ESCC cell lines TE-1 and ECA-109 were purchased from The Cell Resource Centre of Shanghai Institutes for Biological Sciences, Chinese Academy of Science (Shanghai, China) and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., China) supplemented with 10% dialyzed fetal bovine serum (FBS; Biological Industries, Kibbutz Beit-Haemek, Israel), 1% compound antibiotics (Pen Strep; Gibco; Thermo Fisher Scientific, Inc.). Immortalized human normal esophageal epithelial cells, Het-1A (purchased from BeNa Culture Collection, Kunshan, Jiangsu, China), were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc., China), supplemented with 10% FBS as described previously (37). For induction of nutrient starvation, ESCC cells (ESCCs) or Het-1A cells (confluence of 50–60%) were transferred to serum lacking media (supplemented with 1% FBS) for the indicated periods of time. Cells were tested negative for mycoplasma or other infectious agents, and maintained at 37°C in a humidified atmosphere with 5% CO₂. ACSS2 (cat. no. sc-398559; Santa Cruz Biotechnology, Sant Cruz, CA, USA), ATM (product #2873)/p-ATM (product #5883), BRCA1 (product #9010)/p-BRCA1 (product #9009), Bcl-xL (product #2762), γH2AX (product #9718), DNA-PKcs (product #4602), ULK1 (product #8054)/p-ULK1 (product #5869), AMPK (product #5831)/p-AMPK (product #50081), PCNA (product #2586), Ki-67 (product #9449) and β-actin (product #58169) (Cell Signaling Technology, Danvers, MA, USA), Bax (catalog no. AF0120; Affinity, Changzhou, China), Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), and dorsomorphin (an inhibitor of the AMPK pathway; APExBIO, USA) were used following the manufacturer's instructions.

Cancer tissues and paired normal tissues were obtained from 28 patients with ESCC at the Affiliated Hospital of Jiangsu University (Zhenjiang, China) from 2010 to 2018. The Ethics Committee of the Affiliated Hospital of Jiangsu University approved the research. Firstly, the ESCC samples were confirmed by the Pathology Department, and then serial sections were processed and stained with anti-ACSS2 (1:200 dilution; cat. #sc-398559; Santa Cruz Biotechnology), anti-Ki-67 (1:1,000 dilution; product #9449, Cell Signaling Technology) and anti-PCNA (1:4,000 dilution; product #2586, Cell Signaling Technology) antibodies for the immunohistochemistry (IHC) assay. Ki-67 scores ranged from 0 to 100% as the percentage of positive cells within the area of invasive cells. The evaluation criteria for ACSS2 or PCNA were based on our previously published study (13). The proportion of positively staining tumor cells and the staining intensity were examined and scored by five independent pathologists, who were blinded to the patient clinical information as previously described (13). All methods, including the collection and use of patient samples, were performed in accordance with the relevant guidelines and regulations and all patients provided signed informed consent.

Knockdown of human ACSS2 and PCNA were performed using gene-specific siRNAs. These gene-specific siRNA and including control siRNA were purchased from Shanghai GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China), which were reconstituted in sterile DEPC water to a stock concentration of 20 µM, and transfected into TE-1 and ECA-109 cells using Lipofectamine 2000. The siRNA sequences were as follows: siRNA-ACSS2: Sense, 5′-UAUGCUUGGUGACAGGCUCAUCUCC-3′ and antisense, 5′-GGAGAUGAGCCUGUCACCAAGCAUA-3′; siRNA-PCNA: Sense, 5′-UAUGGUAACAGCUUCCUCCTT-3′ and antisense, 5′-GGAGGAAGCUGUUACCAUATT-3′. The inhibitory effect of the siRNA transfection was evaluated with RT-qPCR and western blotting. Upregulation of ACSS2 expression was performed by cloning ACSS2 full-length complementary DNA into a GV141 carrier plasmid, including the control plasmid group, which were purchased from Shanghai Genechem Co., Ltd. (Shanghai, China), and these plasmids were transfected into TE-1 and ECA-109 cells using Lipofectamine 2000. The upregulation effect of the plasmid was evaluated with RT-qPCR and western blotting. After transfection, the cells were subjected to other treatments for the indicated duration and then used for the subsequent assays.

