Targeting the YB-1/PD-L1 Axis to Enhance Chemotherapy and Antitumor Immunity

Hepatocellular carcinoma (HCC) is the second-leading cause of cancer-related death worldwide (1). Despite advances in hepatic resection and transplantation, the long-term survival of HCC patients remains unsatisfactory due to disease recurrence after surgical resection and poor patient response to conventional chemotherapies, including transcatheter arterial chemoembolization (TACE) or the targeted agent sorafenib (2, 3). Chemotherapies, such as doxorubicin, cisplatin, and 5-FU treatments, have low efficacy in HCC compared with other solid cancers because of toxicity and chemoresistance (4). A greater understanding of the molecular mechanisms of tumor chemoresistance, and how to overcome them, is required.

Immunotherapy for HCC involves immune-checkpoint inhibitors, antibody-based therapy, and peptide-based vaccines (5–7). Programmed death-1 (PD-1) is an immunomodulatory receptor expressed in T-cell membranes (8). Programmed death-1 ligand 1 (PD-L1), which binds to the PD-1 receptor, is expressed in tumor and/or macrophage cells (9, 10). The PD-L1/PD-1 interaction inhibits CD8⁺ cytotoxic T-lymphocyte proliferation and survival and affects the function of tumor-infiltrating T cells. This effect, in turn, suppresses the immune system and causes peripheral immune resistance in cancer patients (11). PD-L1 is upregulated in cancer cells in response to chemotherapy agents, resulting in a decrease in T-cell–promoted immune response evasion in these cancer cells (12). However, the mechanism of PD-L1 upregulation remains unclear.

Y-box binding protein 1 (YB-1) is a member of the family of cold-shock binding proteins that bind to the nucleotide sequence invert CCAAT to regulate gene expression (13). Previous studies have shown that YB-1 promotes the expression of multidrug resistance genes, which enhances drug resistance in tumors (13, 14). Here, we demonstrate that tumor chemoresistance induces an immunosuppressive microenvironment and immune evasion through YB-1–mediated PD-L1 upregulation. In response to antitumor chemotherapy, YB-1 drives signaling in the tumor immunosuppressive microenvironment and immune evasion pathway. Furthermore, knockdown of YB-1 reverses HCC chemoresistance via T-cell activation in the tumor microenvironment due to blocked PD-L1 expression.

The HCC cell lines PLC, HCCLM6, and HepG2 cells were purchased from The Cell Bank of Chinese Academy of Science in 2017. HepG2-DoxR cells were purchased from Shanghai GeneChem Co., Ltd., in 2017. Hepa1-6 cells were purchased from China Center for Type Culture Collection in 2017. All the cell lines were authenticated by the original suppliers and routinely authenticated by morphology, growth, antibiotic resistance (where appropriate), and routinely screened for Mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza, Cat. No. LT07-318). Cells were maintained in a medium of RPMI-1640 or DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and were kept in 5% CO₂ incubator at 37°C. All the cells were maintained in culture for less than 4 weeks continuously, and fresh frozen stocks were used at the initiation of each experiment. Human GV248 lentivirus vectors, GV248-shYB-1-1 and GV248-shYB-1-2, were purchased from Shanghai GeneChem Co., Ltd. Human GV341 lentivirus vector and GV341-flag-YB-1 were purchased from Shanghai GeneChem Co., Ltd. Mouse pGCSIL lentivirus vector and pGCSIL-shYB-1-1 and pGCSIL-shYB-1-2 were purchased from Shanghai GeneChem Co., Ltd. Cells infected with GV248-shYB-1-1, GV248-shYB-1-2, pGCSIL-shyb-1, pGCSIL-shyb-2, or GV341-flag-YB-1 following by the selection with puromycin. Human and mouse PD-L1 luciferase reporters containing the promoter region of PD-L1 were purchased from Shanghai GeneChem Co., Ltd.

A normalized mRNA expression data set for HCC was downloaded from the cBioPortal for cancer genomics and used to evaluate expression of YB-1 and PD-L1 transcripts (Liver Hepatocellular Carcinoma (TCGA, Provisional)). This data set includes mRNA profiles for 373 HCC cases including 50 paired normal cases. Overall survival (OS) analysis was calculated for these transcripts for HCC cases. All the detailed information of HCC patients including pathology diagnosis, clinical stage, and survival data can be downloaded from the cBioPortal website (http://www.cbioportal.org/).

