Psoriasin promotes invasion, aggregation and survival of pancreatic cancer cells; association with disease progression

Abstract

Psoriasin (S100A7) is an 11-kDa small calcium binding protein initially isolated from psoriatic skin lesions. It belongs to the S100 family of proteins which play an important role in a range of cell functions including proliferation, differentiation, migration and apoptosis. Aberrant Psoriasin expression has been implicated in a range of cancers and is often associated with poor prognosis. This study examined the role of Psoriasin on pancreatic cancer cell functions and the implication in progression of the disease. Expression of Psoriasin was determined in a cohort of pancreatic tissues comprised of 126 pancreatic tumours and 114 adjacent non-tumour pancreatic tissues. Knockdown and overexpression of Psoriasin in pancreatic cancer cells was performed using specifically constructed plasmids, which either had anti-Psoriasin ribozyme transgene or the full length human Psoriasin coding sequence. Psoriasin knockdown and overexpression was verified using conventional RT-PCR and qPCR. The effect of manipulating Psoriasin expression on pancreatic cancer cell functions was assessed using several in vitro cell function assays. Local invasive pancreatic cancers extended beyond the pancreas expressed higher levels of Psoriasin transcripts compared with the cancers confined to the pancreas. Primary tumours with distant metastases exhibited a reduced expression of Psoriasin. Psoriasin overexpression cell lines exhibited significantly increased growth and migration compared to control cells. In addition, Psoriasin overexpression resulted in increased pancreatic cancer cell invasion which was associated with upregulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. Overexpression of Psoriasin also promoted aggregation and survival of pancreatic cancer cells when they lost anchorage. Taken together, higher expression of Psoriasin was associated with local invasion in pancreatic cancers. Psoriasin expression is associated with pancreatic cancer cell growth, migration, cell-matrix adhesion, and invasion via regulation of MMPs. As such, the proposed implications of Psoriasin in invasion, disease progression and as a potential therapeutic target warrant further investigation.

Figure 1

Expression of Psoriasin in pancreatic cancer and pancreatic cancer cell lines with altered Psoriasin expression. (A) Reduced expression of Psoriasin was seen in a cohort of 45 pancreatic ductal adenocarcinomas in comparison with its expression in the paired adjacent normal pancreatic tissues (GDS4336) (26). (B) An elevated expression of Psoriasin was seen in metastases of pancreatic cancer compared with its expression in primary tumours (GSE71729) (25). (C) Psoriasin and RAGE transcripts were detected in PANC-1, AsPC-1 and MIA-PaCa-2 cell lines using conventional PCR. Knockdown and overexpression of Psoriasin were performed using anti-Psoriasin ribozyme transgenes and a recombinant plasmid vector carrying full-length human Psoriasin coding sequence, respectively. The altered expression of Psoriasin in the transfected pancreatic cancer cell lines (MIA-PaCa-2 and PANC-1) was verified using conventional PCR (D) and real-time PCR (E). ^*p<0.05, ^**p<0.01.

Figure 2

Effect of Psoriasin on proliferation and migration of pancreatic cancer cell lines. The influence on cell proliferation was determined over a period up to five days. Impact on in vitro proliferation was determined for MIA-PaCa-2 (A) and PANC-1 (B) cell lines a colorimetric method. The migration of pancreatic cancer cell lines were measured using ECIS for MIA-PaCa-2 (C) and for PANC-1 (D). Six repeats were included for each cell line and three independent experiments were performed. Growth rate was calculated against the corresponding day 0 control. ^*p<0.05, ^**p<0.01.

Figure 3

Influence on adhesion of pancreatic cancer cell lines. (A) Adhesion of MIA-PaCa-2 cells was altered by the knockdown and overexpression of Psoriasin. (B) Impact on cell adhesion of PANC-1 cell by the differential expression of Psoriasin. (C) Adhesion of PANC-1 cells to the peritoneal mesothelial cells. Adhesion rate was calculated based on the number of cells seeded. Six repeats were included for each cell line and three independent experiments were performed. ^*p<0.05.

Figure 4

Psoriasin and invasion of pancreatic cancer cell lines. The invasion of MIA-PaCa-2 (A) and PANC-1 (B) cell lines were determined using a Transwell invasion assay in triplicates. (C) Expression of MMP-2, p27^Kip1, cyclin D2 and caspase-3 was detected in MIA-PaCa-2 cells with differential expression of Psoriasin using conventional PCR. (D) Active MMP-2 and MMP-9 were determined using gelatine zymography. Invasion rates were calculated against corresponding control in which the same number of cells were seeded at beginning of the experiments. Three independent experiments were performed. ^*p<0.05, ^**p<0.01.

Figure 5

Psoriasin and aggregation of pancreatic cancer cells. Aggregation of MIA-PaCa-2 cells was determined over a duration ≤3 h. Three independent experiments were performed. ^*p<0.05.

Figure 6

Psoriasin and anoikis of pancreatic cancer cells. Apoptosis of suspension MIA-PaCa-2 cells with altered expression of Psoriasin was determined using a flow cytometric method.

Figure 7

Caspase 3 in MIA-PaCa-2 cells were detected using western blot analysis.