RNA was extracted using RNAiso Plus in accordance with the manufacturer's instructions and reverse transcribed into cDNA using a reverse transcription reagent kit, according to the manufacturer's protocol (both from Takara Biotechnology Co., Ltd., Dalian, China). The RT-PCR reactions were prepared using a PCR kit (Takara Biotechnology Co., Ltd.) and performed on an Agilent Mx3000P™ Real-Time PCR System (Stratagene). The primers used were synthesized by Invitrogen (Invitrogen; Thermo Fisher Scientific, Inc.), and the sequences were as follows: ACSS2 primers, 5′-GGATTCCAGCTGCAGTCTTC-3′ (forward) and 5′-CAGCCAGCTCCTTCAGGTT-3′ (reverse); PCNA primers, 5′-CTGAAGCCGAAACCAGCTAGACT-3′ (forward) and 5′-TCGTTGATGAGGTCCTTGAGTGC-3′ (reverse); β-actin primers, 5′-TCACCCACACTGTGCCCATCTACGA-3′ (forward) and 5′-CAGCGGAACCGCTCATTGCCAATGG-3′ (reverse). Relative expression levels were calculated using the 2^−ΔΔCq method (38) and β-actin was used as an internal reference gene.

Cells of different treatment were collected by centrifugation with 1200 rpm in 5 min, processed and lysed in RIPA buffer supplemented with a protease inhibitor cocktail and PMSF (all purchased from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer instructions. Protein concentration was determined by BCA protein assay (CoWin Biotech Co., Ltd, Beijing, China), and 5 µl of protein were loaded and separated by 8 to 12% SDS-PAGE. After transfer to PVDF membranes, 5% BSA in TBST was used to block the membrane at room temperature for 1 h. The membranes were incubated with primary antibodies [ACSS2 (at 1:2,000 dilution); ATM, p-ATM, BRCA1, p-BRCA1, Bcl-xL, γH2AX, DNA-PKcs, ULK1, p-ULK1, AMPK, p-AMPK, PCNA, and Ki-67 (at 1:1,000 dilution); β-actin (at 1:2,000 dilution); Bax (at 1:500 dilution)] saturated with 5% BSA in TBST at 4°C overnight. On the next day, the membranes were incubated with HRP-conjugated anti-rabbit or -mouse antibody (at 1:5,000 dilution, product #7074 and #7076; Cell Signaling Technology) for 1 h at room temperature, and then exposed to enhanced ECL reagent (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China), imaged by an automatic ChemisScope-4300 imager (Clinx Science Instruments Co., Ltd., Shanghai, China) and data were analyzed with Fluor Chem FC3 software (Protein-Simple, USA).

Cell proliferation was examined using the CCK-8 Cell Counting Kit (Nanjing Vazyme Biotech Co., Ltd.) according to the manufacturer's instructions. Briefly, TE-1/ECA-109 and Het-1A cells were grown in a 96-well plate for 24 h, transfected with siRNA-ACSS2 or the negative control, and then cultured in normal medium or under NS. To measure the cell proliferation at 0, 24, 48, or 72 h after transfection, 10 µl of CCK-8 was added to each well for 1 h. Absorbance was measured at a wavelength of 450 nm by a microplate reader (BioTek, USA). Assays were repeated at least three times.

The effect of ACSS2 on cell cycle distribution was determined by flow cytometry. Briefly, cells subjected to the indicated treatments were harvested when they reached 80% confluence, washed with PBS and resuspended in 1 ml of DNA staining solution and 10 µl of permeabilization solution (MultiSciences Biotech Co., Ltd., Hangzhou, China). Following incubation for 30 min in the dark at room temperature, the cells were analyzed by flow cytometry at 72 h after interference. The fractions of cells in the G0/G1, S, and G2/M phases were analyzed. Cell apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection kit (MultiSciences Biotech Co., Ltd., Hangzhou, China) according to the supplier's protocol. Cells were seeded in 12-well plates (6×10⁴/well). The cells were harvested after transfection with siRNA or NC and with or without DDP (5 µg/ml) treatment for 24 h, and washed in cold PBS. Then, cells were resuspended in 500 µl of binding buffer. Next, 5 µl of Annexin V-FITC and 10 µl of PI working solution were added to each reaction system at room temperature for 5 min. Flow cytometric analysis was performed immediately on a flow cytometer (BD FACSCanto II; BD Biosciences) and data were analyzed with BD FACSDiva software (BD Biosciences). Each experiment was repeated at least three times.

