4 and stage 4 s (log2 FC = 0.80, p < 0.001) and between stage 3 and stage 4 s (log2 FC = 0.76, p < 0.05) (Fig. 2a). DLG2 showed no decrease in transcript DLG2 isoform 1 (DLG2-iso1) (ENST00000376104) transcript expression corresponding to the truncated L27-containing SAP-93β or DLG2 isoform 2 (DLG2-iso2) (ENST00000398309) expression, corresponding to non-L27-containing PSD-93α, between the stages. At the total gene expression level (including all DLG2 isoforms) a decrease in expression is observed between stage 4 and stage 4 s (log2 FC = 0.72, p < 0.001) (Fig. 2b). Indicting that isoforms accounting for this difference have not been included in this analysis. DLG4 showed no decrease in isoform 1 expression between the stages. Furthermore, there was no change in total DLG4 expression level between stages (Fig. 2c).
DLG2 isoform 7/8 was downregulated in high stage neuroblastoma
We evaluated the expression of the main DLG2 isoforms, using the transcript data from the TARGET dataset based off GRCh37. We determined that the DLG2 isoforms with the highest expression were DLG2-iso2 (ENST00000398309) and DLG2 L27 only (ENST00000472545), with no or very low expression of isoforms 1 (ENST00000376104), 3 (ENST00000418306) or 4 (ENST00000280241) detected (Fig. 2d). In this chromosome build DLG2-iso7 or 8 are not included and therefore cannot be included in the analysis. The presence of DLG2 L27 only (ENST00000472545) indicates that isoforms 7/8 are likely expressed, but not captured in this expression data using this chromosome build. Using 22 primary neuroblastoma samples, we could confirm by qPCR the unaltered DLG2-iso2 expression observed in the TARGET dataset (Fig. 2e). We could also confirm that there was no expression of isoforms 3 or 4 in our samples. Isoform 1 as a truncated variant of isoforms 7 and 8 (Fig. 1b), cannot be uniquely identified by qPCR when compared to isoforms 7/8, and since the isoform 1/7/8 qPCR result showed the same result as the specific isoform 7/8 qPCR, we concluded that isoform 1 was not expressed in our samples (data not shown). No variation in the expression of DLG2-iso2 (ENST00000398309) was observed between the stages (Fig. 2e), consistent with Fig. 2b. The DLG2-iso7/8 (ENST00000650630) transcript had decreased expression in the stage 4 tumors when compared to the stage 1 and 2 tumors (log2 FC = 3.1, p < 0.05). DLG2-iso7/8 was also decreased compared to DLG2-iso2 in stage 4 (log2 FC = 4.9, p < 0.001) but not between stage 1 + 2 and stage 3 tumors (Fig. 2e). To evaluate the total DLG expression in neuroblastoma we determined the relative expression in Fragments Per Kilobase Million (FPKM) of all DLG family members. DLG1, DLG2 and DLG3 all showed similar expression levels with DLG4 and DLG5 having significantly higher expression (Fig. 2f).
DLG2 expression correlated to LIN7 family gene expression and neuroblastoma samples formed clusters.