Paired cancer and adjacent noncancer paraffin tissue sections were purchased from Shanghai Outdo Biotech., Ltd. (HLiv-HCC180Sur-05 for HCC). Information including pathology diagnosis, clinical stage, and survival data was directly downloaded from online (http://www.superchip.com.cn). IHC was performed with antibodies against YB-1 (20339-1-AP, Proteintech), PD-L1 (17952-1-AP, Proteintech), and CD8A (ab199016, Abcam), then examined by two independent pathologists with no knowledge of the specimens. The immunoreactive score (IRS) gives a range 0–12 as a product of multiplication between positive cells proportion score (0–4) and staining intensity score (0–3). The IRS (0–1, negative; 2–3, mild; 4–8, moderate; 9–12, strong positive; ref. 15) measured expression of YB-1, PD-L1, and CD8A. The IHC data were evaluated by two blinded pathologists.

Cells were lysed in RIPA buffer with proteinase inhibitors. Total protein (20–40 μg) from each sample was electrophoresized on 8% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked in 5% nonfat milk and probed with primary antibodies overnight at 4°C. The membranes were washed and probed with secondary antibody conjugated to horseradish peroxidase and developed with enhanced chemiluminescence (Thermo Scientific).

PD-L1 promoter regions that contained predicted Y-box binding sites were amplified by PCR from genomic DNA, and the PCR fragments were inserted into untranslated region (UTR) downstream of the luciferase gene in the pMIR-reporter luciferase vector (Ambion). Luciferase reporter plasmid, β-galactosidase (β-gal) plasmid, and Flag-YB-1 or negative control were cotransfected into cells using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured 48 hours after transfection using β-gal for normalization.

ChIP assays were performed according to the protocol of the Upstate Biotechnology as described (16). The primer sequence for human PD-L1 promoter is described in the supplementary experimental procedures. The primer sequence for human PD-L1 promoter is P1(−70 to −300): 3′-CAAGGTGCGTTCAGATGTTG-5′, 3′-TCCTGACCTTCGGTGAAATC-5′; P2(−358 to −552): 3′-AGGAAGTCACAGAATCCACGA-5′, 3′-CGTCCCCCTTTCTGATAAAA-5′; P3(−600 to −793): 3′-TTCAGCGCGGGATAATACTT-5′, 3′-AACTTCCCATCCCGAGCTAC-5′; P4(−802 to −951): 3′-GAAGGAAAGGCAAACAACGA-5′, 3′-TGCTCTCTTTTCTCGAACTCC-5′; P5(−965 to −1114): 3′-AAGGCAGTGTCAGCCTCAAT-5′, 3′-CTGCAGTTCAAAATACTGCATAA-5′; P6(−1121 to −1301): 3′-TCACCAAAGTTGGGAAGTCA-5′, 3′-GCAGGAGCATGGAGTTCTCT-5′; P7(−1331 to 1497): 3′-GTTTATGCCCTGGGTCTTGA-5′, 3′-TGTGGCAATACAGTTTACAGGT-5′.

The production of T-cell cytokine was detected by cytokine ELISA using the human or mouse ELISA kit (ExCell Bio) according to the manufacturers' instructions. Enzyme activity was measured at 450 nm using a plate reader.

Blood DCs were isolated from healthy human donors using a Human Blood Dendritic Cell Isolation Kit II (Miltenyi Biotec, No. 130-091-379) following the manufacturer's instruction. Blood DC isolation was approved by the Institutional Review Board of Tianjin Medical University Cancer Institute and Hospital (IRB: E2013139).

The HepG2-DoxR/T cell coculture model was done as described (17). Peripheral blood mononuclear cells (PBMC) were isolated using Lymphoprep density gradient centrifugation (Accurate Chemical). PBMCs were plated at a density of 2 × 106 well-1 in 6-well plates and stimulated with HepG2-DoxR cell lysate, anti-CD3e (10 mg/mL), and anti-CD28 (2 mg/mL) for 48 hours to promote T-cell activation. The T-cell activation protocol was provided by eBioscience (http://www.ebioscience.com/cell-type/t-cells.htm). HepG2-DoxR cells stably expressed YB-1. Stimulated PBMCs were subsequently harvested and purified by Lymphoprep density gradient centrifugation, and cocultured with the HepG2-DoxR cells at a 10:1 ratio for 16 hours. The HepG2-DoxR cells were then sorted by flow cytometry (FCM). The relative expression of PD-L1 in the HepG2-DoxR cells was determined by qRT-PCR assay. Coculture media were assayed for TNFa, IFNg, IL10, IL1b, and TGFb using a cytokine ELISA assay.

