Integration of Bioinformatics Resources Reveals the Therapeutic Benefits of Gemcitabine and Cell Cycle Intervention in SMAD4-Deleted Pancreatic Ductal Adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer. The five-year survival rate of PDAC is very low (less than 8%), which is associated with the late diagnosis, high metastatic potential, and resistance to therapeutic agents. The identification of better prognostic or therapeutic biomarker may have clinical benefits for PDAC treatment. SMAD4, a central mediator of transforming growth factor beta (TGFβ) signaling pathway, is considered a tumor suppressor gene. SMAD4 inactivation is frequently found in PDAC. However, its role in prognosis and therapeutics of PDAC is still unclear. In this study, we applied bioinformatics approaches, and integrated publicly available resources, to investigate the role of SMAD4 gene deletion in PDAC. We found that SMAD4 deletion was associated with poorer disease-free, but not overall, survival in PDAC patients. Cancer hallmark enrichment and pathway analysis suggested that the upregulation of cell cycle-related genes in SMAD4-deleted PDAC. Chemotherapy response profiling of PDAC cell lines and patient-derived organoids revealed that SMAD4-deleted PDAC was sensitive to gemcitabine, the first-line treatment for PDAC, and specific cell cycle-targeting drugs. Taken together, our study provides an insight into the prognostic and therapeutic roles of SMAD4 gene deletion in PDAC, and SMAD4 gene copy numbers may be used as a therapeutic biomarker for PDAC treatment.

Keywords: SMAD4; bioinformatics; cell cycle; in silico; pancreatic ductal adenocarcinoma.

Figure 1

Copy number amplification of SMAD4 gene in pancreatic ductal adenocarcinoma (PDAC). (a) SMAD4 gene was analyzed for gene alterations (mutation status and copy number variation) in various cancer types using “TCGA, PanCancer Atlas” data in the cBioPortal cancer genomics database; (b) A bar code plot for the comparison of SMAD4 gene alterations and mRNA expression in PDAC; (c) A cartoon diagram for the gene and protein structures of SMAD4. MH1 (the Mad homology 1) region is sequence-specific DNA binding domain. MH2 region is responsible for heteromerization and transactivation. This figure was adapted from the image obtained from the cBioPortal website (http://www.cbioportal.org/); (d) A scatter plot for the comparison of SMAD4 copy number amplification and mRNA expression in PDAC. WT, wildtype. Diploid, two alleles present; Gain, low-level gene amplification event; Mut, mutation; (e) The impact of SMAD4 gene alterations on disease-free survival of PDAC patients.

Figure 2

Upregulation of cell cycle-related genes in SMAD4-deleted PDAC. (a) Gene set enrichment analysis (GSEA) for the over- and under-expressed genes in SMAD4-deleted PDAC patients was performed using the WebGestalt online tool; (b) Kyoto Encyclopedia of Genes and Genomes (KEGG) cell cycle pathway mapping was performed using the WebGestalt online tool. The official gene symbols for the mapped genes were annotated and highlighted in red (over-expressed) and blue (under-expressed). No under-expressed genes were enriched in this pathway.

Figure 3

Sensitive and resistant drugs in SMAD4-deleted PDAC cell lines. (a) Chemosensitivity profiles, correlated with SMAD4 copy numbers in PDAC cell lines, were obtained from the CTRP database via an online tool, the CellMinerCDB; (b) The effect of drugs on cell cycle progression.

Figure 4

SMAD4 deletion correlated with an increase in gemcitabine sensitivity in PDAC. (a) The correlations between SMAD4 gene copy numbers (left part)/mRNA expression (right part) and gemcitabine drug activity were obtained from the CTRP database via an online tool, the CellMinerCDB; (b) PDAC cell lines were treated with gemcitabine for 72 h, and then cell viability was determined by an MTT assay. The SMAD4 mutation status was obtained from the CCLE database (missence mutation and frameshift insertion in AsPC-1, and HPAC cells, respectively). BxPC-3 cells harbored a homozygous deletion of SMAD4 gene [16]. The values for SMAD4 copy number variation (CNV) and expression (EXP) were obtained from the CellMinerCDB database (CTRP-Broad-MIT data); (c) Chemosensitivity profiles for PDAC patient-derived organoids were obtained from a previous study. Values shown are the area under the curve (AUC) for each drug. Higher or lower AUC (area under the curve) indicated less or more responsive to each drug, respectively.

Figure 5

Gemcitabine-sensitive genes were closely associated with CDK1 and SMAD4 gene deletion in PDAC. (a) The network between CDK1 and gemcitabine-sensitive genes was constructed using the STRING database; (b) GSEA was performed to analyze the correlation between gemcitabine-sensitive/resistant genes and SMAD4 gene expression.