Abstract Therapeutic resistance and undefined predictive biomarkers severely hinder the clinical popularization of immunotherapy in esophageal squamous cell carcinoma (ESCC). Herein, we identify the glucose transporter 1 (GLUT1) as a critical determinant of immunotherapy resistance. Elevated expression of GLUT1 correlates with poor immunotherapy response and unfavorable prognosis in ESCC patients. GLUT1 deletion or inhibition enhances CD8⁺ T cell infiltration and cytotoxicity, and sensitizes ESCC tumors to anti-PD-1 (α-PD1) therapy. Importantly, dietary glucose restriction exhibits equivalent antitumor efficacy to GLUT1 inhibition when combined with α-PD1. Mechanistically, GLUT1 establishes a positive feedback loop with HAT1 and FOXM1, which epigenetically remodels chromatin accessibility to suppress tumor cell senescence, thereby impeding CD8⁺ T cell-mediated antitumor immunity. Our findings highlight GLUT1 as a predictive biomarker of immunotherapy resistance and suggest dietary glucose restriction as a viable strategy to potentiate immunotherapy efficacy in ESCC. Graphical abstract SHORT SUMMARY Dong et al. identify glucose transporter 1 (GLUT1) as a predictor of poor response to immunotherapy in esophageal squamous cell carcinoma. GLUT1 promotes immune evasion by forming a positive feedback loop with the HAT1/FOXM1 anti-senescence axis, thereby epigenetically remodeling chromatin accessibility. GLUT1 inhibition or dietary glucose restriction restores CD8⁺ T-cell-mediated antitumor immunity and improves the efficacy of anti-PD-1 therapy in preclinical models.

Hepatocellular carcinoma (HCC) frequently recurs after hepatectomy. Transarterial chemoembolization (TACE) is a common adjuvant therapy; however, reliable indicators of its efficacy remain limited. This study aimed to evaluate the clinical significance of matrix metallopeptidase 12 (MMP12) in HCC patients undergoing postoperative adjuvant TACE (PA-TACE) and to explore potential strategies to enhance the efficacy of PA-TACE. A retrospective analysis was conducted on 225 HCC patients who received TACE and were categorized into prophylactic and recurrence TACE groups. Clinical data including liver function, tumor characteristics, and imaging findings were collected. Tissue samples were subjected to MMP12 immunohistochemical staining, and patients were further stratified according to MMP12 expression levels. Univariate and multivariate Cox regression analyses were performed to identify risk factors, and a nomogram was constructed for prognostic evaluation. The role of MMP12 in TACE for HCC was examined using Western blotting, RT-qPCR, mass spectrometry, Transwell, wound-healing, and colony formation assays. Kaplan-Meier curves demonstrated significantly better survival in the low-MMP12-expression group. Microvascular infiltration, alpha-fetoprotein (AFP) levels, and MMP12 expression were identified as independent risk predictors for survival. The nomogram derived from these factors exhibited high predictive accuracy (area under the curve: 0.750-0.959) across multiple time points. In vitro experiments revealed that targeting MMP12 inhibited HCC cell invasion, migration, and colony formation by blocking the MEK/ERK signaling pathway. The MMP12 inhibitor GM6001 enhanced the therapeutic effects of TACE. In conclusion, MMP12 was identified as a key and independent prognostic biomarker for PA-TACE in HCC patients. The prognostic model integrating MMP12, AFP, and microvascular infiltration may help identify patients most likely to benefit from PA-TACE. Targeting MMP12 to block the MEK/ERK pathway and suppress HCC cell malignancy highlights its potential as a therapeutic target to improve PA-TACE efficacy.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignant disease with a poor prognosis. We previously found that p62 presented a marked nuclear-cytoplasmic translocation in ESCC cells as compared that in normal esophageal epithelial cells, but its effects on ESCC cells remain unclear. This study aims to clarify the impacts of different cellular localization of p62 on the function of ESCC cells and the underlying molecular mechanisms. We here demonstrated that cytoplasmic p62 enhances the migration and invasion abilities of esophageal cancer cells, whereas nuclear p62 has no effect. We further explored the interaction protein of p62 by using GST pull-down experiment and identified EPLIN as a potential protein interacting with p62. In addition, reducing EPLIN expression significantly inhibited the migration and invasion of ESCC cells, which were rescued when EPLIN expression was restored after the p62 knockdown. At a molecular level, p62 in cytoplasm positively regulated the expression of EPLIN via enhancing its protein stability. Data from the TCGA and GEO database displayed a significant up-regulation of EPLIN mRNA expression in ESCC tissues compared with corresponding paired esophageal epithelial samples. Our findings present evidence that the nuclear-cytoplasmic translocation of p62 protein contributes to an aggressive malignancy phenotype, providing candidate molecular biomarkers and potential molecular targets for the diagnosis and treatment of ESCC.

