Contribution of microRNAs in understanding the pancreatic tumor microenvironment involving cancer associated stellate and fibroblast cells

Abstract

Understanding of molecular events associated with tumor microenvironment in pancreatic cancer (PC) is an active area of research especially because of the rich desmoplasia seen in human PC. Desmoplasia is contributed by several cell types including cancer-associated fibroblast (CAF) and stellate cells (PSCs), which are believed to play critical roles in conferring aggressiveness to PC. The aberrant expression of microRNAs (miRNAs) in PSCs and CAF cells appears to play a pivotal role in the development and progression of PC. In this study, expression analysis of miR-21/miR-221 in conditioned media derived from PSCs/CAF cells, and from PSCs/CAF cells showed up-regulation of both miRNAs compared to MIAPaCa-2 PC cells. In addition, miR-21 expression in stellate cells derived from normal pancreas was substantially lower when compared to PSCs or CAF cells. COLO-357 PC cells cultured in the presence of conditioned media derived from PSC/CAF cells led to a significant increase in clonogenicity and pancreatosphere formation. Furthermore, inhibition of miR-21 with antisense oligonucleotide (ASO) transfection resulted in decreased migration/invasive capacity of PSCs. Similarly, the effect of ASO-miR-221 transfection in CAF cells reduced the expression of NF-κB and K-Ras (target of miR-221) along with inhibition of migration/invasion. Moreover, miRNA expression profiling of PSCs, MIAPaCa-2, and COLO-357 cells, and further validation by real-time PCR, showed several differentially expressed miRNAs, among which four was significantly up-regulated. Collectively, these results suggest a crosstalk between PSCs/CAF cells and PC cells, resulting in the up-regulation of miR-21/miR-221 expression which in part may confer aggressiveness to PC. We conclude that targeting these miRNAs could be useful for developing precision medicine for the prevention of tumor progression and/or for the treatment of PC.

Keywords: CAF cells; Stellate cells; exosomes; miRNA; pancreatic cancer.

Figure 1

Comparative expression of miR-21 (A) and α-smooth muscle actin (α-SMA) (B) in human PC cell line MIAPaCa-2, cancer associated fibroblast (CAF-19) and four cancer-associated stellate cells (PSCs). There was a significant up-regulation in the expression of miR-21 in all four PSCs tested followed by CAF-19 compared to MIAPaCa-2 cells. RNU48 was used as control miRNA. As expected, activation marker α-smooth muscle actin (α-SMA) was expressed in CAF-19 and in all four PSCs tested, but not in MIAPaCa-2 cells. p values represent comparison between MIAPaCa-2 with CAF-19 and PSCs. **≤ 0.005 and NS = non-significant.

Figure 2

Effect of co-culture of PC cell line COLO-357 with conditioned media derived from PSCs (A) and CAF-19 cells (B). There was a significant increase in colony formation of PC cells cultured with conditioned media derived from both PSCs and CAF-19 cells compared to untreated COLO-357 cells (A and B). Similarly there was an increase in pancreatosphere formation of PC cells cultured with conditioned media derived from both PSCs and CAF-19 cells compared to untreated COLO-357 cells (A and B). p values represent comparison between control cells and cells treated with conditioned media. **≤ 0.01 and NS = non-significant.

Figure 3

Comparative expression of miR-21 in the conditioned media derived from nhPSC and PSCs/CAF-19, and the cells (PSCs, CAF-19 cells and PC cell line MIAPaCa-2). There was a significant up-regulation in the expression of miR-21 in both RNA preparation from intact cells and conditioned media derived from PSCs, followed by CAF-19 cells and MIAPaCa-2 cells compared to all three nhPSCs. RNU48 was used as control miRNA. p values represent data from two different experiments in triplicate and was compared against nhPSCs. **≤ 0.0001.

Figure 4

Treatment of PSCs with ASO miR21, led to a significant reduction in the expression of miR-21 as assessed by qRT-PCR (A), decreased cell migration (B), and decreased cell invasion (C) as assessed by chamber cell invasion assays. p values represent comparison between control ASO and ASO-miR-21. **≤ 0.007 and *≤ 0.05.

Figure 5

Relative expression of miR-221 was compared in MIAPaCa-2, CAF-19 and PSCs both from cells and conditioned media (A), inactivation of miR-221 expression by ASO in CAF-19 cells led to reduced expression of miR-221 as assessed by qRT-PCR (B), decreased K-Ras and NF-κB mRNA expression assessed by qRT-PCR (C), decreased cell migration and invasion of cells by chamber cell invasion assays (D), and decreased K-Ras and NF-κB protein expression as assessed by western blot analysis and were quantified against β-actin (E) Controls used are: for miRNA (RNU48), mRNA (GAPDH), and protein (β-actin). p values represent comparison against MIAPaCa-2 in 5A, comparison against control ASO in 5B-E. **≤ 0.001, *≤ 0.01 and NS = non-significant.

Figure 6

Comparative expression of miR-99a-5p, miR-100-5p, miR-125b-5p, and miR-4488 in MIAPaCa-2, COLO-357, CAF-19, and Ca-hPSC 4 by qRT-PCR. There was a significant up-regulation in the expression of miR-99 and miR-100 in Ca-hPSC-4 compared to CAF-19 and both PC cell lines. The other two miRNAs miR-125 and miR-4488 showed significant up-regulation in both CAF-19 and Ca-hPSC4 compared to both PC cell lines. p values represent comparison between PC cells and Ca-hPSC 4. **≤ 0.0001.

Figure 7

A schematic representation of the tumor microenvironment involving activated PSCs, CAF cells and their interactions with PC cells, EMT, miRNAs and hypoxia-all of which may lead to tumor progression, survival and metastases (tumor aggressiveness).