Growth suppression by MYC inhibition in small cell lung cancer cells with TP53 and RB1 inactivation

Abstract

Small cell lung cancer (SCLC) is the most aggressive type of lung cancer with high mortality. One of the MYC family genes, MYC, MYCL or MYCN, is amplified in ~20% of the SCLCs; therefore, MYC proteins are potential therapeutic targets in SCLC patients. We investigated the therapeutic impact of Omomyc, a MYC dominant negative, in a panel of SCLC cell lines. Strikingly, Omomyc suppressed the growth of all tested cell lines by inducing cell cycle arrest and/or apoptosis. Induction of G1 arrest by Omomyc was found to be dependent on the activation of CDKN1A, in part, through the TP73 pathway. Our results strongly indicate that SCLC cells carrying amplification of MYC, MYCL or MYCN are addicted to MYC function, suggesting that MYC targeting would be an efficient therapeutic option for SCLC patients.

Keywords: CDKN1A; MYC; MYCL; SCLC; small cell lung cancer.

Figure 1. Omomyc induces growth suppression in SCLC cells

A. Status of the MYC family genes, TP53, and RB1 in SCLC cell lines used in this study. mut: mutated. Predominant type of the cell cycle arrest, occurrence of apoptosis and levels of p21, p27 and p16 after MYC inhibition by Omomyc are shown. B. Immunoblot analysis for the expression of MYC, MYCL or MYCN in SCLC cells. Media were changed 24 hr before collection of the cells. C. Growth curve of SCLC cells in the presence or absence of doxycycline (DX). Cumulative population doubling level (PDL) was calculated by adding the PDLs of the previous passages. Data are shown as the mean ± SD of four counts from a single representative experiment. P-values were calculated by unpaired two-tailed t-test. *p<0.05, **p<0.01. D. Percentage of dead cells. E. Representative images of floating aggregates after two (Lu135^omo, H2141^omo, H69^omo) or three (HCC33^omo) passages in culture in the presence or absence of Omomyc. Cells were photographed using phase-contrast microscopy at 5x magnitude.

Figure 2. Omomyc disrupts Myc/MAX heterodimerization

A, B, D, E. Co-immunoprecipitations (co-IP) were carried out using protein extracts from Lu135^omo (A, D) or H2141^omo (B, E) cultured in the presence or absence of Omomyc for 20 hr, using antibodies against tRFP (A, B), MYC (D), or MYCL (E). C. MYC, MYCL, MAX and Omomyc co-IP band intensities were quantified and normalized to corresponding Input band intensities. F. MAX co-IP band intensities were quantified and normalized to corresponding MYC or MYCL IP band intensities. The anti-tRFP antibody was used for Omomyc immunoprecipitation since the antibody for Omomyc crossreacted with MAX.

Figure 3. Effects of Omomyc on cell cycle progression

A–C, Representative population histograms showing the cell cycle profiles and the apoptotic fractions (sub-G1) of Lu135^omo (A), H69^omo (B) and H345^omo (C) with or without Omomyc induction. x axis and y axis corresponds to DNA content and cell number, respectively. Sub-G1 gate is highlighted in green. The ratio of the number of cells in G1 phase versus S plus G2/M phases is shown. D. Cell cycle profile of cells 48 hr (N417^omo, Lu135^omo, H446^omo, H69^omo), 72 hr (H2141^omo), 96 hr (H345^omo), or 1 week (HCC33^omo, H526^omo, H2107^omo) after Omomyc induction.

Figure 4. Omomyc induces CDKN1A and CDKN1B

A. Immunoblot analysis and B. densitometric quantification for the expression of p21, p27 or p16 in SCLC cells with or without DX for 3-7 days. Expression levels of p21, p27 and p16 were normalized to the levels of tubulin, and the ratio of DX+ / DX- is shown. Media were changed 24 hr before collection of the cells.

Figure 5. Activation of CDKN1A by knockdown of MYC or MYCL

A. Immunoblot showing MYC, p21, p27 and PARP1 levels after 72 hr of siMYC transfection in Lu135^omo cells. B, C. Number of Lu135^omo cells and percentage of dead cells after 96 hr of transfection with control siRNA (siCtrl) or siMYC. D. Immunoblot showing MYCL, p21, p27, PARP1 and tRFP levels after 72 hr of doxycycline addition in H2141^shMYCL cells. E, F. Growth curve of H2141^shMYCL cells and percentage of dead cells in the presence or absence of doxycycline. *p<0.05, **p<0.01.

Figure 6. G2/M arrest in H69^omo induced by high levels of Omomyc expression

A, B. Red fluorescence intensity in H69^omo induced for Omomyc-RFP expression with different concentrations of DX for 72 hr. Lu135^omo cells were used as a representative cell line for the induction of Omomyc-RFP in other SCLC cell lines. C. Immunoblot analysis of Omomyc induction, PARP1 cleavage and p21 levels. D, E. Effects of Omomyc on proliferation and death of H69^omo. F. Cell cycle distribution and apoptotic fraction. The ratio of the number of cells in G1 phase versus S plus G2/M phases is shown.

Figure 7. Downregulation of p21 impairs the Omomyc-induced G1 arrest

A. Immunoblot analysis for the knock-down of p21 in N417^omo and Lu135^omo cells. Cells were cultured with or without DX (+/− Omomyc). B. Number of cells after 72 hr (N417^omo) or 96 hr (Lu135^omo) of culture. Data are shown as mean ± SD (n=4). P-values were calculated by unpaired two-tailed t-test. *p<0.01. C. Cell cycle distribution after after 72 hr (N417^omo) or 96 hr (Lu135^omo) of culture. x axis and y axis corresponds to DNA content and cell number, respectively. Sub-G1 gate is highlighted in green. The ratio of the number of cells in G1 phase versus S plus G2/M phases is shown. D. Representative images of floating cell aggregates after 72 hr (N417^omo) or 96 hr (Lu135^omo) of culture. Cells were photographed using phase-contrast microscopy at 5x magnitude. E. Cleavage of PARP1 in Lu135^omo was evaluated by immunoblot analysis. Band intensity was quantified by densitometry and the ratio of cleaved/total (uncleaved+cleaved) was calculated.

Figure 8. Activation of CDKN1A by TP73 in a MYC-amplified SCLC cell line

A. Schematic representation of the CDKN1A gene and sequences amplified after chromatin immunoprecipitation (ChIP). Binding of MYC and MIZ1 has been reported in the proximity of P2 transcription starting site, spanning R2 region. B. ChIPs were carried out using chromatin obtained from Lu135^omo cultured in the presence or absence of Omomyc for 48 hr using antibodies against MYC or MIZ1. R1 and R3 regions were used as negative controls of DNA: protein binding. C. Immunoblot analysis for the expression of TP73 in SCLC cells. D. Immunoblot analysis for the expression of TP73, p21, MYC, and Omomyc in TP73 knock-down Lu135^omo cells 72 hr after Omomyc induction. Protein levels were normalized to the levels of tubulin. E. Flow cytofluorimetric analysis of Omomyc-RFP red fluorescence (575nm ±13) after 96 hr of DX addition. F. Number of cells after 96 hr from the induction of Omomyc. Data are shown as the mean ± SD (n=3). P-values were calculated by unpaired two-tailed t-test. *p<0.05. G. Cell cycle distribution. The ratio of the number of cells in G1 phase versus S plus G2/M phases is shown.

Figure 9. MYC, MYCL and MYCN inhibition by Omomyc induces cell cycle arrest through the activation of p21, in some cases through the TP73 pathway