BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling

Objective Colorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-?B (NF-?B) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour cell survival. Design Immunohistochemistry was carried out on 47 tumour samples and normal tissue from resection margins. The role of BCL-3/NF-?B complexes on cell growth was studied in vivo and in vitro using an siRNA approach and exogenous BCL-3 expression in colorectal adenoma and carcinoma cells. The question whether BCL-3 activated the AKT/protein kinase B (PKB) pathway in colorectal tumour cells was addressed by western blotting and confocal microscopy, and the ability of 5- aminosalicylic acid (5-ASA) to suppress BCL-3 expression was also investigated. Results We report increased BCL-3 expression in human colorectal cancers and demonstrate that BCL-3 expression promotes tumour cell survival in vitro and tumour growth in mouse xenografts in vivo, dependent on interaction with NF-?B p50 or p52 homodimers. We show that BCL-3 promotes cell survival under conditions relevant to the tumour microenvironment, protecting both colorectal adenoma and carcinoma cells from apoptosis via activation of the AKT survival pathway: AKT activation is mediated via both PI3K and mammalian target of rapamycin (mTOR) pathways, leading to phosphorylation of downstream targets GSK- 3 and FoxO1/3a. Treatment with 5-ASA suppressed BCL-3 expression in colorectal cancer cells. Conclusions Our study helps to unravel the mechanism by which BCL-3 is linked to poor prognosis in colorectal cancer; we suggest that targeting BCL-3 activity represents an exciting therapeutic opportunity potentially increasing the sensitivity of tumour cells to conventional therapy

Alternative splicing of TIA-1 in human colon cancer regulates VEGF isoform expression, angiogenesis, tumour growth and bevacizumab resistance

© 2014 The Authors. The angiogenic capability of colorectal carcinomas (CRC), and their susceptibility to anti-angiogenic therapy, is determined by expression of vascular endothelial growth factor (VEGF) isoforms. The intracellular protein T-cell Intracellular Antigen (TIA-1) alters post-transcriptional RNA processing and binds VEGF-A mRNA. We therefore tested the hypothesis that TIA-1 could regulate VEGF-A isoform expression in colorectal cancers. TIA-1 and VEGF-A isoform expression was measured in colorectal cancers and cell lines. We discovered that an endogenous splice variant of TIA-1 encoding a truncated protein, short TIA-1 (sTIA-1) was expressed in CRC tissues and invasive K-Ras mutant colon cancer cells and tissues but not in adenoma cell lines. sTIA-1 was more highly expressed in CRC than in normal tissues and increased with tumour stage. Knockdown of sTIA-1 or over-expression of full length TIA-1 (flTIA-1) induced expression of the anti-angiogenic VEGF isoform VEGF-A 165 b. Whereas flTIA-1 selectively bound VEGF-A 165 mRNA and increased translation of VEGF-A 165 b, sTIA-1 prevented this binding. In nude mice, xenografted colon cancer cells over-expressing flTIA-1 formed smaller, less vascular tumours than those expressing sTIA-1, but flTIA-1 expression inhibited the effect of anti-VEGF antibodies. These results indicate that alternative splicing of an RNA binding protein can regulate isoform specific expression of VEGF providing an added layer of complexity to the angiogenic profile of colorectal cancer and their resistance to anti-angiogenic therapy

Detection of VEGF-A_xxxb Isoforms in Human Tissues

Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls

qRT-PCR using protocols shown in figure 2D and E can detect changes in splicing induced by splicing factor knockdown.

<p>A. C_tmax-C_t for cDNA extracted from prostate cancer (PC3) cells with lentiviral knockdown of SRPK1 or scrambled. B. Amount of VEGF calculated from standard curves in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068399#pone-0068399-g002" target="_blank">Figure 2</a>. C. Amount of VEGF-A₁₆₅b identified by Exon 8b primers (VEGF-A₁₆₅b) or that calculated from mispriming of VEGF-A₁₆₅a. D. Proportion of VEGF that is VEGF-A₁₆₅a or VEGF-A₁₆₅b in control and knockdown cells. Values are Mean±SEM (n = 2). 3E. qPCR for VEGF-A₁₆₅a on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar). 3F qPCR for VEGF-A₁₆₅b on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar).</p

Isoform specific PCR requires positive controls to ensure specificity.

<p>A. Sequence of the VEGF 3′ exon sequence. (i) Exon 7 (red) contains the same last three nucleotides (underlined) as the last three nucleotides of exon 8a (blue, underlined), requiring specific PCR primers that extend into exon 7 (arrow). (ii) mispriming (VEGF-A₁₆₅a -specific primers priming on VEGF-A₁₆₅b, and VEGF-A₁₆₅b -specific primers priming on VEGF-A₁₆₅a) can occur both ways round if the conditions are not tested. B. Published control PCR gels demonstrating specificity of primer conditions. The original description of VEGF-A₁₆₅b describing conditions at which VEGF-A₁₆₅b is not misprimed in the presence of 100ng VEGF-A₁₆₅a (lane highlighted by arrow), but still able to amplify 0.1ng VEGF-A₁₆₅b. C. Annealing temperature dependence of the specificity of the isoform specific primers. Only at >62°C is specificity resolved. D. qPCR using VEGF-A₁₆₅a specific primers on VEGF-A₁₆₅a and VEGF-A₁₆₅b plasmid E. qPCR using VEGF-A₁₆₅b specific primers on VEGF-A₁₆₅a and VEGF-A₁₆₅b plasmid.</p

Positive controls are required to interpret lack of amplification of VEGF-A₁₆₅b by competitive RT-PCR.

