Seong-Ho Park

TERT, the catalytic subunit of telomerase, is aberrantly activated in most cancers and represents an attractive therapeutic target. However, conventional TERT-targeting strategies, including chemical inhibitors and siRNA, are limited by several issues, such as insufficient efficacy and off-target effects. In this study, we investigated whether dCas9-KRAB-mediated CRISPR interference (CRISPRi) could overcome the limitations by transcriptional repression of TERT without DNA cleavage. We first assessed the efficacy of the dCas9-KRAB system by applying it to H1299 non-small-cell lung cancer cells and observed reduction in TERT expression up to approximately 80% and significant decreases in cell viability and growth. Transcriptome-wide analysis showed limited detectable changes in non-target-gene expression under the conditions tested. Together, the results suggest that dCas9-KRAB-mediated CRISPRi could serve as a proof-of-principle approach for targeted repression of TERT in cancer cells with limited detectable effects on non-target-gene expression.

Background: This study aimed to infer a causal gene network associated with bone metastasis in lung cancer and to validate its reliability through experimental gene expression analysis.Methods: Using DNA microarray data from the Gene Expression Omnibus, we analyzed samples from primary lung cancer and those with bone metastasis. Commonly expressed genes in both groups were identified, and a causal network was inferred using Bayesian network inference with Java Objects based on the Bayesian Dirichlet score. To evaluate the network, we predicted the expression changes of downstream genes following knockdown of a key upstream gene and compared these predictions with mRNA expression levels in fatty acid desaturase 1 (FADS1)-knockdown lung cancer cells.Results: The genes FADS1, cardiotrophin-like cytokine factor 1 (CLCF1), chromosome 4 open reading frame 48, sushi, nidogen and EGF like domains 1, FK506-binding protein 15, and coenzyme Q10A (COQ10A) were identified as directly associated with lung cancer bone metastasis. Among them, FADS1 appeared to have a regulatory role, influencing downstream targets. Notably, CLCF1 and COQ10A showed significantly increased expression in FADS1-knockdown cells, consistent with the network’s predictions.Conclusions: These findings suggest that Bayesian network analysis is a reliable machine learning approach for uncovering causal gene relationships in cancer metastasis. Furthermore, FADS1 may serve as a potential therapeutic target in lung cancer bone metastasis. The validity of this network was supported by in vitro experiments using a lung cancer cell line.

Supplementary Figure 1 and Legend

Head and neck squamous cell carcinoma (HNSCC) remains a clinically challenging malignancy with limited targeted therapy options and poor patient outcomes. Thus, identifying unique dependencies, including HNSCC-specific metabolic reprogramming, is imperative for improving treatment strategies for this disease. In this study, we show that HNSCC relies on elevated glucose transporter 1 (GLUT1)–mediated glucose uptake to support redox homeostasis and tumor growth. Analyses of GLUT1 expression data in tumors and cancer cell lines reveal significant upregulation of GLUT1 in HNSCC relative to both normal tissue and other cancer subtypes and that high GLUT1 expression correlates with poorer clinical outcomes. Using a basal epithelial layer–specific GLUT1-knockout mouse model, we demonstrate that GLUT1 ablation in HNSCC cells of origin markedly attenuates tumor initiation and progression, implicating the necessity of GLUT1 in HNSCC tumorigenesis. Building on this observation, combining pharmacologic inhibition of GLUT1 with pro-oxidants such as vitamin C and auranofin induces potent cytotoxicity in vitro and in vivo, partly by precipitating oxidative stress. We further observe that insulin signaling is required to sustain glucose uptake and redox homeostasis, as insulin receptor knockdown decreases proliferation and increases reactive oxygen species levels. Together, these results suggest that although GLUT1 overexpression is a key driver of glucose uptake, insulin signaling also contributes to the metabolic and oncogenic pathways underlying HNSCC progression. Consequently, strategies that co-target GLUT1 and insulin signaling to restrict glucose flux may synergize with pro-oxidant therapies, offering a promising therapeutic avenue for HNSCC.Significance:Enhanced GLUT1 expression and oncogenic insulin signaling drive elevated glucose uptake in HNSCC, which contribute to the maintenance of redox homeostasis and tumor growth. Disrupting both glucose uptake and redox balance may offer a promising therapeutic approach.

