Identification of Genes Involved in EGF-induced Apoptosis Using CRISPR/Cas9 Knockout Screening: Implications for Novel Therapeutic Targets in EGFR-Overexpressing Cancers.

PURPOSE: Exogenous epidermal growth factor (EGF) causes apoptosis in EGF receptor (EGFR)-overexpressing cell lines. The apoptosis-inducing factors could be a therapeutic target. We aimed to determine the mechanism of EGF-induced apoptosis using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-based knockout screen. Materials and Methods: Two-vector system of the human genome-scale CRISPR knockout library v2 was used to target 19,050 genes using 123,411 single guide RNAs (sgRNAs). Recombinant human EGF (100 nM) or distilled water four times was administered to the experimental and control groups, respectively. The read counts of each sgRNA obtained from next-generation sequencing were analyzed using the edgeR algorithm. We used another EGFR-overexpressing cell line (A549) and short hairpin RNAs (shRNAs) targeting five EGF-resistance genes for validation. DUSP1 expression in A431, A549, and HEK293FT cells was calculated using reverse transcription-quantitative polymerase chain reaction. RESULTS: We found 77 enriched and 189 depleted genes in the experimental group using the CRISPR-based knockout screen and identified the top five EGF-resistance genes: DDX20, LHFP, REPS1, DUSP1,<.i> and KRTAP10-12. Transfecting shRNAs targeting these genes into A549 cells significantly increased the surviving fractions after EGF treatment, compared with those observed in the control shRNA-transfected cells. The expression ratio of DUSP1 (inhibits ERK signaling) increased in A431 and A549 cells after EGF treatment. However, DUSP1 expression remained unchanged in HEK293FT cells after EGF treatment. CONCLUSION: The CRISPR-based knockout screen revealed 266 genes possibly responsible for EGF-induced apoptosis. DUSP1 might be a critical component of EGF-induced apoptosis and a novel target for EGFR-overexpressing cancers.

Chemoradiation-Induced Alteration of Programmed Death-Ligand 1 and CD8+ Tumor-Infiltrating Lymphocytes Identified Patients With Poor Prognosis in Rectal Cancer: A Matched Comparison Analysis.

PURPOSE: To evaluate chemoradiotherapy (CRT)-induced changes in the expression levels of programmed death-ligand 1 (PD-L1) and CD8+ tumor-infiltrating lymphocytes (TILs) and prognostic associations in rectal cancer. METHODS AND MATERIALS: We performed a paired analysis using pre-CRT biopsies and the corresponding post-CRT resected tissues of 123 rectal cancer patients undergoing preoperative CRT followed by surgery between 2005 and 2012. Immunohistochemistry of PD-L1 and CD8 was analyzed for the specimens. RESULTS: The expression levels of PD-L1 and density of CD8+ TILs increased after CRT (P<.001 for both). With cutoffs using each median value, sustained higher expression of PD-L1 at pre- and post-CRT (high-to-high) was associated with less increase in the density of CD8+ TILs (P=.020). Patients representing sustained high-to-high PD-L1 expression had poorer overall survival and disease-free interval on univariate Kaplan-Meier analysis (P=.018 and .029, respectively), with inferior disease-free interval in low-to-low density CD8+ TILs (P=.010). On multivariate analysis, 2 subgroups with high baseline PD-L1 expression level, the high-to-low and high-to-high alterations, showed worse overall survival (hazard ratio 8.34, 95% confidence interval 1.85-37.53 and hazard ratio 11.03, 95% confidence interval 2.33-52.29, respectively), with the highest mortality risk observed in the high-to-high group. CONCLUSIONS: This study verified the CRT-induced immunologic shift toward increases in PD-L1 expression and density of CD8+ TILs in rectal cancer patients. The alteration profiles of checkpoint-related molecules identified the patients with poor prognosis, suggesting potential candidates who can benefit from combining CRT and checkpoint inhibitors.

Real-time Tumor Oxygenation Changes After Single High-dose Radiation Therapy in Orthotopic and Subcutaneous Lung Cancer in Mice: Clinical Implication for Stereotactic Ablative Radiation Therapy Schedule Optimization.

