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GAS41 mediates proliferation and GEM chemoresistance via H2A.Z.2 and Notch1 in pancreatic cancer

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Abstract

Purpose

GAS41 is a YEATS domain protein that binds to acetylated histone H3 to promote the chromatin deposition of H2A.Z in non-small cell lung cancer. The role of GAS41 in pancreatic cancer is still unknown. Here, we aimed to reveal this role.

Methods

GAS41 expression in pancreatic cancer tissues and cell lines was examined using qRT-PCR, Western blotting and immunohistochemistry. MTT, colony formation, spheroid formation and in vivo tumorigenesis assays were performed to assess the proliferation, tumorigenesis, stemness and gemcitabine (GEM) resistance of pancreatic cancer cells. Mechanistically, co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP) assays were used to evaluate the roles of GAS41, H2A.Z.2 and Notch1 in pancreatic cancer.

Results

We found that GAS41 is overexpressed in human pancreatic cancer tissues and cell lines, and that its expression increases following the acquisition of GEM resistance. We also found that GAS41 up-regulates Notch, as well as pancreatic cancer cell stemness and GEM resistance in vitro and in vivo. We show that GAS41 binds to H2A.Z.2 and activates Notch and its downstream mediators, thereby regulating stemness and drug resistance. Depletion of GAS41 or H2A.Z.2 was found to down-regulate Notch and to sensitize pancreatic cancer cells to GEM.

Conclusion

Our data indicate that GAS41 mediates proliferation and GEM resistance in pancreatic cancer cells via H2A.Z.2 and Notch1.

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Data availability

The datasets supporting the conclusions of this article are included in the article (and its additional files 1 to 17: Supplementary Material).

Abbreviations

GEM :

Gemcitabine

CSCs:

Cancer stem cells

GAS41:

Glioma amplified sequence 41

GR:

GEM-resistant

BrDs:

Bromodomains

ChIP:

Chromatin immunoprecipitation

NSCLC:

non-small cell lung cancer

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Acknowledgements

The authors are grateful for the support provided by the Tongji University School of Medicine and Shanghai Tenth People’s Hospital.

Funding

This study was supported by the National Natural Science Foundation of China (No. 81801805), the Shanghai Sailing Program (No.16YF1409000) and the Climbing Talent Program of Shanghai Tenth People’s Hospital (No. 2021SYPDRC014).

Author information

Author notes

  1. Shilong Han, Chuanwu Cao, Rui Liu and YiFeng Yuan Equally contributed to this work.

Authors and Affiliations

  1. Department of Intervention and Vascular Surgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, 50 Chifeng Road, Yangpu, Shanghai, 200072, China

    Shilong Han, Chuanwu Cao, YiFeng Yuan, Long Pan, Minjie Xu, Chao Hu, ** Zhang

  2. National Center Clinical Research for Interventional Medicine, Shanghai Tenth People’s Hospital, 50 Chifeng Road, Yangpu, Shanghai, 200072, China

    Shilong Han, Chuanwu Cao, YiFeng Yuan, Long Pan, Minjie Xu, Chao Hu, ** Zhang

  3. Shanghai Tenth People’s Hospital of Tongji University, Tongji University Cancer Center, Shanghai, 200072, China

    Rui Liu

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  1. Shilong Han
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Contributions

Shilong Han, Chuanwu Cao, Rui Liu: Conceptualization, Methodology, Software; Maoquan Li and ** Zhang: Supervision, Project administration; Shilong Han, Chuanwu Cao, Rui Liu: Data curation, Writing-Original draft preparation, Investigation; YifengYuan, Long Pan, Minjie Xu, Chao Hu, ** Zhang: Writing-Reviewing and Editing, Funding acquisition.

Corresponding authors

Correspondence to Maoquan Li or ** Zhang.

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Ethical approval and consent to participate

All participating patients provided written informed consent and the human studies were approved by the Institute Research Ethics Committee of Shanghai Tenth People’s Hospital. The study methodologies conformed to the standards set by the Declaration of Helsinki. The animal studies were performed in accordance with ARRIVE guidelines and were approved by the Institute Research Ethics Committee of the Shanghai Tenth People’s Hospital.

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Supplementary Information

ESM 1.

