SOX4 Transcriptionally Regulates Multiple SEMA3/Plexin Family Members and Promotes Tumor Growth in Pancreatic Cancer

<div><p>Semaphorin signaling through Plexin frequently participates in tumorigenesis and malignant progression in various types of cancer. In particular, the role of semaphorin signaling in pancreatic ductal adenocarcinoma (PDAC) remains unexplored, despite a high likelihood of metastasis and mortality. Unlike other epithelial malignancies that often express a small number of specific genes in the Semaphorin/Plexin family, five or more are often expressed in human PDAC. Such concomitant expression of these SEMA3/Plexin family members is not a result of gene amplification, but (at least partially) from increased gene transcription activated by SOX4 de novo expressed in PDAC. Via chromatin-immunoprecipitation, luciferase promoter activity assay and electrophoresis mobility shift assay, SOX4 is demonstrated to bind to the consensus site at the promoter of each <em>SEMA3</em> and <em>Plexin</em> gene to enhance transcription activity. Conversely, RNAi-knockdown of SOX4 in PDAC cell lines results in decreased expression of SEMA3/Plexin family members and is associated with restricted tumor growth both <em>in vitro</em> and in SCID mice. We further demonstrate that SOX4 levels parallel with the summed expression of SEMA3/Plexin family members (<em>P</em> = 0.033, <em>NPar</em> Kruskal-Wallis <em>one</em>-<em>way</em> analysis), which also correlates with poor survival in human PDAC (<em>P</em> = 0.0409, <em>Kaplan-Meier</em> analysis). Intriguingly, miR-129-2 and miR-335, both of which target SOX4 for degradation, are co-repressed in human PDAC cases associated with up-regulated SOX4 in a statistically significant way. In conclusion, we disclose a miR-129-2(miR-335)/SOX4/Semaphorin-Plexin regulatory axis in the tumorigenesis of pancreatic cancer.</p> </div

Expression of SEMA3, Plexin and NRP in patients with PDAC.

<p>Paired <i>t</i>-test,</p>**<p>: <i>P</i><0.0001.</p

Multiple members of SEMA3/Plexin ligand-receptor complexes expressed in human PDAC tissues and cell lines.

<p>(A) immunohistochemistry of human PDAC cases showing expression of NRP1 and most members of SEMA3/Plexin family. Normal pancreatic tissue did not express any SEMA3/Plexin/NRP (diagonal, scale bar = 50 µm). Scale bar = 100 µm. (B) Bar graph to show distribution of numbers of SEMA3/Plexin/NRP expressed in each PDAC case. Fifty-three out of 62 cases (85.5%) expressed more than 5 members. (C) semi-quantitative RT-PCR analysis of SEMA3-, Plexin- and NRP- transcripts in human PDAC cell lines. GAPDH served as an internal loading control. (D) immunoblot of the whole-cell lysate from human PDAC cell lines to detect endogenous SEMA3, Plexin, and NRP1. (E) representative SEMA3-, Plexin- and NRP1-immunoreactivity detected in PanIN lesions adjacent to invasive carcinoma. Scale bar = 100 µm.</p

SOX4 expression in PDAC correlates with the total number of expression of SEMA3/Plexin/NRP1.

<p>(A) gene loci of each human <i>SEMA3</i>, <i>Plexin</i>, and <i>NRP</i> gene. (B) representative array CGH on chromosome 7 with genomic DNA extracted from PANC-1 and MiaPaCa2 cells, respectively, compared with normal human genomic DNA (blue dots and line: forward experiment, cell line versus control; red dots and line: reverse experiment, control versus cell line). No significant gene amplification (> =  or < =  2-fold change) was observed around <i>SEMA3A</i>, <i>3C</i>, <i>3D</i>, <i>3E</i> and <i>PLXNA4</i> gene loci (indicated by green-colored rectangle and arrow). (C) SOX4 is expressed in PDAC cell lines. Cell lysates were immunoprecipitated using anti-SOX4 antibody and immunoblotted with SOX4 or a control IgG antibody. (D) immunohistochemistry of SOX4 and E2F1 in human PDAC cases. SOX4 and E2F1 were both detected in PanIN of different grades and invasive adenocarcinoma, but not in normal ductal and acinar epithelia or in chronic pancreatitis. Scale bar = 100 µm. (E) Rank sum of staining intensity for SEMA3/Plexin/NRP1 correlates with SOX4 expression in PDAC cases. The staining of each section was scored 1–3 to rank its intensity as weak, medium, or strong. Scores of all SEMA3/Plexin/NRP1 immunostaining for each case were summed and correlated with SOX4 expression (NPar Kruskal-Wallis one-way analysis). The median (line within the box), maximal and minimal values (upper and lower lines outside the box, respectively) for each group are shown in the boxplot.</p

SOX4 trans-activates the transcription of <i>SEMA3</i>, <i>Plexin</i>, and <i>NRP1</i>.

