Journal: Journal of experimental & clinical cancer research : CR

Article Title: Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers.

doi: 10.1186/s13046-025-03338-2

Figure Lengend Snippet: Fig. 1 Identification of immunosuppressive genes via in vivo screen of CRISPR/Cas9 lentivirus gene knockout library. A. The growth curves of PD1 anti body-treated allograft tumors and control tumors in C57BL/6 mice (n = 7/group). B. Images of PD1 antibody-treated allograft tumors and control tumors (left panel) and statistical analysis of the weight of these tumors (right panel). C. Screening process of candidate genes. D. Distribution and identification of PHF8 in different frequencies of genes. E, F. Analysis of PHF8 mRNA expression in CRCs (T) and non-cancerous colon tissues or their adjacent normal colon tissues (N) using TCGA database. G. Analysis of PHF8 protein expression in CRCs (T) and control subjects (N) by UALCAN data platform. H. Correlation analysis between PHF8 protein expression and clinicopathological stage using UALCAN data platform. I. Representative IHC staining of PHF8 (left panel) and the percentage of PHF8-positive cells (right panel) in CRCs (T) and control subjects (N). Scale bars, 200 μm. J. The alterations in the PHF8 gene are found in a total of 6% (35/594) of CRC patients (data from cBioPortal). K. Correlation analysis between PHF8 mRNA expression and the prognosis of CRC patients using the OncoLnc database. L. The pathways modulated by PHF8 were analyzed using the GSEA method. M. Correlation analysis between PHF8 mRNA expression and infiltrating lymphocytes in different types of human cancers. COAD represents colon adenocarcinoma. P value in A was calculated using One-way analysis of variance (ANOVA). P values in B, E-H were calculated using two-tailed unpaired Student’s t-tests. P value in K was determined by the log-rank test. Data were presented as mean ± SD. *, P < 0.05; ***, P < 0.001

Article Snippet: PHF8 inhibitor daminozide was purchased from TargetMol (Boston, MA, USA) [26].

Techniques: In Vivo, CRISPR, Gene Knockout, Control, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers.

doi: 10.1186/s13046-025-03338-2

Figure Lengend Snippet: Fig. 3 Inhibition of PHF8 improves the efficacy of PD1 antibody. A. The growth curves of PHF8-knockdown allograft tumors and control tumors with or without PD1 antibody treatment in C57BL/6 mice (n = 5/group). B. Images of the indicated allograft tumors (left panel) and statistical analysis of tumor weight (right panel). C. The growth curves of allograft tumors in C57BL/6 mice treated with PHF8 inhibitor daminozide and PD1 antibody, individually or in combination (n = 5/group). D. Images of the indicated tumors (left panel) and statistical analysis of tumor weight (right panel). E. Flow cytometry detecting the number of tumor infiltration CD4 +/CD8 + T cells in tumor tissues with the indicated treatments (left panels). Quantification analysis of cell numbers was shown on the right panels. F-H. ELISA assays were performed to measure the levels of TNFα (F), Granzyme B (G), and IFN-γ (H) in tumor tis sues with the indicated treatments. P values in A and C were calculated by two-way ANOVA with Tukey’s multiple comparisons test. P values in B, D, and E-H were calculated using two-tailed unpaired Student’s t-tests. Data were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significance

Article Snippet: PHF8 inhibitor daminozide was purchased from TargetMol (Boston, MA, USA) [26].

Techniques: Inhibition, Knockdown, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers.

doi: 10.1186/s13046-025-03338-2

Figure Lengend Snippet: Fig. 5 The oncogenic role of PHF8 in BRAF- or KRAS-mutant CRC cells. A. MTT assay showing the effect of PHF8 overexpression on the proliferation of LOVO and SW480 cells. B. The effect of PHF8 overexpression on colony formation ability of LOVO and SW480 cells. The left panel shows representative im ages of colony formation, and the right panel shows a quantitative analysis of colony numbers. C. MTT assays were performed to assess the effect of PHF8 knockdown on the proliferation of HCT116 and RKO cells. D. The effect of PHF8 knockdown on colony formation ability of HCT116 and RKO cells. Repre sentative images of colony formation were shown in the left panel, and quantitative analysis of colony numbers was shown in the right panel. E. The effect of PHF8 knockdown on the apoptosis of HCT116 and RKO cells was determined by flow cytometry. F. The growth curves of PHF8-overexpression LOVO cell-derived xenograft tumors and control tumors in nude mice (n = 5/group). G. Images of the indicated xenograft tumors (left panel) and statistical analysis of tumor weight (right panel). H. Left panel shows the representative IHC staining of Ki-67 in the indicated xenograft tumors. Scale bars, 200 μm., The right panel shows a statistical analysis of the percentage of Ki-67-positive cells. I. The growth curves of PHF8-knockdown HCT116 cell-derived xe nograft tumors and control tumors in nude mice (n = 5/group). J. Images of the indicated xenograft tumors (left panel) and statistical analysis of tumor weight (right panel). K. The representative IHC staining of Ki-67 in the indicated xenograft tumors was shown in the left panel, and statistical analysis on the percentage of Ki-67-positive cells was shown in the right panel. Scale bars, 200 μm. P values in A, C, F and I were calculated using One-way analysis of variance (ANOVA). P values in B, D, E, G, H, J, and K were calculated using two-tailed unpaired Student’s t-tests. Data were presented asmean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: PHF8 inhibitor daminozide was purchased from TargetMol (Boston, MA, USA) [26].