Squamous cell carcinomas (SCCs) are common and aggressive malignancies. Immune check point blockade (ICB) therapy using PD-1/PD-L1 antibodies has been approved in several types of advanced SCCs. However, low response rate and treatment resistance are common. Improving the efficacy of ICB therapy requires better understanding of the mechanism of immune evasion. Here, we identify that the SCC-master transcription factor TP63 suppresses interferon-γ (IFNγ) signaling. TP63 inhibition leads to increased CD8 + T cell infiltration and heighten tumor killing in in vivo syngeneic mouse model and ex vivo co-culture system, respectively. Moreover, expression of TP63 is negatively correlated with CD8 + T cell infiltration and activation in patients with SCC. Silencing of TP63 enhances the anti-tumor efficacy of PD-1 blockade by promoting CD8 + T cell infiltration and functionality. Mechanistically, TP63 and STAT1 mutually suppress each other to regulate the IFNγ signaling by co-occupying and co-regulating their own promoters and enhancers. Together, our findings elucidate a tumor-extrinsic function of TP63 in promoting immune evasion of SCC cells. Over-expression of TP63 may serve as a biomarker predicting the outcome of SCC patients treated with ICB therapy, and targeting TP63/STAT/IFNγ axis may enhance the efficacy of ICB therapy for this deadly cancer.

Purpose: To establish a prognostic model of esophageal squamous cell carcinoma (ESCC) patients based on tenascin-C (TNC) expression level and clinicopathological characteristics, and to explore the therapeutic potential of TNC inhibition. Methods: The expression of TNC was detected using immunohistochemistry (IHC) in 326 ESCC specimens and 50 normal esophageal tissues. Prognostic factors were determined by Cox regression analyses and were incorporated to establish the nomogram. The effects of TNC knockdown on ESCC cells were assessed in vitro and in vivo. Transcriptome sequencing (RNA-seq) and gene set enrichment analysis (GSEA) were performed to reveal signaling pathways regulated by TNC knockdown. The therapeutic significance of TNC knockdown combined with small-molecule inhibitors on cell proliferation was examined. Results: TNC protein was highly expressed in 48.77 % of ESCC tissues compared to only 2 % in normal esophageal epithelia (p < 0.001). The established nomogram model, based on TNC expression, pT stage, and lymph node metastasis, showed good performance on prognosis evaluation. More importantly, the reduction of TNC expression inhibited tumor cell proliferation and xenograft growth, and mainly down-regulated signaling pathways involved in tumor growth, hypoxia signaling transduction, metabolism, infection, etc. Knockdown of TNC enhanced the inhibitory effect of inhibitors targeting ErbB, PI3K-Akt, Ras and MAPK signaling pathways. Conclusion: The established nomogram may be a promising model for survival prediction in ESCC. Reducing TNC expression enhanced the sensitivity of ESCC cells to inhibitors of Epidermal Growth Factor Receptor (EGFR) and downstream signaling pathways, providing a novel combination therapy strategy.

