Cancer Res Treat.  2024 Jan;56(1):259-271. 10.4143/crt.2022.1600.

Gemcitabine Inhibits the Progression of Pancreatic Cancer by Restraining the WTAP/MYC Chain in an m6A-Dependent Manner

• Cao P
• Zhang W
• Qiu J
• Tang Z
• Xue X
• Feng T

• KMID: 2550341
• DOI: http://doi.org/10.4143/crt.2022.1600

Abstract

Purpose
Pancreatic cancer (PC) is a common malignant tumor of the digestive system, and its 5-year survival rate is only 4%. N6-methyladenosine (m6A) RNA methylation is the most common post-transcriptional modification and dynamically regulates cancer development, while its role in PC treatment remains unclear.
Materials and Methods
We treated PC cells with gemcitabine and quantified the overall m6A level with m6A methylation quantification. Real-time quantitative reverse transcription polymerase chain reaction and Western blot analyses were used to detect expression changes of m6A regulators. We verified the m6A modification on the target genes through m6A-immunoprecipitation (IP), and further in vivo experiments and immunofluorescence (IF) assays were applied to verify regulation of gemcitabine on Wilms’ tumor 1–associated protein (WTAP) and MYC.
Results
Gemcitabine inhibited the proliferation and migration of PC cells and reduced the overall level of m6A modification. Additionally, the expression of the “writer” WTAP was significantly downregulated after gemcitabine treatment. We knocked down WTAP in cells and found target gene MYC expression was significantly downregulated, m6A-IP also confirmed the m6A modification on MYC. Our experiments showed that m6A-MYC may be recognized by the “reader” IGF2BP1. In vivo experiments revealed gemcitabine inhibited the tumorigenic ability of PC cells. IF analysis also showed that gemcitabine inhibited the expression of WTAP and MYC, which displayed a significant trend of co-expression.
Conclusion
Our study confirmed that gemcitabine interferes with WTAP protein expression in PC, reduces m6A modification on MYC and RNA stability, thereby inhibiting the downstream pathway of MYC, and inhibits the progression of PC.

Keyword

Gemcitabine; Pancreatic neoplasms; RNA methylation; WTAP; MYC

Figure

• 

  Fig. 1. Gemcitabine inhibited pancreatic cancer cells proliferation and migration and overall N6-methyladenosine (m6A) modification. (A) Cell Counting Kit-8 assays were used for detection of PANC1 and CFPAC1 cells proliferation ability after treated with or without 10 µM gemcitabine (GEM). (B) 5-Ethynyl-2-deoxyuridine (EdU) assays was used for detection of PANC1 and CFPAC1 cells proliferation ability after treated with or without Gem, the ratio of EdU-positive to DAPI cells was used to represent the proliferation ability. (C) The number of cells through the transwell chamber was used to represent the migrate ability. (D) Relative m6A regulator WTAP (Wilms’ tumor 1–associated protein) expression level in PANC1 cells treated with or without Gem, ACTB was used as internal reference gene. (E, F) Relative m6A levels in PC cells after treated with or without Gem were assessed. Data are presented as mean±standard error of mean (*p < 0.05).

• 

  Fig. 2. WTAP (Wilms’ tumor 1–associated protein) was raised in pancreatic cancer (PC) and promoted PC cell biological function. (A) WTAP expression level in 179 PC specimens and 171 non-tumor specimens in The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). (B, C) Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) analysis of different genes between WTAP high and low expression groups in TCGA. (D, E) WTAP mRNA (D) and protein (E) expression in PANC1 cells after treated with gemcitabine (GEM), Gem-5 means PC cells were treated with Gem at the concentration of 5 µM, ACTB was used as internal reference gene. (F, G) WTAP mRNA (F) and protein (G) expression after PANC1 cells treated with WTAP-si or si-NC. (H) Proliferation ability of CFPAC1 and PANC1 cells and were detected with Cell Counting Kit-8 (CCK-8) assay at a wavelength of 540 nm after treated with WTAP-si or si-NC. (I) Transwell was used to detect CFPAC1 and PANC1 cells migration ability, cell numbers through the transwell chamber was counted to represent the migrate ability. (J, K) EdU assays was used for detection of PANC1 and CFPAC1 cells proliferation ability after treated with WTP-si or si-NC (*p < 0.05).