After pretreatment with siRNA or NC, and with or without DDP (5 µg/ml) for the indicated time, ESCC monolayers were spun down on slides, and stained by a TUNEL Apoptosis Detection kit (#A113-01; Nanjing Vazyme Biotech Co., Ltd., Nanjing, China) for in situ detection of apoptosis according to the manufacturer's instructions. In brief, slides were further incubated in the prescribed mixture of enzyme and label solution, at 37°C for 1 h in a humidified chamber. The TUNEL-stained cells were counterstained with PBS containing 4′,6-diamidino-2-phenylindole (DAPI, 5 µg/ml) (#422801, BioLegend, USA) for 5 min, and counted to calculate the TUNEL indices. For immunofluorescence, after being washed 3 times with PBS and fixed with 4% paraformaldehyde, the cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min. The fixed cells were blocked in 5% BSA in PBS for 1 h at room temperature and incubated with primary antibodies against γH2AX (at 1:400 dilution; product #9718, Cell Signaling Technology), p-ATM (at 1:500 dilution; product #5883, Cell Signaling Technology) and PCNA (at 1:2,000 dilution; product #2586, Cell Signaling Technology) overnight at 4ºC. FITC-conjugated anti-mouse, Cy3-conjugated anti-rabbit secondary antibodies (1:100 dilution; cat. #SA00003 and #SA00009, Proteintech Group, Wuhan, China) were used to detect the bound primary antibody. Then, the slides were washed 3 times in PBS and incubated with DAPI for 5 min. Immunofluorescence results are representatives of at least three independent experiments. Images were captured on a fluorescence (BX51, Olympus, Japan) or laser confocal microscope (LSM800, ZEISS, Germany) with fixed settings between samples and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

All experimental data are presented as the means ± standard deviation (SD) from three independent experiments. Survival curves were generated by Kaplan-Meier analysis and tested for significance using the Breslow test. The χ²-test was used to explore the relationship between two variables. The differences between two groups were analyzed using Student's t-test, and comparisons in datasets containing multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by the Newman-Keuls post-test. Significant differences were determined using a threshold P-value of 0.05.

IHC staining analysis was used to measure ACSS2 expression in 28 pairs of human ESCC and adjacent normal tissues, and the results revealed that ACSS2 exhibited positive cytoplasmic staining. In cancer tissues, ACSS2 protein was highly expressed in central zones and hypercellular areas, while ACSS2-positive cells in adjacent noncancerous tissues were concentrated in stroma cells and the glandular epithelium at basal and lower levels (Fig. 1A). To investigate the potential role of NS caused by excessive proliferation or poor nutrient supply, we first examined the effect of limited serum on cell viability. As shown in Fig. 1B, the proliferation of TE-1 and ECA-109 cells was significantly decreased at 72 h after the replacement of both media with 1% FBS (NS; P=0.008 and 0.03), while the proliferation of Het-1A cells was decreased more significantly (P<0.01), suggesting that normal esophageal epithelial cells are more sensitive to nutrient supply. To study the role of ACSS2 in the adaptive mechanisms of ESCCs to NS, the transcription levels of ACSS2 were analyzed at different times in limited serum medium. ACSS2 expression showed a sharp increase at 12 h in the TE-1 and ECA-109 cells compared to that in the Het-1A cells (P=0.03) under NS. The mRNA levels reached 9- to 12-fold at 24 h (P<0.01), and the expression levels of ACSS2 mRNA in the TE-1 and ECA-109 cells tended to reach a plateau after 24 h, but the levels were still at a higher level than those in the Het-1A cells (P<0.01) (Fig. 1C). However, the mRNA and protein expression of ACSS2 in the normal esophageal epithelial Het-1A cells did not correlate at all times or under all conditions (Fig. 1C and D), thus effectively adapting to stress and effectively played an extraordinary role, which has been described as a characteristic of cancer cells. When ACSS2 was analyzed in the ESCC cell lines, it was found that ACSS2 protein was upregulated with an increasing period under NS (Fig. 1E). Similarly, ACSS2 was persistently upregulated under NS for more than 1 month (Fig. 1E). ACSS2 has been shown to be upregulated by glucose, lipid deprivation and hypoxia in different cancer cell lines (14,39,40). Together, these data indicate that the proliferation of ESCCs is less restricted than that of human esophageal squamous epithelial cells in response to low serum culture and that the upregulation of ACSS2 may help maintain ESCC cell survival under NS.