Supplementary Table 2. Relationship between NRP1/CRT co-expression and lymph node metastasis in 216 ESCC patients

目的:利用CRISPR/Cas9系统构建稳定敲低膜联蛋白A2(ANXA2)基因的食管鳞癌细胞,研究该基因对食管鳞癌细胞表型的影响.方法:在公共数据库中查找ANXA2基因及对照的向导RNA(gRNA),选取6个ANXA2 gRNA及1个对照gRNA,使用CRISPR/Cas9载体构建表达ANXA2 gRNA的重组质粒,转入HEK293FT细胞制备gRNA/Cas9慢病毒,将慢病毒感染食管鳞癌KYSE30和KYSE150细胞后,使用嘌呤霉素筛选阳性细胞并扩大培养.采用Western blot检测ANXA2蛋白及其下游MYC蛋白的表达情况,逆转录实时荧光定量PCR(RT-qPCR)检测ANXA2 mRNA表达情况,Transwell和集落形成实验分别检测细胞迁移侵袭和集落形成能力的变化.结果:成功构建了CRISPR/Cas9质粒;Sanger测序结果表明,ANXA2基因敲低质粒及对照质粒均构建成功.与对照组比较,病毒感染细胞后,Western blot结果显示,其中两个gRNA靶点病毒感染的KYSE30细胞中ANXA2蛋白及其下游MYC蛋白水平均显著降低(P<0.05);同时RT-qPCR结果证实,上述两个靶点显著下调ANXA2的mRNA表达水平(P<0.05).Transwell和集落形成实验结果显示,敲低ANXA2后,细胞迁移侵袭能力和集落形成能力均显著减弱(P<0.05).结论:ANXA2基因敲低显著抑制了食管鳞癌细胞的恶性表型,为进一步研究ANXA2促进食管鳞癌细胞恶性表型的作用机制提供了细胞模型和实验基础.

Supplementary Data. Supplementary Materials and Methods, Supplementary Figure Legends

High podoplanin (PDPN) expression correlates with poor prognosis in various cancers. However, the expression and clinical value of PDPN in glioma are unclear. In this study, PDPN expression was compared in 227 glioma tissues and 22 paired non-neoplastic tissues, and its association with prognostic factors was statistically analyzed. The effect of PDPN knockdown on the proliferation ability of glioma cells (U87MG and U118MG cell lines) was assessed along with the underlying molecular mechanism. Overexpression of PDPN was observed in the majority of glioma tissues compared with the expression in normal tissues. PDPN overexpression was positively correlated with IDH wild-type status, TERT promoter mutation status, and ATRX retention status, and was negatively correlated with 1p/19q codeletion status. The expression level of PDPN was positively correlated with the glioma grade in the diffuse astrocytoma, IDH wild-type. High PDPN expression was also negatively correlated with survival in astrocytoma patients with IDH mutation or wild-type and in glioblastoma patients with IDH wild-type. Grade, radiochemotherapy, and PDPN overexpression emerged as independent indicators for a poor prognosis of glioma patients. PDPN knockdown suppressed proliferation and reduced p-Akt and p-mTOR protein expression in glioma cells. PDPN is a potential biomarker or therapeutic target for glioma that is closely associated with tumor grade and poor prognosis, which may play a role in enhancing cell proliferation via Akt/mTOR signaling.

Supplementary Figure 3. Western blot analysis in ESCC cells treated with or without 100 μmol/L KN-93 for 24 hours.

目的:检测小分子抑制剂ICG-001对食管鳞癌细胞增殖的抑制作用并探讨其分子机制.方法:使用50和100 μmol/L的小分子抑制剂ICG-001处理食管鳞癌细胞系KYSE410和KYSE450,以溶剂DMSO处理细胞作为对照,进行细胞增殖活力、集落形成能力、细胞周期分布及凋亡检测;分别使用实时荧光定量PCR(qPCR)和Western blot检测ICG-001处理细胞中相关分子(SKP2、Survivin和TCF4)mRNA和蛋白水平的表达变化.结果:与对照组比较,ICG-001处理可显著抑制食管鳞癌细胞系KYSE410和KYSE450的增殖活力和集落形成能力,并可显著诱导细胞发生G0/G1期阻滞(P<0.01);SKP2、Survivin和TCF4的mRNA和蛋白表达水平在ICG-001处理组均显著降低(P<0.01).结论:ICG-001可显著抑制食管鳞癌细胞的增殖活力和集落形成能力,并可诱导细胞发生G0/G1期阻滞,该作用可能与其抑制β-catenin/TCF4的转录活性及其下游分子Survivin和SKP2的表达有关.