The L27-domain enables binding to other L27-domain containing proteins. An important L27-containing scaffolding protein in signaling complex formation is the LIN7 protein family. The relationship between DLG2 and DLG1 and the various LIN7 binding partners was examined using primary tumor data taken from the Z score of 159 tumor data sets on the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). A positive relationship (Y = 0.82x − 0.05, P < 0.001) between DLG2 and LIN7A across tumor datasets could be confirmed (Fig. 3a). Clusters were formed based on the spatial coordinates of DLG2 and LIN7A expression. Medulloblastoma (6/7), Ewings sarcoma (2/2), Glioma (6/7), Pheochromocytomas/Paragangliomas (2/2) and Neuroblastoma (5/5) all showed high DLG2 expression as well as high LIN7A expression. The remaining tumors with similar expression included other tumors of the CNS such as Glioblastoma, Primitive neuroectodermal tumor (PNET) and other brain tumors. Squamous cell carcinoma (2/2) showed high DLG2 expression with low LIN7A expression. The remainder of the tumor dataset consisting of Lung, Colon, Ovarian, Breast and various lymphomas tended to show low expression of both DLG2 and LIN7A (Fig. 3a). A weak linear relationship could be established between DLG1 and LIN7A (Fig. 3b), however no distinct tumor clusters could be formed. A positive relationship (Y = 0.70x + 0.07, P < 0.0001) could be established between DLG2 and LIN7B across tumor datasets (Fig. 3c). Ewing’s sarcoma (2/2) and Neuroblastoma (5/5) clustered with high DLG2 expression as well as high LIN7B expression. No linear relationship (Y = 0.66x + 0.08, P = 0.23) between DLG1 and LIN7B across tumor datasets could be confirmed (Fig. 3d). A positive relationship (Y = 0.97x + 0.04, P < 0.0001) between DLG2 and LIN7C between tumor datasets could be confirmed (Fig. 3e). Ewings sarcoma (2/2) and Neuroblastoma (5/5) clustered with high DLG2 expression as well as high LIN7C expression. Squamous cell carcinoma (2/2) clustered with high DLG2 expression and low LIN7C expression (Fig. 3e). A positive relationship (Y = 1.6x + 0.00, P < 0.05) between DLG1 and LIN7C across tumor datasets could be confirmed (Fig. 3d), however distinct tumor clusters were not formed.
DLG2-isoform 7 expression controlled LIN7A expression and the DLG2-isoform 7 encoded protein can bind LIN7A
To further evaluate the relationship that was established in Fig. 3a between DLG2 and LIN7A gene expression, we determined the expression of LIN7A and DLG2-iso7/8 in neuroblastoma primary samples. A strong positive correlation (R2 = 0.89, Y = 1.1x − 0.06, P < 0.001) between the expression of DLG2-iso7/8 and LIN7A for 22 primary neuroblastoma tumors of varying stages was detected (Fig. 4a). To determine if the relationship was causal we over expressed DLG2-iso7 or knocked down DLG2 expression by siRNA treatment in SKNAS neuroblastoma cells. When DLG2-iso7 was over expressed LIN7A expression increased, and LIN7A expression decreased following DLG2 silencing (Fig. 4b). When LIN7A was over expressed or silenced by siRNA we saw no difference in total DLG2 expression (Fig. 4c). To determine if DLG2-iso7 or DLG2-iso2 bound directly to LIN7A we performed co-immunoprecipitation using co-transfected HEK-293 cells, showing that DLG2-iso7 but not DLG2-iso2 could bind to LIN7A (Fig. 4d).
LIN7A expression was low in high staged tumors and over expression changed the growth behavior of neuroblastoma cells.
To further investigate the importance of LIN7A we evaluated the association of LIN family expression with survival and INSS, using online microarray data in the neuroblastoma patient dataset (GSE49710) obtained from the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). The data was divided into survival outcome; alive or deceased. LIN7A (log2 fc = 1.06, p < 0.001) showed a decrease in expression in the deceased patients compared to the patients that survived (Fig. 5a). LIN7B (log2 fc = 0.43, p = 0.09) and LIN7C (log2 fc = 0.20, p = 0.66) showed no difference in expression (Fig. 5a). The expression of LIN7A was then stratified by INSS stage. Stage 4 tumors showed the lowest expression compared to stage 1 (log2 fc = 0.44, p < 0.01), stage 2 (log2 fc = 0.44, p < 0.001), stage 4 s (log2 fc = 0.25, p < 0.05) and stage 3 (log2 fc = 0.50, p < 0.01) (Fig. 5b). Over expression of LIN7A in neuroblastoma cells (SKNAS) resulted in slower proliferation compared to the control (Fig. 5c, p < 0.001). We observed a decrease in the number of viable cells (Fig. 5d, p < 0.001) and an increase in the non-viable cell fraction (Fig. 5d, p < 0.001) in cells with increased LIN7A expression. LIN7A silencing in SKNAS cells resulted in an increase in cell proliferation (Fig. 5c, p < 0.01), with an associated increase in viable cell number, no effect in the non-viable cell number was observed (Fig. 5d). The LIN7A over expression after expression plasmid transfection, and LIN7A silencing by siRNA treatment of neuroblastoma cells (SKNAS) was confirmed by qPCR (Fig. 5e).