Inflammatory and Senescent Phenotype of Pancreatic Stellate Cells Induced by Sqstm1 Downregulation Facilitates Pancreatic Cancer Progression

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has unique microenvironment with extensive infiltration of fibroblasts, which are mainly derived from the resident pancreatic stellate cells (PaSCs). As activated PaSCs constitute a major contributor to pancreatic cancer progression, the mechanisms underlying their activation have been being intensively studied. Previous studies showed that Sequestosome-1 (sqstm1) can modulate the functional status of fibroblasts in cancer. Here, we further delineated the role of sqstm1 in PaSCs. The analysis of PDAC patient samples revealed reduction of sqstm1 expression in activated PaSCs in both mRNA and protein level. Downregulated sqstm1 via shRNA in PaSCs led to an inflammatory and senescent phenotype with increased IL8, CXCL1, and CXCL2 expression. Further analysis demonstrated that increased intracellular reactive oxygen species level contributed to the senescence in sqstm1-downregulated PaSCs. This was mediated via impaired NRF2 activity since reduced sqstm1 resulted in accumulation of KEAP1. Meanwhile, we found that sqstm1 degradation caused by enhanced autophagy was not associated with transformation of senescent phenotype. At last, the data revealed that sqstm1-downregulated PaSCs promoted pancreatic tumor cell growth, invasion, and macrophage phenotype transformation. Collectively, the current study indicated that sqstm1 controlled transformation of senescent phenotype of PaSCs, which in turn is pro-tumorigenic.

Keywords: pancreatic adenocarcinoma; pancreatic stellate cells; reactive oxygen species; senescence; sqstm1.

Figure 1

Sqstm1 expression was downregulated in cancer-associated fibroblasts (CAFs) of pancreatic cancer. (A) Representative images of immunohistochemistry (IHC) staining for sqstm1 and LC3 in pancreatic cancer tissues. The table shows expression patterns of sqstm1 in tumor and stromal compartment based on IHC staining. Scale bars represent 50 μm. (B) Representative images of double immunofluorescence staining for sqstm1 and α-SMA in pancreatic cancer tissues. Scale bars represent 50 μm. (C) Fluorescence activated cell sorting (FACS) for EpCAM⁺ and FAP⁺ cells and qPCR examining sqstm1 mRNA level for sorted cells. ^* p < 0.05. (D) Examination of sqstm1 mRNA expression by qPCR for pancreatic cell line and cultured primary CAFs line using β-actin and GAPDH as internal control, respectively. (E) Western blot analysis for sqstm1 for pancreatic cell line and cultured primary CAFs line. (F) Representative image of immunofluorescence staining for α-SMA in one cultured CAFs line was showed. Scale bars represent 20 μm. (G) qPCR for sqstm1 in pancreatic stellate cells (PaSCs) following retinoid acid treatment. (H) Western blot analysis for α-SMA in PaSCs following retinoid acid treatment. ^** p < 0.01, ^**** p < 0.0001.

Figure 2

Sqstm1 downregulation induces inflammatory and senescent phenotype of PaSCs. (A) Validation of sqstm1 downregulation following shRNA transfection by western blot in PaSCs. (B) Gene microarray for comparison of transcriptome between negative control and shRNA-transfected PaSCs. (C) Validation of expression change for IL8, CXCL1, and CXCL2 by qPCR. (D) Flow cytometry analysis for FAP expression in negative control and shRNA-transfected PaSCs. (E) β-galactosidase staining for senescence analysis. ^** p < 0.01, ^*** p < 0.001.

Figure 3

Autophagy induces PaSCs activation but not senescence. (A) Western blot analysis for markers of autophagy and fibroblasts activation in PaSCs upon autophagy induction. (B) qPCR analysis for IL8, CXCL1, and CXCL2 in PaSCs upon autophagy induction. (C) β-galactosidase staining for PaSCs upon starvation or rapamycin treatment. Scale bar represents 20 μc.

Figure 4

Sqstm1 regulates ROS balance through KEAP1/NRF2 signaling. (A) ROS measurement via DCFDA probe detection. (B) Examination of IL8, CXCL1, and CXCL2 mRNA level upon NAC treatment in PaSCs. (C) Western blot analysis for KEAP1 and NRF2 in sqstm1-shRNA transfected PaSCs. (D) Immunofluorescence for NRF2 in sqstm1-downregulated PaSCs. Scale bar represents 10 μm. (E) qPCR for Nqo1 between negative control and sqstm1-shRNA transfected PaSCs. (F) Validation of NRF2 expression change upon siRNA transfection in PaSCs by qPCR. (G) β-galactosidase staining for PaSCs upon transfection of NRF2-siRNA. Scale bar represents 20 μm. ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001.

Figure 5

Sqstm1-downregulated PaSCs promotes tumor growth and progression. Comparison of proliferation (A) and invasion (B) of Panc-1 cells upon treatment with conditioned medium (CM) from PaSCs with or without sqstm1 shRNA transfection. (C) Comparison of CD206 expression in monocytes treatment with CM from PaSCs with or without sqstm1 shRNA transfection. (D-E) In vivo tumor xenograft of Panc-1 co-injected with PaSCs or sqstm1-shRNA transfected PaSCs. IHC staining for α-SMA and Ki-67 was analyzed. ^* p < 0.05, ^*** p < 0.001.