Upregulated GDF-15 expression facilitates pancreatic ductal adenocarcinoma progression through orphan receptor GFRAL

Abstract

Growth and differentiation factor 15 (GDF-15) has been studied as an important hallmark of cancer. However, the receptor of GDF-15 in pancreatic cancer cell remains unclear. Here, we investigated its biological effects in pancreatic ductal adenocarcinoma (PDAC). We found that aberrant GDF-15 expression positively correlated with poor survival of PDAC patients. GDF-15 protein enhanced tumor cell proliferation in two pancreatic cancer lines, AsPC-1 and BxPC-3. Knockdown GDF-15 attenuated its biological function in vitro and reduced PDAC cell tumorigenesis upon xenotransplantation into nude mice. Moreover, we identified that glial-derived neurotropic factor family receptor α-like (GFRAL) was upregulated in PDAC tissues and positively correlated with GDF-15 expression. High GFRAL expression was significantly associated with poor survival in PDAC patients. Furthermore, we identified that the biological effects of GDF-15 are mediated by its receptor GFRAL which is present in PDAC cells. After overexpression GFRAL in pancreatic cancer cells, the effect of GDF-15 was significantly enhanced. Overall, our findings demonstrated that the GDF-15 secreted by PDAC cells, binds to GFRAL, itself localized in PDAC cells, to promote cancer cell growth and metastasis through the GDF-15/GFRAL signaling pathway.

Keywords: GDF-15; GFRAL; metastasis; pancreatic ductal adenocarcinoma; proliferation.

Figure 1

GDF-15 is aberrantly elevated in pancreatic cancer tissues and blood, and correlates with disease progression. (A) Detection of GDF-15 protein levels by immunohistochemistry (IHC) in 7 normal pancreatic tissues and 21 pancreatic cancer tissues. (B) GDF-15 protein expression was determined via Enzyme-Linked Immunosorbent Assay (ELISA) in 20 normal blood and 34 pancreatic cancer blood. (C) Kaplan-Meier curves for overall survival time of patients with pancreatic cancer after surgical resection. The high and low GDF-15 protein expression was based on the average value of GDF-15 protein expression in pancreatic cancer patients’ blood samples. ***P<0.0001.

Figure 2

GDF-15 promotes pancreatic cancer cell proliferation in vitro and enhances the chemosensitivity of cells to gemcitabine. (A) The expression level of GDF-15 secreted by pancreatic cancer cell lines (AsPC-1, BxPC-3, Panc-1, Hs766t), was analyzed by ELISA. (B–E) Different concentrations of recombinant human GDF-15 protein, 0ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, were added to the culture of the pancreatic cancer cell lines, AsPC-1, BxPC-3, Panc-1 and Hs766t. (F, G) The effect of recombinant human GDF-15 on pancreatic cancer cell lines (AsPC-1, BxPC-3) was detected by CCK-8 assay. The results are presented as the mean ± s.d. Significance was analyzed using GraphPad Prism. (H, I) The efficiency of lentivirus infection was detected by fluorescence microscope in AsPC-1 cells, and the results from ELISA assay indicated that GDF-15 expression was downregulated significantly. (J, K) The effect of GDF-15 knockdown on the proliferation of AsPC-1 and BxPC-3 cells was detected by CCK-8 assay. (L) WB assay for cleaved PARP in Lv-RNAi-NC- and Lv-RNAi-GDF-15-infected AsPC-1 cells after gemcitabine (20μM) for 48h using GAPDH as a loading control. ***P<0.0001.

Figure 3

GDF-15 promotes subcutaneous pancreatic cancer cell growth in nude mice. Lv-RNAi-NC and Lv-RNAi-GDF-15 AsPC-1 cells were subcutaneously injected into nude mice. (A) Impact of GDF-15 knockdown on AsPC-1 cells growth following xenotransplantation into nude mice by subcutaneous injection. Cancer cells growth were monitored using the IVIS® Spectrum system (Perkin Elmer, USA). The intensity of red fluorescent signal means the size of tumor cells after proliferation. (B) The tumor growth and mean tumor weights were detected after 30 days of inoculation.

Figure 4

GFRAL is overexpressed in pancreatic cancer tissues and pancreatic cancer cell lines, and correlates with disease progression. (A) Detection of GFRAL protein levels by immunohistochemistry (IHC) in 117 pancreatic cancer tissues (clinical stages I, II, III, and IV) and 13 normal pancreatic tissues. (B) WB assay to detect GFRAL protein in different types of pancreatic cancer cell lines and normal pancreatic ductal epithelial cell line. (C) GFRAL expression correlates with overall survival time of patients with pancreatic cancer after surgical resection. (D) A positive relationship between GDF-15 and GFRAL protein was demonstrated in 13 pairs of pancreatic cancer blood and tissues based on Spearman’s correlation.

Figure 5

GFRAL and GDF-15 colocalize in human pancreatic cancer cells. (A–D) H.E images for 4 patient samples with histologically confirmed PDAC; (E–H) Immunofluorescence staining for GFRAL and nuclei counterstained with DAPI; (I–L) Immunofluorescence staining for GDF-15 and nuclei counterstained with DAPI; (M–P) Merged images of GFRAL, GDF-15, and DAPI staining.

Figure 6

GFRAL overexpression enhances the effects of GDF-15 in pancreatic cancer cells. (A) The efficiency of lentivirus infection was detected by fluorescence microscopy in AsPC-1; As(-) means Lv-NC, As(+) means Lv-GFRAL; (B) WB assay indicated that GFRAL expression was significantly upregulated in different pancreatic cancer cell lines(AsPC-1, Panc-1, Hs766t). (C, D) After GFRAL upregulation in AsPC-1 and SW1990 cells, pancreatic cancer cell proliferation ability was enhanced after coculture with 0ng/ml, 10ng/ml, or 20ng/ml GDF-15, with increasing concentrations of GDF-15, the proliferative ability also increased.