<p>(A) representative end-point PCR of chromatin immunoprecipitation (ChIP) using an antibody against SOX4 in PANC-1 cells. Genomic DNAs were amplified with primer sets after bound proteins were digested with proteinase K. The ChIP results showed binding of SOX4 to the genomic region harboring SOX4 binding sites in the predicted promoter of most <i>SEMA3</i> and <i>Plexin</i> genes. (B) quantification of ChIP results in (A), n> = 3. Each lane in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048637#pone-0048637-g003" target="_blank">figure 3A</a> was given a value measured by densitometry and normalized with the value of IgG. ChIP efficiency is defined as percentage of the value derived from PCR of SOX4-immunoprecipitate relative to that of total genomic input. <i>Error bars</i>, standard deviation (SD) from at least triplicate. (C) increased luciferase activity of constructs containing <i>SEMA3</i>- or <i>Plexin</i>- promoter in the absence (white bars) or presence (gray bars) of co-expressed SOX4 in comparison with the promoterless pGL3-Basic control. The constructs harboring a SOX4-consensus binding site were transiently transfected into SOX4-containing HeLa cells. Shown are ratios of firefly luciferase expression to Renilla luciferase expression (expressed from co-transfected plasmid), measured 48 h after transfection, normalized to the mean ratio from the pGL3-Basic. Error bars show SD, n> = 4. Student's <i>t</i>-test, *, <i>P</i><0.01; **, <i>P</i><0.001. In the presence of co-transfected SOX4, luciferase activity driven by each <i>SEMA3</i>- or <i>Plexin</i>- promoter is further augmented (gray bars) compared to mock-transfected (white bars) and controls. (D) Transcriptional activity of <i>PLXNA2</i> promoter driven by SOX4 is attenuated when the consensus SOX4-binding sites are mutated. Boxed DNA sequence, the consensus SOX4-binding site; Underline with shadow in letters, the mutated SOX4-binding sites. Bar graph shows that mutation of either SOX4-binding site caused significant decrease in luciferase activity driven by <i>PLXNA2</i> promoter in the presence of overexpressed SOX4. Error bars, SD, n = 5. Student's <i>t</i>-test, *, <i>P</i><0.01; **, <i>P</i><0.001. (E) EMSA shows that the migration of a radiolabeled DNA fragment derived from the <i>PLXNA2</i> promoter is retarded by SOX4-containing HeLa crude nuclear extract in a concentration-dependent manner. The signal of protein-bound retarded band (arrowheads) was specifically diminished by cold probes containing SOX4-binding consensus sequences and was super-shifted by anti-SOX4 antibody (empty arrowhead). Asterisk, non-specific binding signals; arrow, un-bound radiolabeled probe. (F) effective RNAi knockdown of endogenous SOX4 in PANC-1 cells shown by SOX4 immunoblotting. The value indicated is the normalized relative intensity measured by densitometry (The ratio SOX4/Tubulin for scrambled-siRNA cells is defined as 1.). Two stable siSOX4 clones labeled as siSOX4#1 and siSOX4#2 were constructed with the targeting oligonucleotide directed against different nucleotide sequences in <i>SOX4</i> gene (siSOX4#1, nt.1999–2019 NM_003107.2; siSOX4#2, nt.1362–1382 NM_003107.2). (G) Immunoblotting in SOX4-depleted cells shows a concomitant decrease in the protein amount of NRP1, SEMA3- and Plexin- family members. Tubulin was used as a standard for normalization in the quantification of each lane measured by densitometry. The ratio indicated was the normalized value in siSOX4 cells relative to the normalized value in scrambled-siRNA cells. (H) Real-time PCR to quantify mRNA expression of <i>SEMA3</i> and <i>PLXN</i> genes in SOX4-depleted cells relative to SOX4-abundant scrambled-siRNA cells (shown by fold-change). Student's <i>t</i>-test, *, <i>P</i><0.01; **, <i>P</i><0.001.</p

Co-repressed miR-129-2 and miR-335 are associated with expression of SOX4, which correlates with shorter survival in patients with pancreatic cancer.

