(M1130-04-22) DEC2 Enhancement of Gemcitabine and Oxaliplatin Sensitivity of Pancreatic Cancer Cells through Suppression of IGFBP4

Purpose: Pancreatic Cancer (PC) is characterized by poor prognosis and limited treatment options. The development of resistance to anticancer agents is a significant challenge in PC treatment and poses a major obstacle in the course of treatment. Although the expression of Differentiated Embryonic Chondrocyte-expressed gene 2 (DEC2) affects tumor growth in PC,¹ it has been reported that its impact on the sensitivity to chemotherapy remains unclear. In this study, we aimed to determine the effects of DEC2 on PC chemosensitivity. Considering that the impact of DEC2 on chemosensitivity is likely to be mediated by molecules downstream of the transcription factor DEC2, we analyzed the genes downstream of DEC2, along with chemosensitivity. Understanding this mechanism may contribute to the development of effective therapeutic strategies for overcoming chemoresistance in PC cells.

Methods: We established stable knockdown and overexpression cell lines of DEC2 using human pancreatic cancer cells, PANC-1 and MIAPaca2. The steps are as follows: For the transfection of plasmid vectors, pLKO.1-puro shRNA or pcDNA3.1 vector targeting DEC2 were used. After transfection, the cells were cultured in medium containing antibiotics. Next, we collected formed colonies to obtain the cell lines. The selection markers used either puromycin or G418. The quantification of gene expression was used a two-step operation in RT-qPCR. In chemosensitivity tests using MTT assay, we treated with gemcitabine hydrochloride (GEM), oxaliplatin (L-OHP), paclitaxel (PTX), irinotecan hydrochloride (CPT-11), or 5-fluorouracil (5-FU) to these cell lines to assess the effects of DEC2 on their respective sensitivities. Furthermore, total RNA was extracted from these cell lines and subjected to RNA sequence analysis to confirm changes in the expression of DEC2 downstream genes. RNA sequence analyzes were outsourced to Nippon Genetics Co., Ltd. Finally, we performed that susceptibility tests to identify novel factor affecting sensitivity to anticancer agents using the siRNA method. In this method, we transfected with either siRNA-negative control (NC) or siRNA-IGFBP4, followed by treatment with GEM or L-OHP 24 hours later.

Results: The expression levels of DEC2 in the established cell lines were determined using western blot analysis and RT-qPCR (Figure 1A, B). From cell proliferation rate analysis, difference of cell proliferation rate was not observed between these cell lines (Figure 2A), and from susceptibility tests, their sensitivity to GEM increased in a DEC2 expression level-dependent manner (Figure 2B-a). In DEC2 knocked-down cell lines, the IC₅₀could not be obtained, even when treated with high concentrations of GEM (Figure 2C). Whereas, although slight sensitization was observed with L-OHP in these cell lines, the difference in sensitivity was insignificant compared to that with GEM (Figure 2B-b, C). However, there was no observed effect of DEC2 on sensitivity to PTX, CPT-11, or 5-FU (Figure 2B-c, d, e). Furthermore, in RNA sequence analysis, a volcano plot was used to infer the overall distribution of differentially expressed genes. As a result, IGFBP4 was one of the genes most downregulated by DEC2 (Figure 3A-a, b). After knockdown of IGFBP4, the expression levels were determined using western blot analysis and RT-qPCR (Figure 3B, C). The knockdown of IGFBP4 in GEM-resistant PANC-1 cells and MIAPaca2 cells increased their sensitivity to GEM (Figure 3D-a). Sensitivity to L-OHP was slightly influenced by the knockdown of IGFBP4, which revealed a modest increase in sensitivity, similar to the effect observed with DEC2 (Figure 3D-b).

Conclusion: In this study, we discovered that DEC2 downregulates IGFBP4, and identified IGFBP4 as a novel molecule involved in resistance to GEM and L-OHP in PC cells. These results suggest that DEC2 and IGFBP4 have the potential to be predictive factors for the effectiveness of PC chemotherapy. These findings provide novel insights that may be used to identify potential targets for patients with PC who undergo chemotherapy and offer new avenues for research and potential therapeutic strategies.

References: 1. Inaguma S, Riku M, Hashimoto M, et al. GLI1 Interferes with the DNA Mismatch Repair System in Pancreatic Cancer through BHLHE41-Mediated Suppression of MLH1. Cancer Research. 2013; 73(24): 7313-23.

Acknowledgements: The authors would like to thank Y Kage for providing the experimental facilities and instruments support in the pharmacology support in the pharmacology laboratory, University of Miyazaki. We would like to thank Y Yamada and K Matsusaka in same laboratory for supporting the experimental equipments. Yukio Kato and Katsumi Fujimoto (Hiroshima University) for providing the plasmid vector. This study was supported a clinical research grant from University of Miyazaki Hospital and JSPS KAKENHI grant numbers 21K06691. Ethical approval is not applicable. The authors declare that they have no conflict of interest.


Figure 1. (A) Western blot analysis of DEC2 protein in established PANC-1 and MIAPaca2 cell lines. (B) Quantification of DEC2 expression using RT-qPCR. The results are evaluated as the mean ± SD (n=3). **p < 0.01. The expression level of DEC2 was quantified by β-actin ratio.


Figure 2: Confirmation of cell proliferation rate and chemosensitivity using MTT assay in established PANC-1 and MIAPaca2 cell lines. The results were evaluated as the mean ± SD (n=4 for fig.2A, n=3 for fig.2B and 2C). (A) Comparison of cell proliferation rates. (B) Chemosensitivity analysis of GEM (a), L-OHP (b), PTX (c), CPT-11 (d), 5-FU (e). *p < 0.05, **p < 0.01. (C) IC50 of each anticancer agent.


Figure 3: Comprehensive analysis of downstream genes of DEC2. The results of RT-qPCR and MTT assay were evaluated as the mean ± SD (n=3). *p < 0.05, **p < 0.01. (A) (a) A volcano plot was used to infer the overall distribution of differentially expressed genes. (b) Extraction of IGFBP4, one of the genes whose expression is suppressed by DEC2. (B) Western blot analysis of IGFBP4 protein in PANC-1_shDEC2#2 and MIAPaca2. (C) Quantification of IGFBP4 expression using RT-qPCR. The expression level of IGFBP4 was quantified by β-actin ratio. (D) Chemosensitivity analysis of GEM (a) , L-OHP (b) with the siRNA method.