ASF1A regulates H4^Y72 phosphorylation and promotes autophagy in colon cancer cells via a kinase activity

Figures & data

Table 1 Correlation of ASF1A and clinicopathological parameters in colon cancer

Variables	Case number	ASF1A
LN metastasis
Positive	86	25.13 ± 0.32**
Negative	44	14.22 ± 1.05
Tumour size
>3 cm	68	17.53 ± 0.13**
≤3 cm	62	5.76 ± 1.12
Histological grade (Differentiation)
III grade (Poor)	47	19.75 ± 0.03**
I–II grade (Well + Moderate)	36 + 47	9.22 ± 0.28
TNM stage
III + IV	33 + 35	18.67 ± 1.45**
I + II	28 + 34	8.16 ± 0.11
Age
>50	72	20.05 ± 0.05**
≤50	58	7.89 ± 0.42

Detection was by immunohistochemistry and quantification was with IMAGEJ software. **p < .01. Data are mean ± SD. and were analyzed by Independent-samples t-test.

Figure 1. ASF1A expression is positively correlated with H4^Y72ph levels in colon cancer cells. (A) Expression of H4^Y72ph and ASF1A in different cell lines (LoVo, SW48 and SNU-C1) were detected by western blot. (B and C) Effects of ASF1A overexpression in regulation of H4^Y72ph were detected by western blot. (D) Using interference RNA to silence ASF1A expression and detected the influence on H4^Y72ph in different cell lines (SW48 and SNU-C1) by western blot. All the data were presented by mean + SD (n = 3). ** p < .01; *** p < .001 (to control in H4^Y72ph/H4); ## p < .01 (to control in ASF1A/GAPDH).

Table 2. Relationships between ASF1A expression and H4^Y72ph in specimens of colon cancer.

ASF1A expression
	−	+	++	+++	p Value
H4^Y72ph
−	49	6	3	5
+	12	4	2	5
++	2	5	3	1
+++	9	9	3	12
					<.001

The expression levels were determined by evaluating the extent and intensity of immunopositivity. The expression patterns of two molecules in the 130 colon cancer samples were determined and summarized. The correlation between ASF1A and H4^Y72ph was analyzed using Fisher’s exact test. A p-value of less than 0.05 was set as the criterion for statistical significance.

Figure 2. ASF1A has intrinsic tyrosine kinase activity. (A) H4 or ASF1A was analyzed by western blot using immunoprecipitation with antibodies targeting ASF1A or H4 in SW48 cells. (B) ASF1A and H4 expression was detected by western blot using immunoprecipitation with an anti-HA antibody. (C) H4 was detected using GST pull-down assay with tag in ASF1A full length. (D) An in vitro kinase activity assay was used to detect whether recombinant full-length ASF1A could phosphorylate H4. (E) Samples from the in vitro kinase activity assay were analyzed by western blot WT. wild-type; Y72F, dephosphorylated mutant. All experiments depends on replicates (n = 3).

Figure 3. ASF1A is an essential factor that regulates autophagy in colon cancer cells. (A) In SW48 cells and (B) in SNU-C1 cells, effects of ASF1A silence on the number of autophagosome per field under complete or starved condition. (C) Effects of ASF1A on the expression of LC3-I and LC3-II under complete or starved condition. (D) Effects of ASF1A silence and chloroquine (CQ) on the number of enhanced green fluorescent protein (EGFP)-tagged LC3 under complete or starved condition. (E) Effects of WT and ASF1A silence on the degradation percentage. (F) SW48 cells harbouring doxycycline (Dox)-inducible siRNA targeting autophagy-associated gene (ATG) were transiently transfected with ASF1A-expressing plasmids under complete or starved condition. The results are shown as the mean + SD (n = 3). * p < .05, ** p < .01; *** p < .001.

Figure 4. ASF1A-mediated H4Y72ph engages in autophagy in colon cancer cells. (A) Effects of H4^WT and H4^Y72F on the number of EGFP-LC3 puncta under complete or starved condition. (B) WT and ASF1A^−/− SW48 cells were treated with complete medium or Earle's balanced salt solution (EBSS) for 30 min or 1 h, and whole cell protein samples were then obtained for western blot. (C) Total H4, H4^Y72ph and ASF1A expression was detected by western blot. (D) Effects of H4^WT and H4^Y72F mutation on the number of EGFP-LC3 puncta were detected. The results are shown as the mean + SD (n = 3). * p < .05, ** p < .01.

Figure 5. ASF1A-H4^Y72ph axis promotes colon cancer autophagy via transcriptional regulation of autophagy associated genes (ATG). The mRNA expression levels of (A) ATG5 and (B) ATG7 under ASF1A silence with H4^WT or H4^Y72A or H4^Y72E. (B) The protein expression level of ATG5 and ATG7 were detected by western blot. (D) The relative light of (D) ATG5 and (E) ATG7 under co-treated ASF1A and H4^WT and H4^Y72F. (F) Protein expression level of ATG5 and ATG7 under co-treated ASF1A and H4^WT and H4^Y72F. The results are shown as the mean + SD (n = 3). ** p < .01 and *** p < .001.