• 

  Fig. 3. WTAP (Wilms’ tumor 1–associated protein) knockdown inhibited MYC expression in a N6-methyladenosine (m6A) modification manner. (A) Relative targets RNA expression of WTAP after WTAP knockdown in PANC1, ACTB was used as internal reference gene. (B) Enrichment detection of WTAP targets by WTAP-immunoprecipitation (IP) and m6A-IP in PANC1, the results are presented as fold change of the targets in the WTAP-IP or m6A-IP experimental group. (C) Co-expression trend of WTAP and MYC in pancreatic cancer (PC) patients in The Cancer Genome Atlas. TPM, transcript per million. (D) MYC protein level in PC cells after treated with gemcitabine (Gem) or WTAP-si. (E) WTAP and MYC protein expression in two PC patient specimens by immunofluorescence. (F) Relative m6A levels in PANC1 cells after WTAP knockdown were assessed. (G-L) WTAP and insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) knockdown in PANC1 could enhance the inhibition of Gem on MYC expression (G, I), cell proliferation (H, J), and migration ability (K, L). (M) Relative m6A levels in PANC1 cells after IGF2BP1 knockdown. (N) MYC RNA expression change after IGF2BP1 knockdown in PANC1, ACTB was used as internal reference gene (*p < 0.05).

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  Fig. 4. Gemcitabine inhibited pancreatic cancer (PC) cells tumorigenesis by restraining WTAP (Wilms’ tumor 1–associated protein)/MYC axis through N6-methyladenosine (m6A) modification. (A-C) After 4 weeks of treatment with gemcitabine (Gem) or phosphate buffered saline (PBS), mice were sacrificed and subcutaneous tumors were harvested for detection and comparison of tumor volume (A, B) and weight. (D) Relative WTAP and MYC RNA expression in tumors in Gem group relative to PBS group. (E) Paraffin sections were prepared from subcutaneous tumors of mice in Gem and PBS groups, and the co-expression of WTAP and MYC protein was detected by immunofluorescence. (F) Mechanism of Gem inhibiting PC progression in an m6A modification. (*p < 0.05).

Reference

References

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019; 69:7–34.

2. Neoptolemos JP, Stocken DD, Friess H, Bassi C, Dunn JA, Hickey H, et al. A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. N Engl J Med. 2004; 350:1200–10.

3. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2021. CA Cancer J Clin. 2021; 71:7–33.

4. Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA modifications in gene expression regulation. Cell. 2017; 169:1187–200.

5. Zhao BS, Roundtree IA, He C. Post-transcriptional gene regulation by mRNA modifications. Nat Rev Mol Cell Biol. 2017; 18:31–42.

6. Guo X, Li K, Jiang W, Hu Y, Xiao W, Huang Y, et al. RNA demethylase ALKBH5 prevents pancreatic cancer progression by posttranscriptional activation of PER1 in an m6A-YTHDF2-dependent manner. Mol Cancer. 2020; 19:91.

7. Hu X, Peng WX, Zhou H, Jiang J, Zhou X, Huang D, et al. IGF2BP2 regulates DANCR by serving as an N6-methyladenosine reader. Cell Death Differ. 2020; 27:1782–94.

8. Wang M, Liu J, Zhao Y, He R, Xu X, Guo X, et al. Upregulation of METTL14 mediates the elevation of PERP mRNA N⁶ adenosine methylation promoting the growth and metastasis of pancreatic cancer. Mol Cancer. 2020; 19:130.

9. Taketo K, Konno M, Asai A, Koseki J, Toratani M, Satoh T, et al. The epitranscriptome m6A writer METTL3 promotes chemo- and radioresistance in pancreatic cancer cells. Int J Oncol. 2018; 52:621–9.

10. Hertel LW, Boder GB, Kroin JS, Rinzel SM, Poore GA, Todd GC, et al. Evaluation of the antitumor activity of gemcitabine (2’,2’-difluoro-2’-deoxycytidine). Cancer Res. 1990; 50:4417–22.

11. Xu BQ, Fu ZG, Meng Y, Wu XQ, Wu B, Xu L, et al. Gemcitabine enhances cell invasion via activating HAb18G/CD147-EGFR-pSTAT3 signaling. Oncotarget. 2016; 7:62177–93.