Supplement Figure 1. GAS41 aggravates resistance of GR cells. (A) The IC50 values of GEM with 24-hour treatment. (B) Cell viability after 24-hour treatment with indicated concentrations of GEM was evaluated with the MTT assay. (C-D) The mRNA levels of the stemness-related genes ALDH, ABCG2, CD133, and Nestin by qRT-PCR. (E) The GAS41 mRNA and protein levels in parental and GR BXPC3 cells transfected with GAS41 or vector. (F) The GAS41 mRNA and protein levels in parental and GR MiaPaCa2 cells transfected with shGAS41 or scr. Parental (G) and GR (H) BXPC3 and MiaPaCa2 cells were transfected with GAS41, vector, shGAS41, or scr for indicated hours and indicated concentrations. (I-J) The mRNA and protein levels of the chemoresistance-related genes by qRT-PCR and western blot analysis, respectively. (K-L) The mRNA and protein levels of the stemness-related genes by qRT-PCR and western blot analysis, respectively. n=3, &, #P<0.05, **, &&, ##, $$P<0.01. (PNG 2654 kb)

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Supplement Figure 2. GAS41 aggravates resistance of GR cells by upregulating Notch. (A, B) GR BXPC3 cells were transfected with GAS41 or vector for 24 hours. Untransfected resistant cells were included as control. The mRNA (A) and protein (B) levels of Notch1 and Hes1 were determined with qRT-PCR and western blot analysis, respectively. (C, D) GR MiaPaCa2 cells were transfected with shGAS41 or scr for 24 hours. Untransfected resistant cells were included as control. The mRNA (C) and protein (D) levels of Notch1 and Hes1 were determined with qRT-PCR and western blot analysis, respectively. (E, F) The Notch1 mRNA (E) and protein (F) levels in parental and GR BXPC3 cells transfected with shNotch1 or scr. (G, H) The Notch1 mRNA (G) and protein (H) levels in parental and GR MiaPaCa2 cells transfected with Notch1 or vector. (I) GR BXPC3 cells were co-transfected with GAS41 and scr or GAS41 and shNotch1 for 24 hours. GR MiaPaCa2 cells were co-transfected with shGAS41 and vector or shGAS41 and Notch1 for 24 hours. The cells were treated with GEM at indicated concentrations for 24 hours. The cell viability was determined with the MTT assay. n=3, *P<0.05, **, ##P<0.01. (PNG 519 kb)

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ESM 3.

Supplement Figure 3. H2A.Z.2.1 and H2AZ.1 was up-regulated in human pancreatic cancer (A, B) The H2A.Z.2 mRNA levels in human pancreatic cancer and adjacent normal tissues were examined by qRT-PCR. n=87, **P<0.01. (C-F) H2AZ protein levels after H2AZ knockdown or overexpression by Western Blot. (G) H2AZ protein levels were examined by Western Blot in BXPC3 and MiaPaCa2. (PNG 368 kb)

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ESM 4.

Supplement Figure 4. H2A.Z.2 knockdown in GR cells restores sensitivity to GEM. GR BXPC3 (A, B, E – G) and MiaPaCa2 (C, D, H – J) cells were transfected with shH2A.Z.2-1, shH2A.Z.2-2, or scr for 24 hours. (A, C) The interference efficiency of H2A.Z.2 is tested by qRT-PCR. (B, D) Cell viability after 24-hour treatment with indicated concentrations of GEM by the MTT assay. (E, H) The mRNA levels of the stemness- and chemoresistance-related genes by qRT-PCR. (F-G, I-J) The protein levels of the stemness- and chemoresistance-related genes by western blot analysis. n=3, *, #, $P<0.05, **, &&, ##, $$P<0.01. (PNG 738 kb)

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ESM 5.

Supplement Figure 5. H2A.Z.2 upregulates Notch in GR cells. GR BXPC3 (A, B) and MiaPaCa2 (C, D) cells were transfected with shH2A.Z.2 or scr for 24 hours. Untransfected cells were included as control. (A, C) The mRNA levels of Notch1 and Hes1 by qRT-PCR. n=3, *P<0.05, **, ##P<0.01. (B, D) The protein levels of Notch1 and Hes1 by western blot analysis. (E, F) ChIP analysis was performed with primers targeted to the promoter region of Notch1 and Hes1. IgG was used as control. (PNG 369 kb)

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Han, S., Cao, C., Liu, R. et al. GAS41 mediates proliferation and GEM chemoresistance via H2A.Z.2 and Notch1 in pancreatic cancer. Cell Oncol. 45, 429–446 (2022). https://doi.org/10.1007/s13402-022-00675-8

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