T-cell apoptosis assay was done as described (17). PBMCs were plated at a density of 2 × 10⁶ cells/well in 6-well plates and stimulated with HepG2-DoxR cell lysate, anti-CD3e (10 mg mL/mL), and anti-CD28 (2 mg/mL) for 48 hours to promote T-cell activation. HepG2-DoxR cells stably expressed YB-1. Stimulated PBMCs were subsequently harvested and purified by Lymphoprep density gradient centrifugation, and then cocultured with HepG2-DoxR cells at a 10:1 ratio for 16 hours. Ten micrograms per milliliter anti–PD-L1 were added to the indicated wells to examine PD-1-specific CD8⁺ T-cell apoptosis. The PBMCs were subsequently harvested and stained with PE-conjugated PD-1, Alexa Fluor 488-conjugated annexin V and APC-conjugated CD8 antibodies, respectively. PD-1–dependent CD8⁺ T-cell apoptosis was calculated as a percentage of annexin V⁺ cells in the gated PD-1⁺/CD8⁺ population.

HepG2-DoxR (HepG2-doxorubicin resistance) cells were cultured, harvested, and suspended in PBS. For flank injections, a total of 0.2 mL containing 5 × 10⁶ cells were injected subcutaneously into the flank of 4- to 6-week-old athymic nude mice (Chinese Academy of Sciences at Beijing, China). Mice were housed in groups of five under specific pathogen-free conditions with unlimited access to food and water. Ten days after injection, the mice were treated with doxorubicin. Tumor dimensions were measured every 3 days, and volumes were calculated using the formula: V = L × W2 × 0.52, where L and W are the long and short diameters of the tumor, respectively. All the experimental protocols and animal care were approved by the Institutional Review Board of Tongji Medical College, Huazhong University of Science and Technology.

Hepa1-6 cells were cultured, harvested, and suspended in PBS. For flank injections, a total of 0.2 mL containing 5 × 10⁶ cells were injected subcutaneously into the flank of 6- to 8-week-old C57BL/6 mice (Chinese Academy of Sciences at Beijing, China). Mice were housed in groups of five under specific pathogen-free conditions with unlimited access to food and water. Ten days after injection, the mice were treated with doxorubicin. Tumor growth and survival were assessed. Tumors tissues were used for characterization of myeloid-derived suppressor cells (MDSC), Treg, and CD8⁺ T-cell memory subsets. Mice serum was assayed for IL10, TNFα, IFNγ, and TGFβ by cytokine ELISA assay. All the experimental protocols and animal care were approved by the Institutional Review Board of Tongji Medical College, Huazhong University of Science and Technology.

Data were graphed using GraphPad Prism version 6.01 software. Normally distributed continuous variables are expressed as means ± standard error of the mean (SEM), abnormally distributed continuous variables are expressed as expressed as median. For equivalent variables with a normal distribution, the independent Student t test was utilized to compare two groups. The Mann–Whitney U test was used to compare abnormal distributional variables between two groups. One-way ANOVA followed by Tukey multiple comparison test was used to compare multiple groups. Correlation between variables with normal distribution was assessed using Pearson correlation test, whereas abnormal distributions were assessed using Spearman correlation test. Survival rate was plotted using Kaplan–Meier method and analyzed using the log-rank test. A P < 0.05 was considered statistically significant.

YB-1 is overexpressed in various human cancers, such as breast cancer, colon cancer, lung cancer, gastric cancer, esophageal cancer, and glioblastoma (13). To assess YB-1 expression in HCC, we performed in silico analyses of YB-1 expression using a TCGA data set composed of 373 HCC cases, including 50 paired cases. As shown in Fig. 1A, YB-1 mRNA expression was significantly higher in the HCC tissues than in the normal tissues, and YB-1 expression positively correlated with tumor grade (Spearman rank correlation, r = 0.385, P < 0.001) and stage (Spearman rank correlation, r = 0.419, P < 0.001). A Kaplan–Meier analysis was conducted to determine whether the OS of patients was associated with YB-1 expression in tumors. YB-1 expression was used to assign patients to the high (upper 50th percentile) or low (lower 50th percentile) expression groups. The Kaplan–Meier analysis indicated that the patients with tumors that expressed most YB-1 (upper 50th percentile) had a shorter OS (P = 0.0001; median survival time: high, 41.75 months vs. low, 83.18 months; Fig. 1A).

