Transcriptional splice variants of CD40 and its prognostic value in breast cancer

- Transcriptional splice variants of CD40 and its prognostic value in breast cancer.
- It is expressed on the surface of B cells, macrophages,and dendritic cells, as well as in various cancer cell lines and tissues including non-small cell lung cancer, ovarian, urinary bladder cancer, breast melanoma, pancreatic cancer, colon cancer, and B-lineage malignancies (Ara et al., 2018.
- Argiriadi et al., 2019.
- The interaction between CD40 and its ligand, CD40 ligand (CD40LG), orchestrates dendritic cell–T cell crosstalk, dendritic cell tolerance, and conversion of CD8 + T cell exhaustion (Ara et al., 2018).
- Agonistic antibodies against CD40 display T cell dependent antitumor activity, especially when used in combination with chemotherapy and immune checkpoint inhibitors (Bonaventura et al., 2019)..
- CD40 expression is induced by lipopolysaccharides (LPS) at the transcriptional level via nuclear factor kappa B (NF- κB) and signal transducer and activator of transcription 1 alpha (STAT-1α) in macrophages and microglia (Qin et al., 2005).
- Type 3 variant lacking the cysteine-rich subunit-3/4 (CRD-3 and CRD-4) display low ligand-binding affinity (Tone et al., 2001)..
- CD40–CD40LG interaction stimulates growth inhibition in ovarian, bladder, ovarian, squamous epithelial carcinoma, and cervical cancer cell lines (Tong et al., 2001)..
- Agonistic anti-CD40 antibodies either directly activate macrophages, dendritic cells, or B cells, or indirectly activate them through cytotoxic lymphocytes and natural killer cells (Baxendale et al., 2005).
- CD40 pathway activation is linked with an improved pathologic response to preoperative trastuzumab-plus-T/FEC chemotherapy in breast tumors (Bonaventura et al., 2019).
- Ligation of the trimeric recombinant protein (rCD40LG) with CD40 leads to growth inhibition of breast cancer cells (Slobodova et al., 2011;Gomes et al., 2009)..
- It is broadly expressed on the surface of immune cells and in diverse cancer types, including breast cancer.
- Here, we analyzed both CD40/CD40 ligand expression in breast cancer cells and tissues using public data sets and overall survival analysis in ungrouped breast cancer patients, as well as in the triple-negative breast cancer subtype.
- We detected CD40 gene expression along with its 3 different splice variants (variants 1–3), predominantly in the triple-negative subgroup of breast cancer cell lines.
- The results of the overall survival analysis showed that high CD40 gene expression, particularly in the triple-negative subgroup of breast cancer patients, is associated with better survival.
- In addition to the transcriptional levels of CD40 splice variants, investigation of protein levels of these variants will allow the categorization of breast cancer cells and reveal their potential as an immunotherapeutic target..
- ÜNVER et al.
- CD40 is correlated with a better prognosis in breast cancer (Slobodova et al., 2011).
- Apart from CD40 expression, membrane CD40LG expression has been rarely detected in tumor-infiltrating lymphocytes in breast cancer tissues, supporting their low capacity to inhibit growth of breast cancer cells via the CD40–CD40LG axis (Tong et al., 2001)..
- In contrast, its expression is correlated with an unfavorable patient prognosis in soft tissue sarcomas (Baxendale et al., 2005.
- Ottaiano et al., 2004).CD40 has been associated with a poor prognosis for stages 3 and 4 esophageal squamous cell carcinoma patients as well (Matsumura et al., 2016)..
- In this study, we showed the transcriptional variants of the CD40 gene in different breast cancer cell lines.
- Cell culture:BT-474, HCC-38, MCF-7, MDA-MB-231, MDA-MB-468, SK-BR-3, T-47D, ZR-75-1 breast cancer cell lines and MCF-12A healthy mammary epithelial cell line were cultured in RPMI medium containing L-glutamine (2 mM), 10% fetal bovine serum (Biological Industries, Cromwell, CT, USA), penicillin (100 unit/mL) and streptomycin (100 mg/mL.
- data of the CD40 gene from all of the cancer cell lines were downloaded and a total of 66 breast cancer cell lines were selected.
- Survival analysis: CD40 and its cognate receptor CD40L Gwere evaluated with Kaplan–Meier plotter tool (http://kmplot.com/analysis/index.php)(Gyorffy et al., 2010).
- High CD40 expression is associated with a good prognosis in breast cancer.
- Prognostic value of CD40 gene expression was determined in breast cancer using the Kaplan–Meier plotter.
- Likewise, high CD40LG expression was associated with better survival in all breast cancer (hazard ratios of P = 3.9e-05) and triple negative breast cancer (hazard ratios of P = 0.0018) data set (Figure 1c and Figure 1d)..
- Breast cancer cell lines express CD40 at mRNA levels Gene expression data for cancer cell lines were downloaded from the Cancer Cell Line Encyclopedia (CCLE) database (Barretina et al., 2012) at www.broadinstitute.org/ccle..
- RNA-seq analysis of 66 human breast cancer cell lines derived from the CCLE data setshowed that breast cancer cells display low CD40 expression (Figure 2a).
- Data analysis of these breast cancer cell lines revealed extremely low CD40LG expression as well (Figure 2b)..
- Demonstration of CD40 gene expression with its splice variants.