To elucidate the role of ACSS2 in ESCC cell viability, we first performed siRNA experiments. Data from Fig. 2B show that the specific siRNA treatment observably decreased the protein levels of ACSS2. CCK-8 assays were performed to investigate cell proliferation. The results showed that suppression of ACSS2 expression significantly affected the proliferation of TE-1 and ECA-109 cells compared to that of the control group (NC), regardless of whether the cells were grown in normal medium or under NS (P<0.05, Fig. 2A). As stated above, PCNA acts as a scaffold to recruit proteins for replication, recombination and DNA repair. siRNA-ACSS2 treatment led to a 15 and 18% decrease in PCNA transcript levels which was further decreased 21 and 28% in combination with NS in the TE-1 and ECA-109 cells, respectively. Since PCNA directly participates in proliferation, these data suggest that ACSS2 reactivated PCNA in both cell lines, especially under NS (Fig. 2B). This was further confirmed by using flow cytometry, demonstrating substantial increases in G2/M phase arrest in both cell lines upon siRNA-ACSS2 treatment; the histogram (right) shows that the percentage of cells in the G2/M phase in the siRNA-ACSS2 group was approximately 1.4-fold higher than that in the NC group under NS or not (P<0.01) (Fig. 2C). Notably, the G2/M DNA damage checkpoint is important for repairing DNA after replication (6,41–43); thus, its defect would lead to apoptosis or death. Most cells under apoptosis preferentially are labeled by TUNEL for fragmented DNA at later stages. To examine whether DNA damage increased after siRNA-ACSS2 treatment, TUNEL analysis was performed to determine apoptosis with or without cisplatin (DDP) treatment. At least five viewing fields were utilized to obtain each data point and a representative of three individual dose-dependent experiments is shown. The results showed that there was no difference in the TUNEL-positive cell ratio between the NC group and the siRNA-ACSS2 group in TE-1 or ECA-109 cells, suggesting that the downregulation of ACSS2 did not induce cell death. However, the level of TUNEL-staining was significantly increased on day 3 posttreatment with siRNA-ACSS2 compared with the NC when treated with DDP (10% FBS: 17.78±1.16 vs. 4.09±0.89%; 1% FBS: 27.14±1.23 vs. 5.45±1.07%; P<0.01) (Fig. 2D). Additionally, ACSS2 interference also significantly increased the percentage of early apoptotic cells compared to that in NC groups after DDP treatment, whether under NS or not (P<0.01), but the differences between the siRNA-ACSS2 and NC groups were not statistically significant at 72 h after transfection (Fig. 2E). To further confirm the role of ACSS2 in chemoresistance, plasmid transfection experiment were performed, and the data from Fig. 2F shows that specific plasmid treatment significantly increased the mRNA and protein levels of ACSS2 (P<0.01). Upregulation of ACSS2 expression reduced the percentage of early apoptotic cells in comparison to the NC groups after DDP treatment under NS or not (P<0.01) (Fig. 2G). These preliminary data indicate that ACSS2 is a factor that maintains DNA stability and may be associated with PCNA expression.