12. Gao P, Tchernyshyov I, Chang TC, Lee YS, Kita K, Ochi T, et al. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature. 2009; 458:762–5.

13. Dong P, Maddali MV, Srimani JK, Thelot F, Nevins JR, Mathey-Prevot B, et al. Division of labour between Myc and G1 cyclins in cell cycle commitment and pace control. Nat Commun. 2014; 5:4750.

14. Zhu S, Wang JZ, Chen D, He YT, Meng N, Chen M, et al. An oncopeptide regulates m⁶A recognition by the m⁶A reader IGF2BP1 and tumorigenesis. Nat Commun. 2020; 11:1685.

15. Zhu P, He F, Hou Y, Tu G, Li Q, Jin T, et al. A novel hypoxic long noncoding RNA KB-1980E6.3 maintains breast cancer stem cell stemness via interacting with IGF2BP1 to facilitate c-Myc mRNA stability. Oncogene. 2021; 40:1609–27.

16. Kleeff J, Reiser C, Hinz U, Bachmann J, Debus J, Jaeger D, et al. Surgery for recurrent pancreatic ductal adenocarcinoma. Ann Surg. 2007; 245:566–72.

17. Raimondi S, Maisonneuve P, Lowenfels AB. Epidemiology of pancreatic cancer: an overview. Nat Rev Gastroenterol Hepatol. 2009; 6:699–708.

18. Omura N, Goggins M. Epigenetics and epigenetic alterations in pancreatic cancer. Int J Clin Exp Pathol. 2009; 2:310–26.

19. Huxley R, Ansary-Moghaddam A, Berrington de Gonzalez A, Barzi F, Woodward M. Type-II diabetes and pancreatic cancer: a meta-analysis of 36 studies. Br J Cancer. 2005; 92:2076–83.

20. Duell EJ, Lucenteforte E, Olson SH, Bracci PM, Li D, Risch HA, et al. Pancreatitis and pancreatic cancer risk: a pooled analysis in the International Pancreatic Cancer Case-Control Consortium (PanC4). Ann Oncol. 2012; 23:2964–70.

21. Ligorio M, Sil S, Malagon-Lopez J, Nieman LT, Misale S, Di Pilato M, et al. Stromal microenvironment shapes the intratumoral architecture of pancreatic cancer. Cell. 2019; 178:160–75e27.

22. Zhu H, Li T, Du Y, Li M. Pancreatic cancer: challenges and opportunities. BMC Med. 2018; 16:214.

23. Barbieri I, Kouzarides T. Role of RNA modifications in cancer. Nat Rev Cancer. 2020; 20:303–22.

24. Zhou Z, Lv J, Yu H, Han J, Yang X, Feng D, et al. Mechanism of RNA modification N6-methyladenosine in human cancer. Mol Cancer. 2020; 19:104.

25. Han J, Wang JZ, Yang X, Yu H, Zhou R, Lu HC, et al. METTL3 promote tumor proliferation of bladder cancer by accelerating pri-miR221/222 maturation in m6A-dependent manner. Mol Cancer. 2019; 18:110.

26. Yang X, Zhang S, He C, Xue P, Zhang L, He Z, et al. METTL14 suppresses proliferation and metastasis of colorectal cancer by down-regulating oncogenic long non-coding RNA XIST. Mol Cancer. 2020; 19:46.

27. Liu T, Wei Q, Jin J, Luo Q, Liu Y, Yang Y, et al. The m6A reader YTHDF1 promotes ovarian cancer progression via augmenting EIF3C translation. Nucleic Acids Res. 2020; 48:3816–31.

28. Heinemann V, Quietzsch D, Gieseler F, Gonnermann M, Schonekas H, Rost A, et al. Randomized phase III trial of gemcitabine plus cisplatin compared with gemcitabine alone in advanced pancreatic cancer. J Clin Oncol. 2006; 24:3946–52.

29. Dang CV, O’Donnell KA, Zeller KI, Nguyen T, Osthus RC, Li F. The c-Myc target gene network. Semin Cancer Biol. 2006; 16:253–64.

30. Meyer N, Penn LZ. Reflecting on 25 years with MYC. Nat Rev Cancer. 2008; 8:976–90.