<p>A. Plasmids containing VEGF-A₁₆₅b or VEGF-A₁₆₅a sequence were amplified using primers in exon 8b and exon 7. Two different sized products were generated. B. Densitometric analysis of published RT-PCR gels using plasmids containing VEGF-A₁₆₅b and VEGF-A₁₆₅a and primers in exon 7 and exon 8b. 13/15 show higher intensity for VEGF-A₁₆₅a. p<0.001 paired t test. C. Example of failure of amplification of the VEGF-A₁₆₅b isoform. Two parallel PCR reactions were run on cDNA and plasmid DNA. On the first (at 4mM MgCl₂) no VEGF-A₁₆₅b was generated in the cDNA, or when both VEGF-A₁₆₅a and VEGF-A₁₆₅b plasmids were used as positive controls. In the second (at 5mM MgCl₂) VEGF-A₁₆₅b was generated from cDNA from PC3 cells where SRPK1 was knocked down, and when both templates were included. D. Example of failure of amplification of the VEGF-A₁₆₅b isoform in one PCR machine (and failure of template), but not in a second machine. E. Example of failure of amplification of VEGF-A₁₆₅b product after freeze-thawing of cDNA derived from fresh normal human lung fibroblasts. Chromatogram confirms VEGF-A₁₆₅b sequence from lower band from top gel from patient sample in lane 1 (chromatograms from the other samples also confirmed VEGF-A₁₆₅b sequence).</p

VEGF expression determined by Western blot and immunoprecipitation.

<p>A. Western blot using LiCor Odyssey to simultaneously image pan-VEGF and VEGF-A₁₆₅b probed western blot. Two different podocyte samples, and a primary RPE sample were run on a gel and probed with antibodies to VEGF-A₁₆₅b (mouse monoclonal anti-CTRSLTRKD, and 680nm-donkey anti-mouse, top image) and pan-VEGF (rabbit polyclonal anti-VEGF, and 800nm-donkey anti-rabbit, middle image). The bottom image is the pseudocoloured combined image (600nm green, 800nm red). Note the red VEGF₁₆₅, but yellow VEGF-A₁₆₅b. MWM = molecular weight marker. d = dimer, m = monomer. B. Protein extracted from human cell lines (adenoma and adenocarcinoma(AC)) subjected to immunoprecipitation (IP) for VEGF-A₁₆₅b and immunoblotting (IB) for total VEGF-A. A clear strong band was seen in the IP for both cell types at ∼23kDa and ∼46kDa, consistent with the IP for recombinant human VEGF-A₁₆₅b. A weaker band was seen in the input protein (not subjected to IP), and a second band slightly higher in the AC. A weak band at approximately 56kDa and 28kDa was seen in all lanes subjected to IP, including the VEGF-A₁₆₅a band, but not seen in the recombinant human VEGF-A₁₆₅b not subjected to IP, indicating that this is cross reactivity with the IgG. This band was clearly above the VEGF-A₁₆₅b bands. C. Protein extracted from human cell lines (adenoma and adenocarcinoma(AC)) subjected to immunoprecipitation (IP) for VEGF-A and immunoblotting (IB) for VEGF-A₁₆₅b. A clear strong band was seen in the IP for both cell types at ∼23kDa, the same size as recombinant human VEGF-A₁₆₅b. In the input a band at ∼46Da was seen predominantly, for both cell types, labelled as VEGF-A₁₆₅b dimers. D. Mouse tissues probed with VEGF-A₁₆₅b antibody detect mouse IgG due to the secondary antibody. Top image, western blot of mouse tissues, recombinant mouse IgG or human VEGF-A₁₆₅b or VEGF-A₁₆₅b probed with mouse anti-CTRSLTRKD, and 680nm-donkey anti-mouse IgG. Bottom image blot of same tissues, probed without primary antibody. The same bands are seen in the mouse tissues. Spl = spleen, Col = colon, Hrt = heart, Lng = lung, Liv = liver, Kid = kidney.</p

WT1 Mutants Reveal SRPK1 to Be a Downstream Angiogenesis Target by Altering VEGF Splicing

Angiogenesis is regulated by the balance of proangiogenic VEGF 165 and antiangiogenic VEGF 165b splice isoforms. Mutations in WT1, the Wilms' tumor suppressor gene, suppress VEGF 165b and cause abnormal gonadogenesis, renal failure, and Wilms' tumors. In WT1 mutant cells, reduced VEGF 165b was due to lack of WT1-mediated transcriptional repression of the splicing-factor kinase SRPK1. WT1 bound to the SRPK1 promoter, and repressed expression through a specific WT1 binding site. In WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF and rendered WT1 mutant cells proangiogenic. Altered VEGF splicing was reversed by wild-type WT1, knockdown of SRSF1, or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumor growth. © 2011 Elsevier Inc