PURPOSE: To investigate the serial changes of tumor hypoxia in response to single high-dose irradiation by various clinical and preclinical methods to propose an optimal fractionation schedule for stereotactic ablative radiation therapy. METHODS AND MATERIALS: Syngeneic Lewis lung carcinomas were grown either orthotopically or subcutaneously in C57BL/6 mice and irradiated with a single dose of 15 Gy to mimic stereotactic ablative radiation therapy used in the clinic. Serial [(18)F]-misonidazole (F-MISO) positron emission tomography (PET) imaging, pimonidazole fluorescence-activated cell sorting analyses, hypoxia-responsive element-driven bioluminescence, and Hoechst 33342 perfusion were performed before irradiation (day -1), at 6 hours (day 0), and 2 (day 2) and 6 (day 6) days after irradiation for both subcutaneous and orthotopic lung tumors. For F-MISO, the tumor/brain ratio was analyzed. RESULTS: Hypoxic signals were too low to quantitate for orthotopic tumors using F-MISO PET or hypoxia-responsive element-driven bioluminescence imaging. In subcutaneous tumors, the maximum tumor/brain ratio was 2.87 ± 0.483 at day -1, 1.67 ± 0.116 at day 0, 2.92 ± 0.334 at day 2, and 2.13 ± 0.385 at day 6, indicating that tumor hypoxia was decreased immediately after irradiation and had returned to the pretreatment levels at day 2, followed by a slight decrease by day 6 after radiation. Pimonidazole analysis also revealed similar patterns. Using Hoechst 33342 vascular perfusion dye, CD31, and cleaved caspase 3 co-immunostaining, we found a rapid and transient vascular collapse, which might have resulted in poor intratumor perfusion of F-MISO PET tracer or pimonidazole delivered at day 0, leading to decreased hypoxic signals at day 0 by PET or pimonidazole analyses. CONCLUSIONS: We found tumor hypoxia levels decreased immediately after delivery of a single dose of 15 Gy and had returned to the pretreatment levels 2 days after irradiation and had decreased slightly by day 6. Our results indicate that single high-dose irradiation can produce a rapid, but reversible, vascular collapse in tumors.

Epidermal growth factor-induced cell death and radiosensitization in epidermal growth factor receptor-overexpressing cancer cell lines.

BACKGROUND/AIM: The aim of the present study was to suggest potential mechanisms of epidermal growth factor (EGF)-induced cell death and radiosensitization in EGF receptor (EGFR)-overexpressing cancer cell lines. MATERIALS AND METHODS: Two EGFR-overexpressing cancer cell lines (AMC-HN3, and A431), one EGFR-null cancer cell line (H520) and normal fibroblasts were cultured with 0.01-1000 nM of recombinant human EGF (rhEGF), and a clonogenic assay was performed. After culturing serum-starved cells with 10 nM rhEGF, the expression patterns of two apoptosis-associated proteins (cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP)) and the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway were measured using immunoblotting. The radiosensitizing effect of EGF was also evaluated with a clonogenic assay and phosphorylated H2AX (γH2AX) immunofluorescence. RESULTS: In the clonogenic assay, the number of colonies was decreased in a dose-dependent manner in EGFR-overexpressing cancer cell lines. The expression of cleaved caspase-3 and cleaved PARP was significantly induced in EGFR-overexpressing cancer cell lines. As for the PI3K/AKT/mTOR signaling pathway, EGF paradoxically suppressed the expression of PI3K, AKT and mTOR in a time-dependent manner. As for the radiosensitizing effect, EGF enhanced the radiosensitivity of AMC-HN3 and A431 cells. CONCLUSION: rhEGF treatment induced cell death in the two EGFR-overexpressing cancer cell lines, and the mode of cell death was apoptosis. Moreover, cell death might be associated with the paradoxical suppression of PI3K/AKT/mTOR signaling pathway. rhEGF in combination with radiation augmented the radiation effect in EGFR-overexpressing cancer cell lines via inhibition of DNA damage repair.