YB-1 promotes the expression of multidrug resistance genes, which leads to enhanced drug resistance in tumors (13, 14). However, whether YB-1 is also involved in the induction of tumor immune evasion remains unclear. To identify the relationship between YB-1 and the signaling pathway of HCC cells, we first performed gene set enrichment analysis (GSEA) to analyze the gene sets altered by YB-1 expression in the HCC TCGA data set. Our results showed that the T-cell receptor [normalized enrichment score (NES) = 1.52, FDR = 0.03, P < 0.001], B-cell receptor (NES = 1.38, FDR = 0.098, P = 0.015), and Toll-like receptor (NES = 1.31, FDR = 0.153, P = 0.02) signaling pathways were associated with high YB-1 expression (upper 50th percentile; Fig. 1B), suggesting that YB-1 is involved in the regulation of the tumor immune response in HCC. Using the data from the HCC TCGA database, we evaluated the correlation between YB-1 expression and immune-checkpoint interactions. As shown in Fig. 1C and Supplementary Fig. S1A, YB-1 expression positively correlated with PD-L1 (Pearson rank correlation, r = 0.268, P < 0.001), PD-1 (Pearson rank correlation, r = 0.204, P < 0.001), and CTLA4 (Pearson rank correlation, r = 0.234, P < 0.001) expression. To confirm the interaction between YB-1 and PD-L1, we examined YB-1 and PD-L1 protein expression with IHC staining in HCC specimens. Similar to our previous result, we observed that YB-1 positively correlated with PD-L1 expression (Spearman rank correlation, r = 0.234, P < 0.001; Fig. 1D). YB-1 and PD-L1 protein expression was significantly higher in the HCC tissues than in the normal tissues (Fig. 1D; Supplementary Fig. S1B–S1D). The Kaplan–Meier analysis indicated that patients with tumors that expressed YB-1 highly (IRS > 9) had decreased OS (P = 0.0316; median survival time: high, 17 months vs. low, 43 months; Fig. 1D).

The positive correlation between YB-1 and PD-L1 expression raised the possibility that YB-1 may function through interactions with PD-L1. To test this hypothesis, we performed Western blot analysis using PLC, HCCLM6, and HepG2 cells. YB-1 was stably overexpressed in these cells, and PD-L1 expression was then determined. As shown in Fig. 2A, YB-1 overexpression in these HCC cells increased the protein expression of PD-L1.

It has previously been shown that YB-1 can bind to target gene promoters at the Y-box sequence (13). To further clarify the mechanism of YB-1 in the regulation of PD-L1, we determined that the human PD-L1 proximal promoter region [−1 to −1.5 kb upstream of the transcription start site (TSS)] had a Y-box that could be bound and activated by YB-1 (Fig. 2B). To verify the regulation of PD-L1 by YB-1, we performed luciferase reporter assays. HCCLM6 and HepG2 cells were cotransfected with a YB-1-overexpression vector and a luciferase reporter driven by three different pieces of the PD-L1 promoter region. As shown in Fig. 2B, luciferase expression was higher in cells cotransfected with YB-1/PD-L1–luciferase (C-luc: −1 to −1.5 kb upstream of the TSS, containing the Y-box) than in cells cotransfected with YB-1/PD-L1–luciferase (A-luc: −1 to −0.5 kb upstream of the TSS, lacking the Y-box or B-luc: −1 to −1 kb upstream of the TSS, lacking the Y-box) plasmids or the negative control. Moreover, chromatin immunoprecipitation (ChIP) was performed in HCCLM6 and HepG2 cells (Fig. 2C; Supplementary Fig. S2A) and indicated that YB-1 could activate PD-L1 transcription and revealed that YB-1 could bind to the designated regions of the PD-L1 promoter (P6: −1,121 to −1,301, containing the Y-box).

To identify the relationship between the YB-1 and multidrug resistance in HCC, we used GSEA to analyze the gene sets altered by YB-1 expression in the HCC TCGA data set. Our results showed that doxorubicin-, docetaxel-, cisplatin-, gemcitabine-, dasatinib-, and gefitinib-resistance signaling pathways all positively associated with high YB-1 expression (upper 50th percentile) (Fig. 2D; Supplementary Fig. S2B). The NES demonstrated that the doxorubicin-resistance signaling pathway was the most changed pathway. Therefore, we used the HepG2-DoxR cell model to determine whether YB-1 and PD-L1 were involved in HCC chemoresistance. As shown in Fig. 2D, expression of YB-1 and PD-L1 was significantly increased in the HepG2-DoxR cells compared with the HepG2 cells. YB-1 knockdown in HepG2-DoxR cells resulted in decreased PD-L1 expression (Fig. 2D). FCM analysis demonstrated that cell-surface PD-L1 expression was significantly higher in HepG2-DoxR cells than in HepG2 cells (Fig. 2D). YB-1 knockdown in HepG2-DoxR cells resulted in decreased cell-surface PD-L1 expression (Tukey multiple comparison test, α = 0.05; ***, P < 0.001; ns, no significance; Fig. 2D).

