human sclc cell lines h446  (ATCC)

Journal: Cancer Science

Article Title: Ligand‐independent EphA2 contributes to chemoresistance in small‐cell lung cancer by enhancing PRMT1 ‐mediated SOX2 methylation

doi: 10.1111/cas.15653

Figure Lengend Snippet: Ligand‐independent phosphorylation of EphA2 is related to chemoresistance of SCLC. (A) mRNA expression of EPHA2 was compared in normal lung tissue ( n = 20) and SCLC ( n = 150) FFPE samples by RT‐qPCR. Data are presented in the form of mean ± SD based on independent biological replicates, **** p ≤ 0.0001. (B) Kaplan–Meier analysis of the overall survival of 150 patients with SCLC divided into high ( n = 75) and low groups ( n = 75), separated by the median level, based on EPHA2 expression levels. * p ≤ 0.05. (C) Western blot analysis shows that the expression of EphA2 (total and pS897) was enhanced in drug‐resistant sublines H69AR and H446CDDP in comparison with drug‐sensitive sublines H69 and H446, while EphA2‐pY588 showed the opposite. (D) Western blots analysis shows that the expression of ephrin‐A1 was decreased in H69AR and H446CDDP compared with H69 and H446. The expression of Akt‐pS473 showed the opposite. The expression of Akt showed no significant difference between H69 and H69AR, and between H446 and H446CDDP. (E) Western blots analysis shows that EphA2‐pS897 expression was decreased in H69AR and H446CDDP after ephrin‐A1 treatment, while EphA2‐pY588 showed the opposite. EphA2 (total) expression remained unaltered after ephrin‐A1 treatment. (F) Western blot analysis showed that Akt‐pS473 was decreased in H69AR and H446CDDP after MK2206 treatment. Akt (total) expression remained unaltered. (G) Western blot analysis shows that EphA2‐pS897 expression was decreased in H69AR and H446CDDP after MK2206 treatment. EphA2 (total) expression remained unaltered. Western blot analysis shows inhibition of Akt expression by Akt siRNA suppressed phosphorylation of EphA2‐S897 in H69AR (H) and H446CDDP (I), while EphA2 (total) expression remained unaltered. (J) Expression levels of CD44, Myc, and SOX2 were higher in drug‐resistant sublines (H69AR and H446CDDP) compared with drug‐sensitive sublines (H69 and H446). (K) Western blots show that the expression levels of N‐cadherin and vimentin were higher in drug‐resistant sublines compared with drug‐sensitive sublines, while E‐cadherin expression was the opposite. These experiments were repeated at least three times and representative images are shown.

Article Snippet: Human SCLC cell lines (NCI‐H69, NCI‐H69AR, and NCI‐H446) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Phospho-proteomics, Expressing, Quantitative RT-PCR, Western Blot, Comparison, Inhibition

Journal: Cancer Science

Article Title: Ligand‐independent EphA2 contributes to chemoresistance in small‐cell lung cancer by enhancing PRMT1 ‐mediated SOX2 methylation

doi: 10.1111/cas.15653

Figure Lengend Snippet: Wild‐type EphA2 enhances the cancer stemness and chemoresistance of SCLC, whereas the P817H mutation neutralizes intrinsic function. (A) Western blots show that the expression levels of EphA2 (total and pS897) were significantly reduced in H69AR cells, which were stably transfected with sh EPHA2 . (B) IC 50 values of epirubicin (ADM), cisplatin (CDDP), and etoposide (VP‐16) were significantly reduced in response to sh EPHA2 transfection compared with the control group in H69AR cells. The number of independent biological replicates = 3, *** p ≤ 0.001, **** p ≤ 0.0001. (C) Western blots show that the expression levels of CD44, Myc, and SOX2 were decreased in H69AR cells with silenced EPHA2 . (D) Western blots show that the expression levels of N‐cadherin and vimentin were reduced in H69AR cells with silenced EPHA2 , while E‐cadherin expression was increased. (E) SFE of sh EPHA2 groups in H69AR cells. Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (F) Western blots show the changes in expression levels of EphA2 (total and pS897) in H69 cells, which were stably transfected with wild‐type EPHA2 (WT), A785S, P817H, or Y930D mutant EPHA2 . (G) IC 50 values of ADM, CDDP, and VP‐16 in H69 cells with upregulated wild‐type or mutant EphA2, respectively. The number of independent biological replicates = 3, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (H) Western blots show that the expression levels of CD44, Myc, and SOX2 were increased in H69 cells overexpressing wild‐type, A785S, or Y930S mutant EphA2. (I) Western blots showed the expression levels of N‐cadherin and vimentin were increased in H69 cells overexpressing wild‐type, A785S, or Y930S mutant EphA2, while E‐cadherin expression was reduced. (J) SFE of wild‐type and mutant EphA2 groups in H69 cells. Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (K) H69 cells stably overexpressed wild‐type EphA2, P817H mutant EphA2, or the control. (L) H69AR cells were stably transfected with sh EPHA2 or shNC. Each group of cells was injected into mice, followed by chemotherapy (CDDP + VP‐16) or empty vehicles were injected intraperitoneally as indicated ( n = 5 mice for each group). (M) Tumor weights of EphA2 WT groups and EphA2 P817H groups. Data are displayed as the mean ± SD, n = 5, * p ≤ 0.05, ** p ≤ 0.01. (N) Tumor weights of sh EPHA2 groups. Data are displayed as the mean ± SD, n = 5, *** p ≤ 0.001. Each group of cells was injected into mice, followed by chemotherapy (CDDP + VP‐16) or empty vehicles were injected intraperitoneally as indicated ( n = 5 mice for each group). These experiments were repeated at least three times and representative images are shown.

