Activation of Kv11.1 potassium channel suppresses non-small cell lung cancer growth by promoting c-Myc degradation

Figures

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  Fig. 1. KCNH2 is a prognostic factor for LuAd. A Violin plots, and B bar chart of KCNH2 gene expression in LuAd comparing paired tumor and normal gene array (GEO database). The bars represent the proportions of tumor samples exhibiting higher expression of the selected gene compared to noncancerous samples at each of the quantile cut-off values (min=minimum, Q1=1st quartile, Med=median, Q3=3rd quartile, Max=maximum). Specificity was calculated by dividing the number of tumor samples by the sum of tumor and normal samples below each given cut-off. In cases where the fold change was greater than 1, “over” was used instead of “below”. C Survival analysis (Kaplan-Mayer plot) and D progression free survival for LuAd patients expressing high KCNH2 level (red) vs low level (black). E Violin plots and F bar chart of KCNH2 gene expression in LuAd comparing paired tumor and normal gene array (GEO database). G Kaplan-Mayer plot and H progression free survival for SSC patients expressing high KCNH2 level (red) vs low level (black).

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  Fig. 2. Kv11.1 activation arrests proliferation in lung cancer cells. A Western blot showing expression of the Kv11.1 ion channel protein in human derived A549, H1299, H1437, H1650 lung cancer cell lines. B Cell imaging of H1299 or H1650 lung cancer cell lines stained with FITC-Kv11.1 (Alomone; APC-109-F) vs unstained (Control). Images were obtained via wide-field fluorescent imaging with a 20× objective lens (blue = DAPI nuclear counterstain, green = GFP-tubulin). C Cell cycle analysis (flow cytometry) of A549 or D) H1299 lung cancer cell line treated with NS1643 (50 mM; 24–48 h). Numeric breakdown (data for NS1643 refers to 48 h of treatment); A459: control G0/G1 phase = 56.66% ± 0.66%; NS1643 G0/G1 phase = 70.53% ± 0.90%; control S phase = 20.0% ± 1.15%; NS1643 S phase = 10.13% ± 0.59%; control G2/M phase = 20.66% ± 0.66%; NS1643 G2/M phase =16.0% ± 0.57%. H1299: control G0/G1 phase = 56.93% ± 0.7%; NS1643 G0/G1 phase = 64.83% ± 0.44%; control S phase = 10.1% ± 1.45%; NS1643 S phase = 9.86% ± 0.52%; control G2/M phase = 26.56% ± 0.58%; NS1643 G2/M phase = 24.0% ± 0.72%. A smaller concentration of NS1643 produced the same effect but for a longer time. E Western blot showing the effects…

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  Fig. 3. NS1643 does not promote lung cancer cells’ death. A Cell death assay via Annexin V detection (flow cytometry) in A549 or H1299 lung cancer cell line treated with NS1643 (50 mM; 24–48 h).

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  Fig. 4. KCNH2 inversely correlates with MYC. A KCNH2 mRNA expression (RNA sequence V2 RSEM in log2) showed negative co-relation with MYC. B Survival analysis (cBioPortal) for LuAd patients expressing high KCNH2 level (orange) vs low level (green), C high KCNH2 (orange) and high MYC (purple) or D comparison between high KCNH2, Low KCNH2 and MYC. E–H Survival analysis (cBioPortal) for SSC patients expressing high KCNH2 level (orange) vs low level (green), high KCNH2 (orange) and high MYC (purple) or comparison between high KCNH2, Low KCNH2 and MYC.

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  Fig. 5. Kv11.1 reduces the steady-state levels of the c-Myc protein and increases expression of genes repressed by c-Myc. A Western blot showing the effect of NS1643 treatment (50 mM; 0-30-60-120 min) on c-Myc protein level in A549, H1299 or B H1437, H1650 lung cancer cell lines. Bar graph showing quantification of the events in (A, B). C Bar graph shows mRNA expression for FASN, DUSP1, and GaDD45A in A549 or D H1299 lung cancer cell lines after 24 h NS1643 treatment.

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  Fig. 6. Kv11.1 activator NS1643 arrests lung tumor growth. A Proliferation assay performed with Lewis lung carcinoma cells exposed to NS1643 (25–50 mM/24 h). Inset Western blot indicates expression of the Kv11.1 channel. B Effects of NS1643 injection (6 mg/Kg; 3 days interval of intraperitoneal injection) on LLC tumor growth in LLC syngeneic model. C, D Size and weight comparison of LLC tumors dissected from control (DMSO) or test (NS1643) mice. E Representative images of IHC staining of Ki-67 and F c-Myc (A) in control vs NS1643 treated LLC tumor model. Scale bar = 50 µm, magnification ×400. *, ** statistically significant (P < 0.05 or P < 0.01, respectively).

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  Fig. 7. Kv11.1 activation destabilizes c-Myc protein. A Western blot showing the effect of NS1643 alone, cycloheximide alone (CHX) or NS1643 and CHX at different time points (0, 30, 60, 90 min) on c-Myc protein density in A549 lung cancer cells. Intensities were normalized for the loading control histone H3. B Quantification of the events in (A) for the 0, 30, 60 min time points. C Western blot showing the effect of NS1643 (50 mM; 24 h) alone or with the proteasome inhibitor MG132 in A549 cells. The bar graph indicates quantification of (C). D Western blot showing the effect of NS1643 (50 mM; 24 h) on phosphorylation of Serine 62 or Threonine 58 of c-Myc in A549 cell line. E Western blot showing the effect of NS1643 (50 mM; 24 h) on the doxycycline-induced c-MycT58A in A549 cells. F Western blot showing the effect of NS1643 (50 mM; 24 h) on c-Myc in A549 cells transiently transfected with either OTUD6B1 or OTUD6B2 in pCDNA3. Similar results were obtained in H1299. *, **statistically significant (P < 0.05 or P < 0.01, respectively).