Previous studies have shown that YB-1 enhances drug resistance in tumors. To confirm the function of YB-1 in doxorubicin resistance, we knocked down YB-1 in HepG2-DoxR cells followed by doxorubicin treatment. As shown in Supplementary Fig. S2C, HepG2-DoxR cells are refractory to doxorubicin treatment. However, YB-1 knockdown induced inhibition of growth to doxorubicin in HepG2-DoxR cells (Tukey multiple comparison test, α = 0.05, ***P < 0.001; Supplementary Fig. S2C), indicating that YB-1 knockdown could revert doxorubicin resistance of HepG2-DoxR cells. Next, we transplanted HepG2-DoxR cells with stable knockdown of YB-1 into athymic nude mice. The athymic nude mice were then treated with doxorubicin. A similar result was observed. As shown in Supplementary Fig. S2D, HepG2-DoxR tumors are refractory to doxorubicin treatment. YB-1 knockdown induced inhibition of HepG2-DoxR tumor growth to doxorubicin (Tukey multiple comparison test, α = 0.05; *, P < 0.05; ***, P < 0.001; ns, no significance; Supplementary Fig. S2D).

The PD-L1/PD-1 immune-checkpoint pathway preferentially modulates the secretion of regulator cytokines in the tumor microenvironment (18). Therefore, we first assessed whether YB-1–induced PD-L1 overexpression in HCC affected the tumor microenvironment. We established a HepG2-DoxR/T-cell coculture model. Cocultured T cells with HepG2-DoxR cells with stably knockdown YB-1 increased TNFα and IFNγ expression and decreased IL1β, IL10, and TGFβ secretion in the media. Furthermore, inhibition of PD-L1 activity with a PD-L1 blocking antibody (anti–PD-L1) abolished YB-1–mediated cytokine secretion (Fig. 3A). These findings suggested that YB-1 modulated cytokine secretion in the tumor microenvironment via promotion of a PD-L1–dependent pathway.

IFNγ, which is produced by inflammatory cells in the tumor microenvironment, stimulates PD-L1 expression in tumor cells (19). To determine whether YB-1 affected T-cell function via inhibition of PD-L1 expression, we used the HepG2-DoxR/T-cell coculture model. Coculture of T cells with IFNγ pretreated HepG2-DoxR cells increased apoptosis in PD-1⁺/CD8⁺ T cells compared with T cells cultured with untreated HepG2-DoxR (Fig. 3B). However, in HepG2-DoxR cells with stable knockdown of YB-1, apoptosis in PD-1⁺/CD8⁺ T cells was abolished (Fig. 3B). The effect of anti–PD-L1 was similar to the effect of YB-1 knockdown-mediated apoptosis in PD-1⁺/CD8⁺ T cells, which suggests that YB-1 knockdown repressed IFNγ-induced PD-L1–associated CD8⁺ T-cell apoptosis by blocking PD-L1. These findings also suggest that chemoresistance-induced immune resistance could be reversed by YB-1 knockdown.

CD8⁺ T cells function in antitumor immunity in many types of cancers (20, 21). CD8A (cytotoxic/suppressor T-cell marker) identified cytotoxic/suppressor T cells that interact with major histocompatibility complex class I–bearing targets. To evaluate the abundance of CD8⁺ T cells in HCC, we performed in silico analyses of CD8A expression using the HCC TCGA data set. As shown in Fig. 4A, CD8A mRNA was lower in HCC tissues than in normal tissues. Kaplan–Meier analysis indicated that patients with tumors that highly expressed CD8A (upper 50th percentile) had a greater OS (P = 0.0133; median survival time: high, 81.87 months vs. low, 46.20 months; Fig. 4A). We then examined CD8A protein in HCC specimens. Similar to the mRNA analysis, CD8A protein was significantly less in HCC tissues (Fig. 4B). The Kaplan–Meier analysis indicated that patients with tumors that expressed CD8A highly had a better OS (P = 0.035; median survival time: high, 46 months vs. low, 16 months; Fig. 4B; Supplementary Fig. S3A). We also observed that CD8A negatively correlated with YB-1 (Spearman rank correlation, r = −0.2227, P = 0.03) and PD-L1 (Spearman rank correlation, r = −0.2856, P = 0.01) expression (Fig. 4B). These findings indicated that CD8⁺ T-cell expression positively correlated with HCC prognosis.