Article Snippet: Human SCLC cell lines (NCI‐H69, NCI‐H69AR, and NCI‐H446) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Mutagenesis, Western Blot, Expressing, Stable Transfection, Transfection, Control, Injection

Journal: Cancer Science

Article Title: Ligand‐independent EphA2 contributes to chemoresistance in small‐cell lung cancer by enhancing PRMT1 ‐mediated SOX2 methylation

doi: 10.1111/cas.15653

Figure Lengend Snippet: PRMT1 augments stemness and chemoresistance in SCLC. (A) Western blots show the changes in expression levels of PRMT1 in H69 cells, which were stably transfected with His‐tagged PRMT1 . (B) Western blots show that the expression levels of CD44, Myc, and SOX2 were increased in H69 cells overexpressing PRMT1. (C) Western blots show that the expression levels of N‐cadherin and vimentin were increased in H69 cells overexpressing PRMT1, while E‐cadherin expression was reduced. (D) SFE of PRMT1 groups in H69 cells. Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (E) IC 50 values of ADM, CDDP, and VP‐16 in H69 cells with upregulated PRMT1. The number of independent biological replicates = 3, *** p ≤ 0.001. (F) Western blots show that the expression levels of PRMT1 were reduced in H69 cells with co‐transfected wild‐type or P817H mutant EPHA2 and sh PRMT1 . (G) Western blots show that the expression levels of CD44, Myc, and SOX2 were reduced in H69 cells with co‐transfected wild‐type or mutant EPHA2 and sh PRMT1 . (H) Western blots show that the expression levels of N‐cadherin and vimentin were reduced in H69 cells with co‐transfected wild‐type or mutant EPHA2 and sh PRMT1 , whereas E‐cadherin expression was increased. (I) SFE of sh PRMT1 groups in H69 cells with wild‐type or mutant EPHA2 . Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (J) IC 50 values of ADM, CDDP, and VP‐16 in H69 cells with co‐transfected wild‐type or mutant EPHA2 and sh PRMT1 . The number of independent biological replicates = 3, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. These experiments were repeated at least three times and representative images are shown.

Article Snippet: Human SCLC cell lines (NCI‐H69, NCI‐H69AR, and NCI‐H446) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Mutagenesis

Journal: Cancer Science

Article Title: Ligand‐independent EphA2 contributes to chemoresistance in small‐cell lung cancer by enhancing PRMT1 ‐mediated SOX2 methylation

doi: 10.1111/cas.15653

Figure Lengend Snippet: Inhibiting ligand‐independent EphA2 suppresses the growth of SCLC in vitro and in vivo. (A) Western blots show that the expression level of EphA2‐pS897 was reduced in response to treatment with ALW‐II‐41‐27 (ALW) for 2 h in a dose‐dependent manner in H69AR cells. The total EphA2 expression remained unaltered. (B) Western blots show that the expression level of EphA2‐pS897 was reduced in response to treatment with dasatinib (Dasa) for 24 h in a dose‐dependent manner in H69AR cells. The total EphA2 expression remained unaltered. (C) IC 50 values of ADM, CDDP, and VP‐16 were reduced in H69 and H69AR cells. H69 and H69AR cells were treated with empty vehicle, or 0.75 or 1.5 μmol/L of ALW‐II‐41‐27. The number of independent biological replicates = 3, *** p ≤ 0.001, **** p ≤ 0.0001. (D) IC 50 values for ADM, CDDP, and VP‐16 were reduced in H69 and H69AR cells. H69 and H69AR cells were treated with empty vehicle, 2.5 or 5 μmol/L of dasatinib. The number of independent biological replicates = 3, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (E) Western blots show that the expression levels of N‐cadherin and vimentin were reduced in response to treatment with ALW‐II‐41‐27, in H69AR cells, while E‐cadherin expression was increased. (F) Western blots show the changes in expression levels of CD44, Myc, SOX2, and PRMT1, in response to ALW‐II‐41‐27 treatment, in H69AR cells. (G) Western blots show that the expression levels of N‐cadherin and vimentin were reduced in response to the treatment with AMI‐1, in H69AR cells, while E‐cadherin expression was increased. (H) Western blots show the changes in expression levels of CD44, Myc, and SOX2, in response to treatment with AMI‐1, in H69AR cells. (I) SFE of H69AR cells, which were treated with ALW‐II‐41‐27. Data are presented in the form of mean ± SD based on three independent biological replicates, **** p ≤ 0.0001. (J) Immunofluorescence shows the changes in subcellular localization of EphA2 and EphA2‐pS897 in response to treatment with ALW‐II‐41‐27, in H446 cells overexpressing wild‐type or P817H EphA2. Scale bars = 10 μm. (K) Effect of chemotherapy (CDDP + VP‐16), ALW, or the combination of ALW‐II‐41‐27 and chemotherapy on tumor growth using SCLC PDX models. (L) Effect of chemotherapy (CDDP + VP‐16), ALW‐II‐41‐27, or the combination of ALW‐II‐41‐27 and chemotherapy on tumor growth in vivo. Nude mice were engrafted with H69AR cells subcutaneously. These experiments were repeated at least three times and representative images are shown.

Article Snippet: Human SCLC cell lines (NCI‐H69, NCI‐H69AR, and NCI‐H446) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Immunofluorescence