Impact of Sox11 over-expression in Ba/F3 cells

In mantle cell lymphoma (MCL) there is controversy as to whether SOX11 acts as an oncogene or a tumor suppressor. To investigate its function as a potential oncogene, Sox11 was induced in the pro-B cell line Ba/F3, a cell-line previously used for the evaluation of the transformation capability of genes. In these cells Sox11 expression reduced proliferation and induced cell aggregation. 872 genes were differentially expressed, including cell adhesion genes. Among the upregulated genes was an enrichment of gene sets related to forwarding the cell cycle, notably, they also contained cru cial checkpoint genes, consistent with the observed reduction in cell proliferation.

The Sox11 gene belongs to the Sry-related high-mobility group (HMG) box (Sox) transcription factor family, and together with Sox4 and Sox12, constitutes the SoxC group. The SoxC genes are of vital importance during organogenesis as they regulate developmentally important genes, such as protocadherin β, PCDHB.1 Postnatally, Sox11 expression is mainly restricted to neuronal tissue,2 whereas Sox4 is involved in early B-cell development and is necessary for the survival of pro-B cells.3 SOX11 has no identified role in hematopoiesis or lymphopoiesis and is epigenetically silenced in most mature B cells, but is expressed in MCL and in rare reactive lymphocytes.4 SOX11 is also expressed in non-lymphoid malignancies, such as glioma, breast cancer and ovarian cancer. Both the oncogenic and tumor suppressor function of SOX11 has been reported in epithelial malignancies.65 In MCL, it is proposed that SOX11 acts as an oncogene, mainly by inducing cell proliferation, enforcing PAX5 expression and inhibiting terminal B-cell differentiation into plasma cells via PRDM1 and BCL6.87 SOX11 overexpression has also been associated with increased B-cell receptor (BCR) signaling and promotion of oncogenic transformation of B cells in a murine model.9 However, other studies report reduced cell proliferation upon SOX11 expression in MCL cells.1110 The non-malignant, IL-3 dependent pro-B cell line Ba/F3, which does not express immunoglobulins,12 has previously been used for evaluating the transformation capability of potential oncogenes.13 Herein we used the Ba/F3 cell line to investigate the functional and transcriptional changes resulting from induced Sox11 expression.

Sox11 was expressed in the Ba/F3 cell line for 72 h (Sox11-ON) (Figure 1A). In contrast to the non-induced cells (Sox11-OFF), Sox11-ON cells began to form small clusters at 12 h (Online Supplementary Figure S1) with large and pronounced aggregates observed at 72 hours (Figure 1B and Online Supplementary Video). Conditioned medium from Sox11-ON cells did not induce cell aggregation in non-transduced Ba/F3 cells. We detected lower proliferation in Sox11-ON cells (Online Supplementary Figure S2A). Further experiments showed that Sox11-ON cells displayed significantly lower 3H-Thymidine incorporation at 24, 48 and 72 hours (72 hours shown in Figure 1C) and lower metabolic activity than Sox11-OFF cells (Figure 1D). Re-suspending cell aggregates every 24 h did not affect 3H-Thymidine incorporation at 72 h (Online Supplementary Figure S2B).

Figure 1.Phenotypical, proliferative and transcriptional changes in Ba/F3 cells upon 72 h of induced Sox11 expression. (A) Western Blot of SOX11 protein expression in Sox11-ON (doxycycline supplemented medium) and Sox11-OFF (control medium) cells at 72 h, detected with the rabbit polyclonal anti-SOX11 antibody HPA000536, Sigma-Aldrich. B) Bright field microscopy images of cell aggregates following 72 h of continuous Sox11 expression (10x), imaged by Nikon Ti-E microscope. C) Sox11-ON cells incorporates less ³H-Thymidine at 72 h of Sox11 induction following a 4 h pulse, as compared to Sox11-OFF cells, measured in counts per minute, error bars represent the standard deviation (P=0.0086, n=3). D) Mean relative absorption measured by XTT (n=3). Metabolic activity is significantly reduced (P=0.0124) in Sox11-ON cells as compared to Sox11-OFF cells. E) Volcano plot representation of transcript level differences by Affymetrix MTA-1 mouse arrays (Microarray data has been made available through the GEO database with accession number GSE108419). Names are shown for the genes with the largest transcript level fold change (log2FC ≥1.3 or ≤−1.3). Blue and red: genes with significantly altered transcript levels in Sox11-ON cells and a fold change below -2 (blue) (FDR q-value ≤0.05 and log2FC ≥−1) or above 2 (red) (FDR q-value ≤0.05 and log2FC ≥1); gray: genes significantly changed at the transcript level (FDR q-value ≤0.05); black: genes with non-significant transcript level changes. F) Expression levels after Sox11 induction in genes specifically expressed at different stages of B-cell development. Only the pro-B restricted genes Id1 and Tal1 had significantly altered transcript levels in Sox11-ON cells (FDR q-value: 0.006 and 0.016, respectively). None of the other investigated pro-B and pre-B cell associated genes were altered at the transcript level. Genes associated with later B-cell developmental stages are shown for comparison. Transcript levels are presented as a gene-wise standardized expression (Z-score). FC: fold change.

