Aram Ko, Ji-Young Shin, Jinho Seo, Kang-Duck Lee, Eun-Woo Lee, Min-Sik Lee,
Han-Woong Lee, Il-Ju Choi, Jin Sook Jeong, Kyung-Hee Chun, Jaewhan
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei
University, Seoul (AK, Ji-S, E-WL, M-SL, H-WL, Ja-S); National Cancer Center,
Gastric Cancer Branch, Goyang-si, Kyunggi-do (J-YS, K-DL, I-JC); Department of
Biochemistry & Molecular Biology, Yonsei University College of Medicine,
Seoul, Korea (K-HC); Department of Pathology, Dong-A University Medical School,
Busan, Korea (JSJ).
*Correspondence to: Jaewhan Song, PhD, Department of Biochemistry,
Yonsei University, Sinchon-dong, Seodaemun-gu, Seoul 120.749, Korea
Background We investigated whether Makorin ring finger protein 1
(MKRN1), an E3 ligase, affects p14ARF-associated cellular senescence and
tumorigenesis by posttranslational modification in gastric tumorigenesis.
Methods A link between MKRN1 and ARF was examined in MKRN1 null mouse
embryonic fibroblasts (MEFs) and in human fibroblasts and gastric cancer cells
by silencing MKRN1 using small interfering RNA (siRNA) and short hairpin RNA
(shRNA). Ubiquitination and proteasomal degradation assays were used to assess
p14ARF degradation associated with MKRN1. MKRN1 and p14ARF expression levels
were analyzed with immunohistochemistry in malignant and normal tissues from
gastric cancer patients and with χ2 tests. The tumor growth of
gastric cancer cells stably expressing MKRN1 shRNA, p14ARF shRNA, or both was
examined in mouse xenograft models (n = 4-6) and analyzed with unpaired t
tests. All statistical tests were two-sided.
Results MKRN1 knockout MEFs exhibited premature senescence and growth
retardation with increased p19ARF protein expression. Similar results were
obtained for human fibroblasts or gastric cancer cell lines by MKRN1 knockdown.
Biochemical analyses confirmed that MKRN1 targets p14ARF for ubiquitination and
subsequent proteasome-dependent degradation. A statistically significant
association was shown between MKRN1 overexpression and p14ARF underexpression
(P = .016). Xenograft analyses using p53-functional AGS or -dysfunctional
SNU601 cells displayed statistically significant tumor growth retardation by
silencing MKRN1, which was reversed under depletion of p14ARF (AGS cells, MKRN1
knockdown tumors vs MKRN1 and p14ARF knockdown tumors: 164.6 vs
464.8mm3, difference = 300.2mm3, 95% CI = 189.1 to
411.3mm3, P < .001).
Conclusions We demonstrated that MKRN1 functions as a novel E3 ligase of
p14ARF and that it potentially regulates cellular senescence and tumorigenesis
in gastric cancer.