To confirm the function of YB-1 in vivo, we transplanted Hepa1-6 cells with stable knockdown of YB-1 into C57BL/6 mice. The C57BL/6 mice were then treated with anti-CD8. The survival rate of the C57BL/6 mice was shown in Supplementary Fig. S3B. Mice with YB-1–knockdown tumors survived significantly longer than the control group, whereas anti-CD8 treatment abrogated the YB-1 knockdown-mediated survival benefit (log-rank test; *, P < 0.05; **, P < 0.01; ns, no significance). We next determined the tumor volume in these mice. As shown in Fig. 4C, YB-1 knockdown significantly decreased the tumor volume compared with control group, whereas anti-CD8 treatment impaired the efficacy of YB-1 knockdown (Tukey multiple comparison test, α = 0.05, *, P < 0.05; **, P < 0.01; ns, no significance). These findings suggested that CD8⁺ T cells are required for the efficacy of YB-1 knockdown.

We also evaluate the function of YB-1 in Hepa1-6 tumor-bearing athymic nude mice, which are deficient in mature T lymphocytes. We observed that knockdown of YB-1 in Hepal-6 cells reduced tumor volume in athymic nude mice (Fig. 4D). However, there was no effect of anti-CD8 treatment on tumor formation, indicating that the function of YB-1 in tumor partly dependent on immune response. Finally, we conducted MTT assays in Hepal-6 cells with stable knockdown of YB-1 and found that knockdown of YB-1 in Hepal-6 cells decreased cell viability (Supplementary Fig. S3C).

We analyzed PD-L1 protein expression in mouse Hepa1-6 HCC cells with stable YB-1 knockdown. As shown in Supplementary Fig. S4A, YB-1 knockdown decreased PD-L1 in Hepa1-6 cells. Meanwhile, FCM analysis indicated that cell-surface PD-L1 expression was lower in Hepa1-6 cells with YB-1 knockdown (Tukey multiple comparison test, α = 0.05, **, P < 0.01; ns, no significance; Supplementary Fig. S4B). Next, we found that the mouse PD-L1 proximal promoter region (−1 to −2 kb upstream of the TSS) also had a Y-box that could be bound and activated by mouse YB-1 during transcription (Supplementary Fig. S4C). To verify the regulation of PD-L1 by YB-1, we performed luciferase reporter assays. As shown in Supplementary Fig. S4C, luciferase expression was lower in cells cotransfected with the YB-1–knockdown vector/PD-L1–luciferase plasmid (B-luc: −1 to −1.7 kb from the TSS, containing the Y-box) than in cells cotransfected with the YB-1–knockdown vector/PD-L1–luciferase plasmid (A-luc: −1 to −1 kb from the TSS, lacking the Y-box) or the negative control (Tukey multiple comparison test, α = 0.05, *, P < 0.05; **, P < 0.01;ns, no significance). Moreover, ChIP in Hepa1-6 cells (Supplementary Fig. S4D) illustrated that mouse YB-1 could activate the transcription of mouse PD-L1 and revealed that mouse YB-1 could bind to the designated regions of the mouse PD-L1 promoter (P5: −1,432 to −1,654 upstream of the TSS, containing the Y-box).

Next, to investigate the effects of YB-1 knockdown treatment in HCC, we injected YB-1–knockdown Hepa1-6 cells into syngeneic C57BL/6 mice and then administered doxorubicin or anti–PD-L1 treatment. The tumor volumes are shown in Fig. 5A and Supplementary Fig. S5A; doxorubicin treatment significantly decreased the tumor volumes in C57BL/7 mice with YB-1–knockdown tumors, which indicated that YB-1 knockdown might enhance the efficacy of doxorubicin (Tukey multiple comparison test, α = 0.05; ***, P < 0.001; Fig. 5A; Supplementary Fig. S5A). YB-1 knockdown alone exhibited inhibitory effects on tumor growth, which indicated that YB-1 might function as a tumor oncogene in HCC (Tukey multiple comparison test, α = 0.05; ***, P < 0.001; Fig. 5A; Supplementary Fig. S5A). Furthermore, anti–PD-L1 treatment impaired the YB-1 knockdown-mediated benefit in the doxorubicin treatment group, demonstrating that PD-L1 was required for the efficacy of the YB-1 knockdown treatment (Tukey multiple comparison test, α = 0.05, *, P < 0.05; Fig. 5A; Supplementary Fig. S5A). To address whether combination therapy resulted in an induction of sustained antitumor immunity, C57BL/6 mice were rechallenged 30 days after complete tumor rejection with a higher dose of Hepa1-6 tumor cells on the opposite flank. No palpable tumors were detected on the treated mice after a few weeks, whereas tumors on naïve mice were palpable after 10 days (Fig. 5B).