Supplementary Figure 3 and legend

Combination of GLUT1 inhibition and ascorbate suppresses HNSCC tumor growth. A and B,In vitro proliferation (top) and intracellular H2O2 levels (bottom) of FaDu (A) or SCC-61 (B) cells treated with WZB117 (20 μmol/L) or BAY-876 (100 nmol/L) and vitamin C (0.25 mmol/L) or combination of GLUT1 inhibitor and vitamin C (n = 5 for each group). Two-way ANOVA (top) or two-tailed t test (bottom). ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05. C,In vivo tumor growth of FaDu xenografts treated with vehicle PBS/DMSO, WZB117 (20 mg/kg i.p. daily), or BAY-876 (4.5 mg/kg of corn oil/DMSO p.o. daily; n = 6 for each group). Two-way ANOVA. ****, P < 0.0001. D,In vivo tumor growth of FaDu xenograft tumors treated with vehicle PBS/DMSO (n = 16), WZB117 (20 mg/kg i.p. daily; n = 16), vitamin C (4 g/kg i.p. daily; n = 7), and a combination of WZB117 and vitamin C (n = 7). Two-way ANOVA. ****, P < 0.0001; **, P < 0.01; *, P < 0.05. E–G,In vivo tumor growth of SCC-61 xenografts (E), tumor weights (F), IHC analysis of 4HNE, Ki67, and pH2A.X (G) in SCC-61 xenograft tumors treated with vehicle PBS/DMSO (n = 6), WZB117 (20 mg/kg i.p. daily; n = 5), vitamin C (4 g/kg i.p. daily; n = 5), or combination of WZB117 and vitamin C (n = 6). Two-way ANOVA. **, P < 0.01; *, P < 0.05. Scale bars, 1 mm. H, IHC analysis quantification of % area of 4HNE (lipid peroxidation marker), pH2A.X (DNA damage marker), and Ki67 (nuclear/cell proliferation marker) in xenografted tumors. Four to six images of each tumor were captured and analyzed for quantification. Two-tailed t test. *, P < 0.05. All error bars represent mean ± SEM. BAY, BAY-876; H&E, hematoxylin and eosin; Vit C, vitamin C; RLU, relative light unit; WZB, WZB117.

Supplementary Figure 2 and legend

GLUT1 overexpression in TCGA HNSCC tumor samples. A, TCGA mRNA-seq analyses [normalized transcripts per million (TPM) mappable reads] of GLUT1 and metabolic genes associated with glycolysis, PPP, and de novo serine biosynthesis pathway in the patients of the TCGA HNSCC cohort (n = 520) and matched normal (n = 44) tissue samples. Each dot represents one patient. Error bars represent the median ± quartile range, Mann–Whitney U test. ****, P < 0.0001. B, Top five Hallmark gene sets enriched in patients of the TCGA HNSCC cohort are compared with matched normal tissue samples by gene set enrichment analysis. C, Violin plots depicting SLC2A1 (GLUT1) mRNA expression in TCGA tumor samples stratified by lineage. Lineages are ordered by descending mean SLC2A1 expression, and HNSCC is highlighted in red. D, Violin plot comparing SLC2A1 (GLUT1) mRNA expression in TCGA HNSCC tumors with GTEx normal esophageal mucosa. Dots represent individual cell lines. Welch t test. ****, P < 0.0001. E, Heatmap indicating expression of genes enriched in the patients of the TCGA HNSCC cohort when compared with matched normal tissue samples. F and G, Immunoblot analysis and quantification of GLUT1 expression in human HNSCC tumor (n = 5) and matched normal head and neck epithelial (n = 5) tissue samples. **, P < 0.01. Error bars represent the median ± quartile range. Mann–Whitney U test. H,SLC2A1 mRNA expression from human HNSCC tumor samples (n = 8) and matched normal head and neck epithelial tissue samples (n = 7). Error bars represent the mean ± SEM. Mann–Whitney U test. ***, P < 0.001. I, Kaplan–Meier survival curves depicting OS of patients with HNSCC (n = 519) up to 120 months, stratified by SLC2A1 mRNA expression. High- and low-SLC2A1 expression groups were determined using the maximally selected rank statistic. A significant difference in OS between groups was assessed using the log-rank test, and a Cox proportional hazards model was applied to calculate the HR. ENO1, enolase 1; G6PD, glucose-6-phosphate dehydrogenase; HK2, hexokinase 2; PKM, pyruvate kinase M; PSAT1, phosphoserine aminotransferase 1; PSPH, phosphoserine phosphatase; SHMT2, serine hydroxymethyltransferase 2; SLC2A1, solute carrier family 2 member 1.