- Six out of 8 breast cancer cell lines expressed CD40 variant/.
- a) Survival curves for all patients with breast cancer (n = 3951).
- b) Survival curves for patients with triple negative breast cancer (n = 255).
- c) Survival curves for all patients with breast cancer (n=3951).
- D) Survival curves for patients with triple negative breast cancer (n=255)..
- a) RNA-seq data analysis of CD40 mRNA in breast cancer cell lines (n=66).
- b) RNA-seq data analysis of CD40LG mRNA in breast cancer cell lines (n=66).
- c) Correlation between CD40 and CD40LG gene expression based on the TCGA breast cancer data set..
- Similarly, MCF-12A normal breast cells showed a similar transcriptional pattern as these breast cancer cells for CD40 splice variants.
- a) Schematic demonstrations of CD40 splice variants.
- d) Expression of CD40 splice variants in breast cancer cell lines cultured with or without LPS.
- immune system, coexpression of CD40 and its ligand CD40L mediates oncogenic effects on immortalized human epithelial cells in vitro (Baxendale et al., 2005)..
- Early phase trials regarding CD154 gene transfer have shown various cellular consequences, including cytokine production and enhanced Fas-mediated tumor apoptosis, following CD40 ligation(Weiss et al., 2009;Tong and Stone, 2003)..
- CD40 expression exists on the basal part of stratified squamous epithelium, suggesting its modulatory role in cell growth and differentiation (Young et al., 1998)..
- In murine fibroblasts, constitutive CD40 signaling plays a pivotal role in the transformation of the cells (Esteva et al., 2007).
- However, its ligand CD40LG has barely been detected in normal and malignant epithelial cells (Tong et al., 2001.
- CD40 has been detected in all compartments, namely membrane, cytoplasm, and nucleus, in breast cancer (Makhoul et al., 2018).
- Although Her-2/neu breast tumors show significantly higher cytoplasmic expression of CD40 compared to the Luminal A/B type, its expression is higher on the membrane surface of MDA-MD-231, a highly aggressive TNBC cell line (Makhoul et al., 2018)..
- According to the data analysis, breast cancer cell lines (n=66) displayed extremely low CD40LG expression, indicating that the main source of CD40LG expression in breast cancer could be immune cells, most likely on the cell surface of the activated T cells..
- Higher CD40 expression has been associated with a poor prognosis in gastric cancer, esophageal squamous cell carcinoma, low-grade gliomas, and glioblastoma (GBM)(Li et al., 2009.
- Matsumura et al., 2016.
- Werner et al., 2019).
- 0.045 (Mu et al., 2015).
- Since soluble CD40 expression would suppress CD40LG + T cells and contribute to immune escape mechanisms (Kim et al., 2015), it is critical to explore this splice variant in cancer cells..
- Overall, the survival analysis that we performed, in contrast to the studies on solid tumors mentioned earlier, has shown that high CD40 expression is associated with good prognosis in triple-negative breast cancer.
- Similarly, tumor-associated CD40 expression has a favorable prognostic effect in renal cell carcinoma and diffuse large B-cell lymphoma following chemotherapy (Weiss et al., 2014;Rydstrom et al., 2010).
- CD40 expression is accompanied by CD8 T cell infiltration in renal cell carcinoma, indicating that it may be useful as a biomarker for patient survival (Weiss et al., 2014).
- 4 triple-negative breast cancer cell lines.
- In this study, we aimed to show CD40 splice variants under normal conditions, as well as in the presence of LPS, a strong transcriptional inducer of CD40 gene (Dong et al., 2008).
- Furthermore, the activation of NF-κB has been shown to be essential for CD40 expression stimulated with LPS on human monocytes, as determined in murine macrophages and microglia (Pearson et al., 2001.
- Wu et al., 2009)..
- (Yu et al., 2017).
- In the earlier study, consistent with our results, BT-20 was also identified as CD40-positive, while MCF- 7 and ZR-75-1 were CD40-negative (Esteva et al., 2007)..
- Treatment with CD40 ligand decreased proliferation of BT-20 and T-47D cells which are CD40-positive, but did not change the growth of CD40-negative breast cancer cell lines, as detected in MCF-7 or ZR-75-1 cells (Esteva et al., 2007), supporting the idea that CD40–CD40LG axis modulates proliferation of cancer cells.
- Interestingly, the ER + /PR + /HER2 + breast cancer cell line BT-474 does express Variant 3 in the presence of LPS.
- High CD40 expression was detected in the cytoplasm of hormone- receptor–positive breast cancer, whereas it was detected on the surface membrane of triple-negative breast cancer (Kim et al., 2015.
- Slobodova et al., 2011).
- When the demonstration of CD40 variant expressions is technically considered, it can be mentioned that not showing the variants by sequencing and not quantifying the expression differences of CD40 variants using quantitative real-time PCR is among the handicaps of the experimental assay.Therefore, no further quantitative evaluation of gene expression has been performed due to the absence of CD40 expression in some of the breast cancer cell lines and for the purpose of displaying the existence of CD40 variants (V1–3) only..
- In conclusion, investigation of CD40 splice variants’.
- expression among different types of breast cancer cells is.
- of CD40 mRNA.
- analysis of the genetic structure and functionality of CD40 and CD40LG would allow the development of more useful prognostic factors and prognostic prediction tools in breast cancer subtypes..
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