Techniques: Mutagenesis, MTT Assay, Over Expression, Knockdown, Flow Cytometry, Derivative Assay, Control, Immunohistochemistry, Two Tailed Test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers.

doi: 10.1186/s13046-025-03338-2

Figure Lengend Snippet: Fig. 6 PHF8 enhances the metastatic ability of BRAF- or KRAS-mutant CRC cells. A. The effect of PHF8 overexpression on the migration and invasion po tential of LOVO and SW480 cells. Representative images of migrated/invaded cells were shown in the left panel, and statistical analysis on the number of migrated/invaded cells was shown in the right panel. B. The effect of PHF8 knockdown on the migration and invasion potential of HCT116 and RKO cells. The left panel shows the representative images of migrated/invaded cells, and the right panel shows statistical data on the number of migrated/invaded cells. C. qRT-PCR assays were performed to assess the effect of ectopic expression of PHF8 on mRNA expression of N-cadherin, Slug, Snail Vimentin, MMP2, and MMP9 in LOVO and SW480 cells. D. The effect of PHF8 knockdown on mRNA expression of the above metastasis-related genes was determined by qRT-PCR assays in HCT116 and RKO cells. β-actin was used as the normalized control. E. The systemic metastasis of mice after tail vein injection of PHF8-overexpression SW480 cells and control cells was evaluated by an in vivo imaging system (left panel), and statistical results of bioluminescence in mice were shown on the right panel. F. The effect of tail vein injection of PHF8-overexpression SW480 cells and control cells on the survival of mice. G. Representative H&E images of metastatic tumors in lung, liver, and kidney tissues of mice after tail vein injection of PHF8-overexpression SW480 cells and control cells. The yellow rectangles were used to mark the location of the tumor. P values in A-E were calculated using two-tailed unpaired Student’s t-tests. P value in F was determined by the log-rank test. Data were presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001

Article Snippet: PHF8 inhibitor daminozide was purchased from TargetMol (Boston, MA, USA) [26].

Techniques: Mutagenesis, Over Expression, Migration, Knockdown, Quantitative RT-PCR, Expressing, Control, Injection, In Vivo Imaging, Two Tailed Test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers.

doi: 10.1186/s13046-025-03338-2

Figure Lengend Snippet: Fig. 8 c-Myc upregulates PHF8 by inhibiting miR-22-3p. A, B. c-Myc was knocked down in LOVO and SW480 cells, and western blotting (A) and qRT-PCR (B) assays were used to determine its effect on protein and mRNA expression of PHF8. C. LOVO and SW480 cells were treated with 50 µM c-Myc inhibi tor 10,074-G5 for 48 h, and its effect on miR-22-3p expression was then evaluated by qRT-PCR assay. D-E. LOVO and SW480 cells were transfected with miR-22-3p inhibitor (D), and its effect on cell proliferation was then determined by MTT assays (E). U6 was used to normalize miR-22-3p expression. F, G. qRT-PCR (F) and western blotting (G) assays were used to evaluate the effect of miR-22-3p inhibitor on mRNA and protein expression of PHF8 in LOVO and SW480 cells. β-actin was used as a normalized control for qRT-PCR assay, and GAPDH was used as a loading control for western blotting analysis. H. Western blotting analysis was performed to evaluate the effect of miR-22-3p inhibitor on PHF8 expression in LOVO and SW480 cells pretreated with 50 µM c-Myc inhibitor 10,074-G5. P value in E was calculated using One-way analysis of variance (ANOVA). P values in B-D and F were calculated using two-tailed unpaired Student’s t-tests. Data were presented as mean ± SD. ***, P < 0.001; ns, no significance

Article Snippet: PHF8 inhibitor daminozide was purchased from TargetMol (Boston, MA, USA) [26].

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Transfection, Control, Two Tailed Test

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers.

doi: 10.1186/s13046-025-03338-2

Figure Lengend Snippet: Fig. 9 A schematic model of the mechanism by which PHF8 promotes malignant phenotypes and immune escape of KRAS- or BRAF-mutant CRC cells. Briefly, PHF8 downregulates the expression of MHC and interferon regulatory genes. On the other hand, PHF8 upregulates PD-L1 by remodeling histone modifications within its promoter, thereby leading to immune escape. Similarly, PHF8 also upregulates the expression of KRAS, BRAF, and c-Myc by remod eling histone modifications within their promoters to activate the MAPK/ERK/c-Myc signaling pathway. Thus, PHF8 may be a potential therapeutic target for KRAS- or BRAF-mutant CRCs. In addition, c-Myc can upregulate PHF8 at the post-transcriptional levels by downregulating miR-22-3p, while PHF8 in turn promotes c-Myc expression to form a positive feedback loop, ultimately promoting the malignant phenotypes and immune escape of KRAS- or BRAF-mutant CRC cells. Red asterisk represents mutant KRAS or BRAF.

Article Snippet: PHF8 inhibitor daminozide was purchased from TargetMol (Boston, MA, USA) [26].

Techniques: Mutagenesis, Expressing