GLUT1 ablation attenuates tumorigenesis in 4NQO-induced mouse model of human HNSCC. A, Schematic showing timeline for treatment of mice with 4NQO. 4NQO was administered in drinking water for 8 weeks and replaced with normal drinking water for the remainder of the study. Tongues were collected every 2 weeks after the first exposure to 4NQO for analyses. For Cre-mediated GLUT1 ablation in basal epithelial cells, tamoxifen was injected (100 mg/kg i.p. daily for 5 consecutive days) 4 weeks after the final administration of 4NQO. Tumors were harvested 4 weeks after the final tamoxifen injection. B, Representative IHC analyses of GLUT1 expression in CK5+ and p63+ basal epithelial cells of untreated and 4NQO-treated mice. C, Representative images of tumor-bearing tongues from 4NQO-treated mice; black arrowheads depict squamous tongue tumors (left). Graph shows quantification of the number of visible tongue tumors identified in 4NQO-treated WT and Krt5+ basal epithelial–specific GLUT1 CKO mice (n = 6 for each group). Mann–Whitney U test. P = 0.06 (right). D, Representative H&E images of 4NQO-treated WT and CKO mouse tongues. Arrows depict 4NQO-induced squamous tumors (left). Graph shows quantification of 4NQO-induced tongue tumor area (μm2) in GLUT1 WT and CKO mice (n = 6 for each group). Mann–Whitney U test. **, P < 0.01 (right). E, Representative co-immunofluorescent staining of GLUT1 and p63 in 4NQO-induced squamous tongue tumors of GLUT1 WT and GLUT1 CKO mice. DAPI, 4',6-diamidino-2-phenylindole. F, Representative IHC analyses of p63, GLUT1, and Ki67 expression in 4NQO-induced squamous tongue tumors. All error bars represent mean ± SEM. All scale bars, 1 mm unless otherwise noted. H&E, hematoxylin and eosin; KO, knockout.

GLUT1 inhibition potentiates oxidative cytotoxicity of radiation in human HNSCC cell lines. A and B,In vitro proliferation (top) and intracellular H2O2 levels (bottom) of FaDu cells treated with WZB117 (20 μmol/L; A) or BAY-876 (100 nmol/L; B) with or without radiotherapy (RT, 4 Gy; n = 6 for each group). Two-way ANOVA (top) or two-tailed t test (bottom). ****, P < 0.0001; **, P < 0.01. C and D,In vitro proliferation (top) and intracellular H2O2 levels (bottom) of SCC-61 cells treated with WZB117 (20 μmol/L; C) or BAY-876 (100 nmol/L; D) with or without radiation (RT, 4 Gy; n = 6 for each group). Two-way ANOVA (top) or two-tailed t test (bottom). ****, P < 0.0001; ***, P < 0.001. E–J, Colony formation of HNSCC cells FaDu (E, F, and I) and Detroit 562 (G, H, and J) treated with WZB117 (20 μmol/L) or BAY-876 (100 nmol/L) with or without RT (4 Gy). Colonies were stained with crystal violet, and the colony area was measured using ImageJ (n = 3). Two-tailed t test. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05. BAY, BAY-876; RLU, relative light unit; WZB, WZB117.