TM4SF1 Promotes Gemcitabine Resistance of Pancreatic Cancer In Vitro and In Vivo

Abstract

Background: TM4SF1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and affects the development of this cancer. Also, multidrug resistance (MDR) is generally associated with tumor chemoresistance in pancreatic cancer. However, the correlation between TM4SF1 and MDR remains unknown. This research aims to investigate the effect of TM4SF1 on gemcitabine resistance in PDAC and explore the possible molecular mechanism between TM4SF1 and MDR.

Methods: The expression of TM4SF1 was evaluated in pancreatic cancer cell lines and human pancreatic duct epithelial (HPDE) cell lines by quantitative RT-PCR. TM4SF1 siRNA transfection was carried out using Hiperfect transfection reagent to knock down TM4SF1. The transcripts were analyzed by quantitative RT-PCR, RT-PCR and western blotting for further study. The cell proliferation and apoptosis were obtained to investigate the sensitivity to gemcitabine of pancreatic cancer cells after silencing TM4SF1 in vitro. We demonstrated that cell signaling of TM4SF1 mediated chemoresistance in cancer cells by assessing the expression of multidrug resistance (MDR) genes using quantitative RT-PCR. In vivo, we used orthotopic pancreatic tumor models to investigate the effect of proliferation after silencing TM4SF1 by a lentivirus-mediated shRNA in MIA PaCa-2 cell lines.

Results: The mRNA expression of TM4SF1 was higher in seven pancreatic cancer cell lines than in HPDE cell lines. In three gemcitabine-sensitive cell lines (L3.6pl, BxPC-3, SU86.86), the expression of TM4SF1 was lower than that in four gemcitabine-resistant cell lines (MIA PaCa-2, PANC-1, Hs766T, AsPC-1). We evaluated that TM4SF1 was a putative target for gemcitabine resistance in pancreatic cancer cells. Using AsPC-1, MIA PaCa-2 and PANC-1, we investigated that TM4SF1 silencing affected cell proliferation and increased the percentages of cell apoptosis mediated by treatment with gemcitabine compared with cells which were treated with negative control. This resistance was associated with the expression of multidrug resistance genes including ABCB1 and ABCC1. In vivo, silencing of TM4SF1 in MIA PaCa-2 cell lines increased the effectiveness of gemcitabine-based treatment in orthotopic pancreatic tumor models evaluated using noninvasive bioluminescent imaging.

Conclusion: These findings suggest that TM4SF1 is a surface membrane antigen that is highly expressed in pancreatic cancer cells and increases the chemoresistance to gemcitabine. Thus, TM4SF1 may be a promising target to overcome the chemoresistance of pancreatic cancer.

Fig 1. Expression of TM4SF1 in pancreatic cancer cell lines.

Quantitative RT-PCR analysis resulted regarding the expression of TM4SF1 mRNA in human pancreatic cancer cell lines.TM4SF1 mRNA was more highly expressed in the cancer cell lines than in HPDE cells. The mRNA expression of TM4SF1 was lower in three gemcitabine-sensitive cell lines (L3.6pl, BxPC-3, SU86.86) than that in four gemcitabine-resistant cell lines (MIA PaCa-2, PANC-1, Hs766T, AsPC-1).

Fig 2. For proliferation assays and apoptosis studies, AsPC-1, MIA PaCa-2, and PANC-1 cells were transfected with siControl or siTM4SF1 and then treated with or without gemcitabine.

(A) Quantitative RT-PCR and western blotting demonstrated the silencing of TM4SF1 in AsPC-1, MIA PaCa-2 and PANC-1 cell lines. The cell lines were transiently transfected with siControl or siTM4SF1, and mRNA was isolated from them after 48 hours and protein was isolated after 72 hours. (B) AsPC-1, PANC-1 and MIA PaCa-2 cells were incubated with various concentrations of gemcitabine, respectively. The results of MTS assay showed that the proliferation ability of siTM4SF1 cells was lower than that of siControl cells after treating with gemcitabine. (C) After 72 hours, the percentage of cells with sub-G0/G1 DNA content was identified via FACS. TM4SF1 silencing resulted in increased sensitivity of the cells to treatment with gemcitabine, resulting in increased apoptosis rates. (Columns, means for three experiments; bars, standard deviation. *P < 0.05 versus control.).

Fig 3. TM4SF1-silencing pancreatic cancer cells mediated the expression of MDR genes.

Quantitative RT-PCR data showing fold changes in the mRNA level of ABCB1 and ABCC1 genes after silencing TM4SF1 in AsPC-1, MIA PaCa-2 and PANC-1 cell lines.

Fig 4. Increased sensitivity of pancreatic cancer cells to treatment with gemcitabine in vivo resulting from TM4SF1 silencing.

Quantitative RT-PCR and western blotting showed the silencing of TM4SF1 in MIA PaCa-2 stably transfected with shControl or shTM4SF1. (B) Tumors induced by MIA PaCa-2 cells stably expressing shControl or shTM4SF1 and expressing the luciferase gene developed in nude mice. At the end of the experiment, the animals were analyzed using bioluminescent imaging. Weights of tumors induced by MIA PaCa-2 cells stably expressing shRNA with or without gemcitabine (100mg/kg) treatment in nude mice.The tumor weights (g) of shCtrl, shCtrl+GEM, shTM4SF1 and shTM4SF1+GEM were 0.934±0.132, 0.792±0.101, 0.750±0.149 and 0.398±0.080. TM4SF1 silencing by gemcitabine resulted in markedly lower tumor weights than in the control mice (*P < 0.05). (C) In vivo proliferation was measured by PCNA assay on paraffin sections from shControl and shTM4SF1 animals treated with gemcitabine. A representative image at a magnification of ×200 indicating cell proliferation is shown.