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. 2021 Apr 7:12:635808.
doi: 10.3389/fgene.2021.635808. eCollection 2021.

Human ALKBH6 Is Required for Maintenance of Genomic Stability and Promoting Cell Survival During Exposure of Alkylating Agents in Pancreatic Cancer

Shengyuan Zhao  1 , Rodan Devega  1 , Aaliyah Francois  1 , Dawit Kidane  1
Affiliations

Human ALKBH6 Is Required for Maintenance of Genomic Stability and Promoting Cell Survival During Exposure of Alkylating Agents in Pancreatic Cancer

Shengyuan Zhao et al. Front Genet. .

Abstract

Alpha-ketoglutarate-dependent dioxygenase (ALKBH) is a DNA repair gene involved in the repair of alkylating DNA damage. There are nine types of ALKBH (ALKBH1-8 and FTO) identified in humans. In particular, certain types of ALKBH enzymes are dioxygenases that directly reverse DNA methylation damage via transfer of a methyl group from the DNA adduct onto α-ketoglutarate and release of metabolic products including succinate and formaldehyde. Here, we tested whether ALKBH6 plays a significant role in preventing alkylating DNA damage and decreasing genomic instability in pancreatic cancer cells. Using an E. coli strain deficient with ALKB, we found that ALKBH6 complements ALKB deficiency and increases resistance after alkylating agent treatment. In particular, the loss of ALKBH6 in human pancreatic cancer cells increases alkylating agent-induced DNA damage and significantly decreases cell survival. Furthermore, in silico analysis from The Cancer Genome Atlas (TCGA) database suggests that overexpression of ALKBH6 provides better survival outcomes in patients with pancreatic cancer. Overall, our data suggest that ALKBH6 is required to maintain the integrity of the genome and promote cell survival of pancreatic cancer cells.

Keywords: Alkb E. coli; DNA repair; alkbh6; alkylating DNA damage; pancreatic cancer.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
ALKBH6 partially restores growth and increases resistance to alkylating DNA-damaging agents. (A) Model of ALKBH6 (238 aa) based on SWISS-MODEL indicating metal binding sites labeled in red (HIS114, ASP116, and HIS182). The Fe2+ ion in the active site is shown in purple; protein residues (His114 represented by blue/gray ring; His182 represented by blue/gray ring; D116 represented by gray/red) are labeled in white. (B) Schematic representation and results of colony-forming assay using ALKB-proficient strain (WT), ALKB-deficient strain (ALKB–), and ALKBH6-complemented E. coli strain (ALKB– + pBAD-ALKBH6). Results were normalized with untreated groups. Student’s t-test was used to determine whether there was significant difference between deficient and complemented strains. (C) Schematic representation and results of growth curve assay using the same three strains mentioned above. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 represent t-test results. N.S represents non-significant difference, three independent experiments.
FIGURE 2
FIGURE 2
Loss of ALKBH6 induces DSBs. (A) Western blot of siRNA-mediated silencing of ALKBH6 on MIA-PaCa-2 cells; (B) Representative image of siRNA-mediated silencing of BXPC3 and MIA-PaCa-2 cells treated with MMS and examined with co-localization of 53BP1 and γH2AX; note that blue color represents DAPI staining of the cell nuclei; scale bar represents 10 μm; (C) Percent of cells positive for co-localization of 53BP1 and γH2AX treated with MMS/MNU; (D) Representative image of ALKBH6 knockout; (D) results of tail moment of BxPC3 and MIA-PaCa-2 cells treated with MMS and examined with neutral comet assay; (E) Cell cycle profile of cells deficient in ALKBH6. Student’s t-test was applied for data analysis. *p < 0.05, **p < 0.01), ****p < 0.0001, N.S represents non-significant difference, three independent experiments.
FIGURE 3
FIGURE 3
ALKBH6 protects pancreatic cells from alkylating agents. (A,B) MTT assay results of MIA-PaCa-2 and BxPC-3 cells treated with a different concentration of MNU for 24 h following ALKBH6 knockout; (C,D) MTT assay results of MIA-PaCa-2 and BxPC-3 cells treated with a different concentration of MMS for 24 h following siRNA silencing of ALKBH6 cells; Student’s t-test was applied for data analysis. *p < 0.05, **p < 0.01, ***p < 0.001), ****p < 0.0001, N.S represents non-significant difference, three independent experiments.
FIGURE 4
FIGURE 4
ALKBH6 overexpressed in pancreatic cancer patients positively correlates with patient survival. (A) Schematic representation of ALKBH6 gene amplification and expression levels of ALKBH6 mRNA in normal versus tumor samples. mRNA expression was analyzed from TCGA database and the RNA Seq dataset was analyzed using the log scale expression. An expression of >0.5 denotes the high expressed group (n = 76) versus < –0.5 for the low expressed group (n = 71); (B) Survival curve of pancreatic cancer patients with high ALKBH6-expressing versus low ALKBH6-expressing pancreatic tumors. Kaplan-Meier survival graph of patients with high and low expression of ALKBH6; (C) Survival of patients with (n = 56) and without (n = 20) TP53 mutation-driven ALKBH6-overexpressing tumors promotes worse prognosis in pancreatic cancer.
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