MDSCs and regulatory T cells (Treg) are components of the tumor immunosuppressive microenvironment (22). Therefore, we examined the abundance of CD8⁺ T cells, MDSCs, and Tregs in Hepa1-6 tumors. As shown in Fig. 5C, doxorubicin treatment with YB-1–knockdown tumors significantly increased CD8⁺ T-cell and decreased MDSCs and Tregs (Tukey multiple comparison test, α = 0.05; **, P < 0.01). The addition of anti–PD-L1 did not bring extra antitumor effect because YB-1 knockdown-mediated benefit partly depended on the PD-L1 pathway (Tukey multiple comparison test, α = 0.05; ns, no significance).

To further demonstrate that PD-L1 was required for the efficacy of the YB-1 knockdown treatment, we also overexpressed PD-L1 with simultaneous knockdown of YB-1 in Hepa1-6 cells. Tumor volumes were shown in Supplementary Fig. S5B. YB-1 knockdown alone significantly decreased the tumor volume in experimental mice compared with control mice, whereas overexpression of PD-L1 impaired the efficacy of YB-1 knockdown (Tukey multiple comparison test, α = 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance). Therefore, we examined the abundance of CD8⁺ T cells in Hepa1-6 tumors using a FACScan flow cytometer. As shown in Supplementary Fig. S5B, YB-1 knockdown alone significantly increased the CD8⁺ T-cell expression. Overexpression of PD-L1 impaired the efficacy of YB-1 on CD8⁺ T-cell expression (Tukey multiple comparison test, α = 0.05, ***, P < 0.001; ns, no significance). These findings suggested that PD-L1 was required for the efficacy of YB-1 in Hepa1-6 tumors. Furthermore, FCM analysis showed that YB-1 knockdown in Heap1-6 tumors resulted in decreased PD-L1 (Tukey multiple comparison test, α = 0.05; ***, P < 0.001; ns, no significance).

IFNγ expression is a surrogate marker for cytotoxic T cells (CTL; ref. 23). As shown in Fig. 6A, doxorubicin treatment significantly increased the number of IFNγ⁺/CD8⁺ T cells in C57BL/6 mice with YB-1–knockdown tumors (Tukey multiple comparison test, α = 0.05; **, P < 0.01). Furthermore, anti–PD-L1 treatment impaired the YB-1 knockdown–mediated benefit in the doxorubicin treatment group (Tukey multiple comparison test, α = 0.05; ns, no significance; Fig. 6A). FCM analysis showed that PD-L1 expression was significantly increased following doxorubicin treatment in Hepa1-6 tumors (Fig. 6B). YB-1 knockdown in doxorubicin-treated Hepa1-6 tumors resulted in decreased PD-L1 (Tukey multiple comparison test, α = 0.05; ***, P < 0.01; Fig. 6B). Furthermore, as shown in Fig. 6C, doxorubicin treatment did not inhibit the secretion of IL10 or TGFβ in mice with tumors (Tukey multiple comparison test, α = 0.05; ns, no significance). However, doxorubicin treatment significantly inhibited the secretion of IL10 and TGFβ in the mice with YB-1–knockdown tumors (Tukey multiple comparison test, α = 0.05; **, P < 0.01). Anti–PD-L1 treatment impaired the YB-1 knockdown–mediated benefit in the doxorubicin treatment group (Tukey multiple comparison test, α = 0.05; ns, no significance). Meanwhile, doxorubicin treatment in mice with YB-1–knockdown tumors significantly increased the secretion of TNFα and IFNγ (Tukey multiple comparison test, α = 0.05; **, P < 0.01), whereas the addition of anti–PD-L1 does not contribute to the synergistic effect of the combination group (Tukey multiple comparison test, α = 0.05, ns, no significance; Fig. 6C). This finding suggested that YB-1 knockdown enhanced the efficacy of chemotherapy by regulating the secretions of cytokines. Taken together, these findings demonstrate that YB-1 knockdown may improve T-cell immune response (Fig. 6D).