To investigate the potential mechanism of ACSS2-dependent ESCC chemoresistance under NS, the levels of PCNA, p-ATM, γH2AX and related proteins were assessed. The cooperation of ATM protein kinase and DNA-PKcs plays critical roles in the regulation of the DNA damage response and cellular homeostasis (44,45). As expected, among the siRNA-ACSS2 groups, the expression levels of p-ATM increased 4.5-fold under NS combined with DDP compared to 2.0-fold following DDP treatment alone. However, the downregulation of ACSS2 had no significant effects on DNA-PKcs or p-BRCA1 levels. Importantly, we also observed that both siRNA-ACSS2 and DDP influenced the accumulation of γH2AX, exhibiting obvious synergistic effects under NS. B-cell lymphoma-extra large (Bcl-xL) is a member of the Bcl-2 family, an anti-apoptotic protein in mitochondria that prevents the release of cytochrome c. As an apoptosis activator, Bax leads to a loss in membrane potential and the release of cytochrome c. Considering that the lower ACSS2 under NS is much more vulnerable to DNA damage, we further detected variations in Bcl-xL and Bax. However, the suppression of ACSS2 expression in the ESCCs had no significant effect on the protein content of Bcl-xL or Bax, whether under sufficient nutrition or deficiency. Interestingly, despite its inhibitory effect on Bcl-xL, the presence of DDP had no influence on Bax expression (Fig. 3A). As shown in Fig. 3B and C, changes in PCNA and p-ATM and γH2AX foci were detected in the ESCCs, represented by ECA-109 cells, after treatment with cisplatin. In regards to the p-ATM and PCNA signals, although the percentages of p-ATM-positive cells increased steadily after cisplatin, the frequencies of p-ATM foci peaked at 24 h after downregulation of the expression of ACSS2; 89 to 95% of ESCCs with increased p-ATM showed decreased PCNA staining after low-serum culture for 48 h (Fig. 3B). The changes in the γH2AX signals were similar to those of the p-ATM foci, and the percentages of PCNA were steadily depended on the levels of ACSS2, but the intensity of γH2AX was stronger at 24 h post damage (Fig. 3C). These data suggest that nuclear-wide γH2AX expression, p-ATM responses and PCNA expression are related to ACSS2 expression in ESCCs after cisplatin treatment. More importantly, the effective interference of PCNA combined with DDP treatment also increased γH2AX and p-ATM expression especially during NS (Fig. 3D), indicating that PCNA plays a vital role as a scaffold associated with ACSS2-regulated DNA repair in ESCCs. The formation of PCNA, γH2AX, and p-ATM in the DNA damage response is related to the activation of the AMPK signaling pathway in multiple cancer cells (1,46,47). Similarly, our results showed that the significant phosphorylation of AMPK was induced by culture of TE-1 and ECA-109 cells under NS. The inhibition of ACSS2 accompanied by the significant downregulation of p-AMPK showed a decreasing tendency, whereas there was no significant difference in p-ULK1, which mediates autophagy (Fig. 3E). Furthermore, the levels of PCNA and ACSS2 were significantly affected after dorsomorphin treatment, showing an even stronger inhibition of ACSS2 (Fig. 3F). These results indicated that the interaction of ACSS2 with AMPK signaling mediates the stabilization of PCNA and DNA repair.

To explore the association between ACSS2 and ESCC development, we further examined the association of ACSS2 expression with the clinicopathological characteristics of ESCC patients. The results indicated that the level of ACSS2 was higher in ESCC tissues, than that in the adjacent normal tissues (3.23±0.55 vs. 1.56±1.36; P<0.05, Figs. 1A and 4A). To determine the correlation of ACSS2 and PCNA expression, we analyzed samples from two independent cohorts of ESCC patients. An ACSS2 staining score >3 in the cohort was considered high, while a score £3 in the cohort was considered low, and cohorts 1 and 2 included 15 and 13 patients, respectively. We found that the strong intensity of ACSS2 increased the expression of PCNA in ESCC tumors (Fig. 4B). However, the clinicopathological correlation analysis revealed no positive correlation between elevated ACSS2 levels and age, gender, tumor diameter, lymph node metastasis, TNM stage and differentiation. Notably, the expression of ACSS2 was higher in tumors with high PCNA expression compared to those with low PCNA expression (P=0.0163) and was higher in tumors with Ki-67 overexpression (P<0.01, Fig. 4C, Table I). Furthermore, to establish a more sensitive model for predicting the outcomes of patients with ESCC, we combined ACSS2 expression and PCNA or Ki-67 intensity to create a prognostic scoring system. Based on the IHC scores, the patients were stratified into two subgroups (Ki-67 and PCNA, respectively): A low subgroup for low IHC-positive rates or scores (<50% or ≤3) and a high subgroup for high IHC-positive rates or scores (≥ 50% or >3). The analysis showed that the predictive value of ACSS2 alone was higher than that of PCNA (P=0.0311 vs. P=0.4012; Fig. 4D and E), and a combination of ACSS2 and PCNA revealed a better prognostic value (P=0.0446; Fig. 4G). In addition, the combination of Ki-67 and ACSS2 was better than that for Ki-67 or ACSS2 alone (P=0.0166; Fig. 4F and H). These results suggest that the combination of ACSS2 and PCNA expression could establish a better predictive model for the overall survival of ESCC patients.