目的:探讨P4HA2在食管鳞癌中的表达情况及作用机制.方法:利用基因表达综合数据库(GEO)的RNA表达谱数据及免疫组织化学染色和Western blot技术分析食管鳞癌组织及正常食管组织中P4HA2的mRNA和蛋白表达情况;通过细胞增殖实验、集落形成实验和裸鼠移植瘤实验,在体外和体内检测P4HA2对食管鳞癌细胞恶性表型的影响;利用Western blot检测P4HA2调控的下游通路及关键分子.结果:与正常食管组织相比,食管鳞癌组织中P4HA2的mRNA和蛋白水平均显著升高(P<0.01);体外和体内实验结果显示,与对照组相比,敲降P4HA2可显著抑制食管鳞癌细胞的增殖、集落形成以及裸鼠移植瘤的生长(P<0.01);分子水平检测表明,敲降P4HA2显著降低EGFR的蛋白水平,以及AKT和S6的磷酸化水平(P<0.05);在食管鳞癌细胞中敲降P4HA2后回复EGFR的表达,AKT和S6的磷酸化水平以及细胞增殖和集落形成表型均得以恢复(P<0.01).结论:P4HA2在食管鳞癌组织中高表达,且P4HA2通过激活EGFR/AKT/S6信号通路促进食管鳞癌细胞的增殖和肿瘤生长.

Supplementary Figures S1-S5.

Amplification and overexpression of MCM7 in ESCC. A. Amplification of MCM7 in array CGH study. B. Validation of MCM7 amplification by FISH. Left: two MCM7 signals in peripheral blood cells; Middle: two MCM7 signals in ESCC cells; Right: more than four MCM7 signals in ESCC cells. C. MCM7 overexpressed in ESCC by semi-quantitative RT-PCR. D. MCM7 overexpressed in ESCC by Western blotting. E. MCM7 overexpressed in ESCC by IHC.

Effect of ANO1 on cell proliferation. Knock-down the expression of ANO1 inhibited the cell proliferation of KYSE30 (A) and KYSE510 cells (B). The absorbance at 450 nm was measured.

Venn diagrams comparing differences in expression of our gene/microRNA expression assay and other microarray experiments. A. Comparison of differentially expressed genes in different experiments. B. Comparison of differentially expressed microRNAs in different experiments. â†', overexpressed; â†", underexpressed.

mRNA expression of ANO1 in ESCC cell lines. X. Name of ESCC cell lines. Y. The relative mRNA expression of ANO1 compared with GAPDH.

Supplementary Table 1. NRP1 expression positively correlates with CRT in ESCC

Supplementary Data from Suppression of Anoikis by SKP2 Amplification and Overexpression Promotes Metastasis of Esophageal Squamous Cell Carcinoma

PDF file - 1061K, S Figure 1. Down-regulation of CRT expression reduces PTP1B mRNA and protein expression in KYSE510 cells. S2. PTP1B promotes ESCC cell invasion and migration in KYSE510 cells. KYSE510 cells were transfected with PTP1B-specific siRNA (siRNA PTP1B) or non-targeting siRNA control (siRNA cont). S3. CRT regulates cell motility through PTP1B in ESCC cells. (A-C) 1x105 KYSE150 cells were seeded on 24-well plates with three replicates. S4. Cancer-inflammatory response was minimal in mouse model. S5. CRT regulates PTP1B transcription through Stat5a. S6. The expression of CRT and PTP1B in 176 ESCC tissues was evaluated by immunohistochemistry. S7. Correlation of PTP1B expression with overall survival in patients with CRT high-expressing tumors. S8. Model of CRT-dependent signaling pathway in ESCC cell metastasis. Supplementary Table 1. The candidate genes altered in CRT-siRNA KYSE150 cells as compared with non-targeting siRNA cells detected by cDNA array