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. 2019 Apr;8(4):1779-1792.
doi: 10.1002/cam4.2056. Epub 2019 Mar 24.

SALL4 induces radioresistance in nasopharyngeal carcinoma via the ATM/Chk2/p53 pathway

Xin Nie  1 , Ergang Guo  1 , Cheng Wu  1 , Dongbo Liu  1 , Wei Sun  1 , Linli Zhang  1 , Guoxian Long  1 , Qi Mei  1 , Kongming Wu  1 , Huihua Xiong  1 , Guoqing Hu  1
Affiliations

SALL4 induces radioresistance in nasopharyngeal carcinoma via the ATM/Chk2/p53 pathway

Xin Nie et al. Cancer Med. 2019 Apr.

Abstract

Radiotherapy is the mainstay and primary curative treatment modality in nasopharyngeal carcinoma (NPC), whose efficacy is limited by the development of intrinsic and acquired radioresistance. Thus, deciphering new molecular targets and pathways is essential for enhancing the radiosensitivity of NPC. SALL4 is a vital factor in the development and prognosis of various cancers, but its role in radioresistance remains elusive. This study aimed to explore the association of SALL4 expression with radioresistance of NPC. It was revealed that SALL4 expression was closely correlated with advanced T classification of NPC patients. Inhibition of SALL4 reduced proliferation and sensitized cells to radiation both in vitro and in vivo. Furthermore, SALL4 silencing increased radiation-induced DNA damage, apoptosis, and G2/M arrest in CNE2 and CNE2R cells. Moreover, knockdown of SALL4 impaired the expression of p-ATM, p-Chk2, p-p53, and anti-apoptosis protein Bcl-2, while pro-apoptosis protein was upregulated. These findings indicate that SALL4 could induce radioresistance via ATM/Chk2/p53 pathway and its downstream proteins related to apoptosis. Targeting SALL4 might be a promising approach for the development of novel radiosensitizing therapeutic agents for radioresistant NPC patients.

Keywords: SALL4; apoptosis; cell cycle; nasopharyngeal carcinoma; radioresistance.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Representative immunohistochemistry staining image for SALL4 expression in NPC tissue (original magnification × 400; Bar, 20 μm). A, Representative image for negative expression in noncancerous tissues (nasal polyp). B, Representative images for different staining intensity of SALL4 in NPC tissues. a: negative; b: weakly positive (light yellow); c: moderately positive (yellow‐brown), and d: strongly positive (dark brown)
Figure 2
Figure 2
Construction of acquired radioresistant cell line CNE2R and interference of SALL4 expression in NPC cell lines. A, Representative photograph of colony formation assay. B, Survival fraction in CNE2R cells was markedly increased compared with those in CNE2 cells. C and D, Western blot assay and quantitative real‐time PCR were used to detect SALL4 expression in MCF7, A549 cells and NPC cell lines. GAPDH was used as a loading control. E and F, SALL4 expression was detected by western blot to analyze the effect of lentivirus transfection in CNE2 (E) and CNE2R (F) cells. β‐Actin was used as a loading control
Figure 3
Figure 3
The effect of interference of SALL4 expression on proliferation and radiosensitivity. A and B, CCK8 assays were used to detect the proliferation ability. A, Silencing SALL4 reduced proliferation and SALL4 overexpression increased proliferation in CNE2 cells. B, Silencing SALL4 reduced the proliferation ability of CNE2R cells. Diagrams were from three independent experiments (mean ± SD, n = 3). ***P < 0.001. C and E, Representative photographs of colony formation assays. D, Survival fractions of CNE2 cells were increased by overexpressing SALL4, and decreased by SALL4 silencing, compared to the control group. F, SALL4 silencing remarkably decreased the survival fraction of CNE2R cells, compared to control group
Figure 4
Figure 4
SALL4 is correlated with the γH2AX‐mediated repair of DNA double‐strand breaks (DSBs), apoptosis, and cell cycle arrest induced by radiation. A and B, Images were captured using confocal microscopy. Cell nucleus was stained with DAPI (blue) and antibody to γ‐H2AX (red). Bar, 5 μm. The number of γH2AX foci per cell was determined by analyzing 100 randomly selected cells. C and D, The apoptotic rates were determined by flow cytometry. Compared with control groups, the apoptotic rate decreased in SALL4 overexpression group, while that in SALL4 silencing groups increased. E and F, The cell cycle proportion was detected by flow cytometry. Knockdown of SALLL4 induced G2/M arrest, and SALL4 overexpression decreased G2/M proportion. All histograms were from three independent experiments (mean ± SD, n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001. ns: not significant
Figure 5
Figure 5
SALL4 correlates with the expression of proteins related to the ATM/CHK2/P53 pathway. Western blot was applied to analyze the expression levels of related proteins. β‐actin was used as a loading control. A, Before and after irradiation, SALL4 silencing reduced the expressions of p‐ATM, p‐Chk2, and p‐p53 in CNE2 and CNE2R cells. B, SALL4 overexpression increased expression of p‐ATM, p‐Chk2, and p‐p53 in CNE2 cells. C, Expressions of pro‐apoptosis proteins (cleaved‐caspase‐3 and Bax) were increased and expressions of anti‐apoptosis protein (Bcl‐2) were decreased in CNE2‐shSALL4 and CNE2R‐shSALL4 cells after irradiation. D, SALL4 silencing increased the Bax/Bcl‐2 ratio. E, Expressions of cleaved‐caspase‐3 and Bax were decreased and expression of Bcl‐2 was increased in CNE2‐SALL4 cells after irradiation. F, SALL4 overexpression reduced Bax/Bcl‐2 ratio in CNE2 cells after radiation ***P < 0.001. ns: not significant
Figure 6
Figure 6
SALL4 affects the tumorigenesis and radiosensitivity of nasopharyngeal carcinoma in vivo. Human NPC xenografts in nude mice model are used. A, Representative tumor xenografts of each group. B, Representative immunohistochemistry staining image for SALL4 expression in tumor tissue. Original magnification × 400. Bar. 20 μm. C, The volumes of tumor in CNE2R‐CTL and CNE2R‐shSALL4 groups treated with or without radiation. D, The volumes of tumor in CNE2‐CTL and CNE2‐SALL4 groups treated with or without radiation. E, The tumor weight (left) and the tumor weight inhibition rate (TWI %) (right) of CNE2R‐CTL and CNE2R‐shSALL4 groups treated with or without radiation. F, The tumor weight (left) and the tumor weight inhibition rate (TWI %) (right) of CNE2‐CTL and CNE2‐SALL4 groups treated with or without radiation. Quantifications of tumor volumes were showed with means ± SD from three independent experiments. ***P < 0.001, **P < 0.01
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