Chemoresistance is a clinical problem that compromises treatment efficiency and ultimately results in cancer treatment failure (24). Studies have demonstrated an activation of the PD-1/PD-L1 pathway in chemoresistant tumor cells, which results in decreased tumor-specific T-cell activity that potentially promotes tumor immune evasion (25–30). However, it is still unknown how the signaling events regulate the PD-1/PD-L1 pathway in resistant cancer cells, or how chemotherapy affects antitumor immunity and causes chemoresistance. YB-1, characterized by the presence of the cold-shock domain, has been reported to induce chemoresistance in cancer therapy (16). YB-1 is upregulated in chemoresistant tumor cells, and YB-1 overexpression in drug-sensitive tumor cells induced multidrug resistance (31). However, the potential function of YB-1 in the induction of chemotherapy-based immune evasion was unclear. In this study, we demonstrated that high YB-1 and PD-L1 expression was both associated with a chemoresistant phenotype in HCC. YB-1 directly regulated PD-L1 transcription. Knockdown of YB-1 in HCC chemoresistant cells resulted in decreased PD-L1 expression. YB-1 promoted PD-L1–associated CD8⁺ T-cell apoptosis and regulated T-cell cytokine secretions via upregulation of PD-L1. These findings indicate that endogenous YB-1 plays a role in the development of chemoresistance associated with an induced tumor immunosuppressive microenvironment and immune evasion. Suppression of PD-L1 may be possible by decreasing endogenous YB-1 expression, which would improve the therapeutic index of the chemotherapy-based tumor immunosuppressive microenvironment and immune evasion. Targeting the YB-1 signaling axis, which would simultaneously reverse both tumor immune evasion and multidrug resistance, may improve the antitumor response.

YB-1 plays a role in the acquisition of multidrug resistance via the upregulation of P-glycoproteins, which are MDR1 gene products (13, 14). Previous evidence indicates that high expression of P-glycoproteins is a mechanism of HCC chemoresistance to a range of chemotherapeutic drugs (32). In this study, we used GSEA to analyze the gene sets altered by YB-1 expression in the HCC TCGA data set. Our results showed that doxorubicin-resistance signaling pathway was the most changed signaling pathway. Therefore, we used the HepG2-doxorubicin-resistance cell model to determine the function and mechanism of YB-1 in a chemoresistance-based tumor immunosuppressive microenvironment and in immune evasion.

HCC is characterized by exhaustion of the tumor-specific CD8⁺ T-cell infiltration (33), which involves negative costimulatory molecules, such as PD-1 and CTLA4 (33). Therapies that disrupt negative signaling mechanisms may be therapeutic tools with the potential to restore activity of the tumor-specific CD8⁺ T-cell infiltration (33). In this study, we found that CD8⁺ T cells, which positively correlated with HCC prognosis, were required for the efficacy of YB-1 knockdown in HCC. YB-1 promoted PD-L1–associated CD8⁺ T-cell apoptosis. Knockdown of YB-1 enhanced the efficiency of chemotherapy due to increased CD8⁺ T-cell abundance. These findings indicate that YB-1 confers chemotherapy-based immune evasion by inhibiting CD8⁺ T-cell activation.

A parallel increase in MDSCs and Tregs contributed to the tumor immune-suppressive environment (34, 35). In this study, YB-1 knockdown treatment was likely a result of the downregulation of PD-L1 expression after YB-1 knockdown in Hepa1-6 tumor cells. The present findings indicate that PD-L1 is correlated with tumor-infiltrating lymphocytes (TIL) in an HCC tumor model, its upregulation suppressed T-cell activation. Our finding regarding the high frequency of Tregs and MDSCs in tumors that lack TILs is consistent with an association between Tregs and MDSCs with a poor outcome in human HCC. Although YB-1 knockdown resulted in moderate TIL proliferation, the Tregs and MDSCs were only reduced 5%–10% following YB-1 knockdown. Thereby, YB-1 knockdown resets the tumor immunosuppressive environment, which allowed for the activation of CD8⁺ T cells, the inhibition of MDSCs and Tregs, and the induction of proinflammatory cytokines, such as IFNγ and TNFα.

We identified a mechanism where YB-1 was the key signaling molecule in chemoresistance-based tumor treatment. Changes in YB-1 expression resulted in alterations in the tumor immunosuppressive environment. This study provides evidence for a targeting strategy that involves the functional interaction between YB-1 and chemotherapy-based immune evasion to treat chemoresistant HCC.

No potential conflicts of interest were disclosed.

Conception and design: Z. Tao, T. Wang, K. Chen

Development of methodology: Z. Tao, L. Sun, T. Wang, K. Chen

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Z. Tao, H. Ruan, L. Sun, K. Chen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Z. Tao, H. Ruan, L. Sun, D. Kuang, Y. Song, Q. Wang, K. Chen

Writing, review, and/or revision of the manuscript: Z. Tao, H. Ruan, Y. Hao, K. Chen

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Z. Tao, H. Ruan, K. Chen

Study supervision: Z. Tao, T. Wang, K. Chen

This work was supported by grants from the National Natural Sciences Foundation of China (81672524 and 81602678), the Hubei Provincial Natural Science Foundation of China (2018CFA038), and the Natural Science Foundation of Tianjin (17JCQNJC12300).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.