The global gene expression profile for Sox11-ON cells following 72 h of Sox11 expression was distinct from both Sox11-OFF and non-transduced Ba/F3 cells (Online Supplementary Figure S3) with 872 significantly changed genes (534 genes up-regulated (fold change (FC) ≥1.5) and 338 genes down-regulated (FC ≤−1.5) in Sox11-ON cells false discovery rate (FDR) q-value ≤0.05) as shown in Figure 1E and Online Supplementary Table S1. The gene with the highest FC, Mmp8, is involved in proteolysis and is induced in peripheral blood mononuclear cells following incubation with S100A8 and S100A9 proteins, both of whose corresponding transcripts were among the most increased in the Sox11-ON cells. Additionally, the immunomodulatory cytokine, Ccl3, was down-regulated in Sox11-ON cells. In cancer, S100A8/A9 over-expression has been associated with increased adhesion, reduced migration, impaired tumor growth and reduced transcript levels of CCL3.14 Among the most differentially expressed genes were several adhesion-associated genes retrieved from the curated cell-adhesion database OKCAM. Stfa1 and the SOX11 regulated protocadherin β genes1 (Pcdhb16 and Pcdhb17) were up-regulated, while Sell and Itgam were down-regulated (Figure 1E). Down-regulation of Sell, which is involved in leukocyte rolling, has also been reported in MCL.15

To investigate whether Sox11 expression in Ba/F3 cells would influence the gene expression profile of B-cell developmental genes, the expression of genes characteristic for different stages of B-cell development was analyzed as described in the Online Supplementary Materials and Methods. Among those, Sox11 induction increased transcript levels for two pro-B-cell restricted genes, Id1 and Tal in Sox11-ON (FC: 1.2 and 1.3, respectively), but not any of the other genes typically associated with specific stages of B-cell development (Figure 1F). Even though expression of many genes was affected by Sox11 expression, no significant changes in expression were observed for other investigated pro-B cell associated genes other than Id1 and Tal, nor for other B-cell development associated genes that were included for comparison, such as Prdm1 and Bcl6. Additionally, we observed no transcript level change for the previously reported Sox11 target Pax5.8

To functionally classify genes with changed transcript levels a Gene Set Enrichment Analysis (GSEA) of Gene Ontology terms for biological processes resulted in 234 gene sets enriched for Sox11-induced genes and 54 gene sets enriched for Sox11 down-regulated genes (FDR q-value ≤ 0.05, Online Supplementary Table S2). Two hundred and forty out of the 288 functional categories could be divided into 27 clusters (Figure 2, details in Online Supplementary Table S2 and Table S3). The majority of clusters associated with genes that had increased transcript levels in Sox11-ON cells represented basal cellular functions. Several of those clusters are associated with aspects of the cell cycle: c1, c3, c7, c10, c16, c17 and c18. An analysis of the leading-edge genes in these clusters revealed a pattern largely associated with forwarding the cell cycle, but notably these clusters also contain important cell cycle checkpoint genes such as Chek1 and Chek2, as well as DNA damage response genes such as Trp53, Brca1 and Brca2 (Online Supplementary Table S3). The functional outcome was reduced proliferation. Other clusters were indicative of epigenetic modifications (c9 and c13) and changes in transcription (c2, c14, c22 and c24). Only three clusters represent genes with lower transcript levels in Sox11-ON cells: c5, c11 and c15. These gene sets, associated with leukocyte function, contained genes encoding for cytokines and immunomodulatory pathways, including Ccl3, Ccl4, Il6 and Tnf.

Figure 2.Significantly enriched gene ontology terms from the gene set enrichment analysis (GSEA) clustered based on similarity in gene content. (A) 240 of the 288 significant functional categories were divided into 27 clusters (containing at least three functional categories). Functional categories representing up-regulated genes in Sox11-ON cells are presented in red, while gene sets representing down-regulated genes are presented in blue. Gene set sizes are represented by circle size, where gene sets containing few genes are represented by small circles and functional categories with more genes have larger circles. A darker shade is indicative of lower GSEA q-value. Darker edges represent a greater similarity between functional category nodes. Clusters are ranked according to size, where c1 contains the most functional categories (n=41) and c27 (n=3) the fewest. Functional categories have been positioned manually for increased legibility. B) Detailed information about clusters in A. The columns represent: 1. keywords, which are the most prevalent words among the gene ontology term names in each cluster, as generated by the Cytoscape plugin AutoAnnotate, 2. nodes, which indicate the number of functional categories in each cluster, 3. genes, which represent the number of unique genes for the unified list of genes from all functional categories in the cluster, 4. significant, which indicates the number of unique genes in the cluster that were differentially expressed (FDR q-value ≤0.05) and 5. leading edge, which indicates the number of unique genes per cluster that were present in the GSEA leading edge.

In conclusion, SOX11 has been reported to have oncogenic properties in MCL,8 however this has not been confirmed in other reports.161110 Oncogenic transformation associated with increased BCR signaling has been reported in murine B cells overexpressing Sox11.9 While the pro-B cell line in the present study lack the BCR, Sox11 was nevertheless able to significantly alter the global gene expression pattern, indicating that the implications of Sox11 expression can be highly context dependent. In the context of a non-malignant pro-B cell line, Sox11 expression markedly up-regulated transcript levels of genes involved in basal cell functions and down-regulated transcript levels of genes associated with leukocyte responses. The net results of induced Sox11 expression in Ba/F3 cells was reduced proliferation and a marked cell aggregation. However, these results cannot be directly extrapolated to MCL, a lymphoma which is characterized by high genomic complexity. Consequently, the lack of oncogenic effects upon induced Sox11 expression in the Ba/F3 cells does not exclude the possibility that SOX11 exhibits oncogenic activity in other cell contexts where crucial cell cycle checkpoint genes are absent, or perhaps by cooperating with oncogenes, tumor suppressor genes or ongoing BCR-signaling mechanisms that are already deregulated in lymphoma.