<p>(A) time-course change of ERK1/2 phosphorylation and the cytoplasm-nucleus distribution of SOX4 after PD98059 treatment. The whole cell lysates at the indicated time after treatment with 20 µM PD98059 were subjected fractionation, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. At 40 min after PD98059 treatment when ERK1/2 phosphorylation was inhibited, no difference was observed in the total amount of SOX4 protein, or the cytoplasm-nuclear distribution of SOX4. (B) <i>Left</i> and <i>Central</i> plots: Strong inverse correlation between miR-129-2 expression/miR-335 expression and SOX4 expression in PDAC cases as analyzed by Pearson correlation with log transformation for normality in scatter plots. n, the number of cases analyzed; Negative value in R indicates inverse correlation between X- and Y-variables. <i>Right</i> plot: the expression level of miR-335 in pancreatic carcinoma samples is positively correlated with the level of miR-129-2 in a linear regression way. (C) Marginal effect on suppressing SOX4 expression by transient transfection of miR-129-2 in PANC-1 cells as assessed by quantitative real-time PCR analysis in triplicate using a paired <i>t</i>-test. (D) Kaplan-Meier curves were shown as a function of SOX4 or E2F1 immunohistochemistry. <i>Left</i>: SOX4 expression in human PDAC correlates with shorter patient survival. <i>Right</i>: E2F1 expression did not correlate with patient survival. The <i>P</i>-value corresponds to the log-rank test by comparing the survival curves.</p

Reduced <i>in vitro</i> and <i>in vivo</i> tumor growth by SOX4 suppression.

<p>(A) Cell proliferation is affected in PANC-1 cells with RNAi-suppression of SOX4. 30000 cells were seeded into each well in a 12-well plate. At 24, 48 or 72 hours after seeding, cells were harvested and counted by trypan blue exclusion assay. <i>Error bars</i>, SD from six independent experiments, Student's <i>t</i>-test. (B) Representative flow cytometry of siSOX4 cells stained with propidium iodide shows no significant variation from scrambled-siRNA cells in apoptosis (depicted by sub-G1) or in cell cycle progression. The percentage of cells in sub-G1, G0/G1, S and G2/M phase was shown at the right downward corner of each plot. (C) representative TUNEL-labeling of cells challenged by the genotoxic agent cisplatin (10 µg/ml, 12-h). Differences in apoptosis was not observed between SOX4-knockdown (siSOX4#1 and siSOX4#2) and control (scrambled-siRNA) cells. (D) lower cell proliferation rate and decreased number of cells staying at M phase in siSOX4 cells. siSOX4 and scrambled-siRNA cells were plated on coverslips and incubated for 48 hours. Before harvest and fixation with paraformaldehyde, cells were incubated with 10 µM of BrdU for 3 hours and were then subjected to anti-Ki-67 or anti-BrdU immunofluorescent staining. Twenty randomly selected high power fields (x400) were counted for statistical analysis (Student's <i>t</i>-test). (E) Tumor xenograft of siSOX4/PANC-1 cells grows smaller than that of control cells (scrambled-siRNA) in a SCID mouse. The mice were sacrificed 30 days after subcutaneous inoculation of the control cells on the left and the siSOX4 cells on the right side of the flank. (F) Smaller and lighter tumor xenografts of siSOX4 cells compared to those of control (n = 9, Student's <i>t</i>-test). (G) representative H&E, Ki-67, PHH3 immunostain, and TUNEL labeling in tissue sections from tumor xenografts. Scale bars = 50 µm. (H, I, and J) quantification of proliferation (Ki-67 labeling), apoptosis (TUNEL stain) and mitosis (PHH3-immunostain). Eighteen randomly selected fields (x200) in each Ki-67-stained section and cells in twenty-seven randomly selected fields (x400) for TUNEL labeling or PHH3 immunostain were counted in each xenograft tumor. The proliferating index and the mitotic counts of siSOX4 tumor cells are significantly lower than that of control cells, while the percentage of apoptotic cells did not differ (Student's <i>t</i>-test). n, the total number of cells counted.</p