Tumor Growth Suppression and Enhanced Radioresponse by an Exogenous Epidermal Growth Factor in Mouse Xenograft Models with A431 Cells.

PURPOSE: The purpose of this study was to evaluate whether an exogenous epidermal growth factor (EGF) could induce anti-tumor and radiosensitizing effects in vivo. MATERIALS AND METHODS: BALB/c-nu mice that were inoculated with A431 (human squamous cell carcinoma) cells in the right hind legs were divided into five groups: I (no treatment), II (EGF for 6 days), III (EGF for 20 days), IV (radiotherapy [RT]), and V (RT plus concomitant EGF). EGF was administered intraperitoneally (5 mg/kg) once a day and the RT dose was 30 Gy in six fractions. Hematoxylin and eosin (H&E) stained sections of tumor, liver, lung, and kidney tissues were investigated. Additionally, tumors were subjected to immunohistochemistry staining with caspase-3. RESULTS: EGF for 6 days decreased tumor volume, but it approached the level of the control group at the end of follow-up (p=0.550). The duration of tumor shrinkage was prolonged in group V while the slope of tumor re-growth phase was steeper in group IV (p=0.034). EGF for 20 days decreased tumor volume until the end of the observation period (p < 0.001). Immunohistochemistry revealed that mice in group V showed stronger intensity than those in group IV. There were no abnormal histological findings upon H&E staining of the normal organs. CONCLUSION: EGF-induced anti-tumor effect was ascertained in the xenograft mouse models with A431 cells. Concomitant use of EGF has the potential role as a radiosensitizer in the design of fractionated irradiation.

Novel Podoviridae family bacteriophage infecting Weissella cibaria isolated from Kimchi.

The first complete genome sequence of a phage infecting Weissella cibaria (Weissella kimchii) is presented. The bacteriophage YS61 was isolated from kimchi, a Korean fermented vegetable dish. Bacteriophages are recognized as a serious problem in industrial fermentations; however, YS61 differed from many virulent phages associated with food fermentations since it was difficult to propagate and was very susceptible to resistance development. Sequence analysis revealed that YS61 resembles Podoviridae of the subfamily Picovirinae. Within the subfamily Picovirinae, the 29-like phages have been extensively studied, and their terminal protein-primed DNA replication is well characterized. Our data strongly suggest that YS61 also replicates by a protein-primed mechanism. Weissella phage YS61 is, however, markedly different from members of the Picovirinae with respect to genome size and morphology. Picovirinae are characterized by small (approximately 20-kb) genomes which contrasts with the 33,594-bp genome of YS61. Based on electron microscopy analysis, YS61 was classified as a member of the Podoviridae of morphotype C2, similar to the 29-like phages, but its capsid dimensions are significantly larger than those reported for these phages. The novelty of YS61 was also emphasized by the low number of open reading frames (ORFs) showing significant similarity to database sequences. We propose that the bacteriophage YS61 should represent a new subfamily within the family Podoviridae.

Proteomic identification of radiation response markers in mouse intestine and brain.

Increasing efforts are being made to develop more sensitive and faster molecular methodologies at the genomic and proteomic levels for the identification of protein markers after exposure to ionizing radiation (IR). However, few specific protein markers, especially organ-specific markers, have been identified. In this study, we analyzed altered protein expressions in various tissues, namely, brain, lung, spleen, and intestine, from 1 Gy-irradiated mice by employing 2-DE analysis. MALDI-TOF MS and peptide mapping identified 25 proteins that showed greater than twofold expressional changes. In order to confirm significant differences between control and IR-treated samples, ten identified proteins with available commercial antibodies were selected for immunoblotting. Of these, only five showed protein expression patterns that were similar to 2-DE data. These were heat shock protein 5 (HSP 5), HSP 90 kDa ß, HSP 1, transaldolase 1 (TA1), and phosphoglycerate kinase 1 (PGK1). In particular, PGK1 was specifically upregulated in mouse intestine, and TA1 was specifically downregulated in brain by irradiation. TA1 expression was unaltered in other tissues. Based on these data, we suggest that TA1 and PGK1 can be considered as candidate tissue-specific protein markers of IR exposure.