Mol. Cells 2020; 43(10): 889-897
Published online October 26, 2020
https://doi.org/10.14348/molcells.2020.0182
© The Korean Society for Molecular and Cellular Biology
Correspondence to : scbae@chungbuk.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
K-RAS is frequently mutated in human lung adenocarcinomas (ADCs), and the p53 pathway plays a central role in cellular defense against oncogenic K-RAS mutation. However, in mouse lung cancer models, oncogenic K-RAS mutation alone can induce ADCs without p53 mutation, and loss of p53 does not have a significant impact on early K-RAS–induced lung tumorigenesis. These results raise the question of how K-RAS–activated cells evade oncogene surveillance mechanisms and develop into lung ADCs. RUNX3 plays a key role at the restriction (R)-point, which governs multiple tumor suppressor pathways including the p14ARF–p53 pathway. In this study, we found that K-RAS activation in a very limited number of cells, alone or in combination with p53 inactivation, failed to induce any pathologic lesions for up to 1 year. By contrast, when Runx3 was inactivated and K-RAS was activated by the same targeting method, lung ADCs and other tumors were rapidly induced. In a urethane-induced mouse lung tumor model that recapitulates the features of K-RAS–driven human lung tumors, Runx3 was inactivated in both adenomas (ADs) and ADCs, whereas K-RAS was activated only in ADCs. Together, these results demonstrate that the R-point–associated oncogene surveillance mechanism is abrogated by Runx3 inactivation in AD cells and these cells cannot defend against K-RAS activation, resulting in the transition from AD to ADC. Therefore, K-RAS–activated lung epithelial cells do not evade oncogene surveillance mechanisms; instead, they are selected if they occur in AD cells in which Runx3 has been inactivated.
Keywords cancer initiation, K-Ras, lung cancer, p53, Runx3
Lung adenocarcinoma (ADC) is the most frequent subtype of lung cancer. Most lung ADCs develop through stepwise progression from atypical adenomatous hyperplasia (AAH) to bronchio-alveolar carcinoma (BAC), and ultimately to multiple types of invasive ADCs (Subramanian and Govindan, 2008; Wistuba and Gazdar, 2006). Human AAH and BAC are considered to be equivalent to mouse lung adenoma (AD). Approximately 25% of human lung ADC cases harbor activating mutations in the
However, in mouse lung tumor models, oncogenic
Adenovirus carrying Cre recombinase (
To determine the order of
H&E staining was conducted according to a standard protocol. Briefly, slides were rehydrated by ethanol, xylene, and water to remove the paraffin. The nuclei were stained with hematoxylin (#S3309; DAKO, USA) for 3 min, and the cytoplasm was stained with eosin (HT110280; Sigma) for 30 s. Slides were mounted with Permount (SP15-500; Thermo Fisher Scientific, USA) after the dehydration and clearing steps.
For histological analysis, lungs were inflated with 4% paraformaldehyde or formalin (3.7% formaldehyde) and fixed for 36 h. Fixed paraffin sections were rehydrated, subjected to antigen retrieval, and blocked in Tris-buffered saline (0.1% Triton X-100 containing 1% bovine serum albumin) or protein-free blocking solution (DAKO), and sequentially incubated with specific primary antibodies and biotinylated (DAKO) or Alexa Fluor-conjugated secondary antibodies (Invitrogen, USA). Images were produced with a conventional microscope mounted with a DP71 digital camera (Olympus, Japan), an LSM 710 T-PMT confocal microscope (Carl Zeiss, Germany), and an AXIO Zoom.V16 and ApoTome.2 (Carl Zeiss). Images were processed with equivalent parameters using the ZEN Light Edition software (Carl Zeiss).
Standard exome-capture libraries were generated from 1 µg input DNA using the Agilent SureSelect Target Enrichment protocol for Illumina paired-end sequencing library (ver. B.3, June 2015). Probe sets were SureSelect Human All-Exon V6 or SureSelect Mouse All-Exon (Agilent, USA). DNA was quantitated using PicoGreen, and DNA quality was assessed by agarose gel electrophoresis. One microgram of DNA from each cell line was diluted in EB buffer and sheared to a target peak size of 150 to 200 bp using the Covaris LE220 focused ultrasonicator (Covaris, USA). The 8-microTUBE Strips were loaded into the tube holder of the ultrasonicator, and the DNA was sheared using the following settings: mode, frequency sweeping; duty cycle, 10%; intensity, 5; cycles per burst, 200; duration, 60 s × 6 cycles; temperature, 4°C to 7°C. The fragmented DNA was repaired, an ‘A’ was ligated to the 3′ end, and Agilent adapters were ligated to the fragments. Once ligation was assessed, the adapter ligated product was amplified by polymerase chain reaction (PCR). The final purified product was quantified using TapeStation DNA ScreenTape D1000 (Agilent). For exome capture, 250 ng of DNA library was mixed with hybridization buffers, blocking mixes, RNase block, and 5 µl of SureSelect All-Exon capture library according to the standard Agilent SureSelect Target Enrichment protocol. Hybridization to the capture baits was conducted for 24 h at 65°C in a PCR machine, with the thermal cycler lid heated to 105°C. The captured DNA was washed and amplified. The final purified product was quantified by qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and quantified again using the TapeStation DNA ScreenTape D1000 (Agilent). Finally, the DNA was sequenced on the HiSeq 2500 platform (Illumina, USA).
To determine whether the oncogenic
Currently, all available genetically engineered
All
To determine whether the same thing would happen when
Similar to what we observed with
Overall, these findings indicate that
Leaky activation of Cretm/ERT1 occurs in a wide range of tissues (Kemp et al., 2004). Indeed, tumors in
Notably, thymic lymphoma cells infiltrated into the lung along the broncho-vascular bundle in some
Mouse lung tumors induced by urethane, a tobacco carcinogen, recapitulate the natural history of smoking-associated,
Although
To confirm the existence of this defense mechanism and identify the critical genes involved, we need to understand why tumor development in animal models requires activation of oncogenes in a large number of cells. For example,
The Arf–p53 pathway is the primary defense against oncogenic
Targeted therapies that inhibit activated oncogenes have yielded clinical responses but eventually lead to cancer recurrence with secondary oncogene activation in almost all malignancies (Janne et al., 2009; Podsypanina et al., 2008). Therefore, it would be of great therapeutic value to understand why cancers recur after the failure of therapies that target activated oncogenes. Our results explain why cancers recur: inhibition of an activated oncogene causes the cancer to regress, but the regressed cells remain cancer-prone because their oncogene surveillance mechanisms are inactivated.
S-C Bae is supported by a Creative Research Grant (2014R1A3A2030690) through the National Research Foundation (NRF) of Korea. Y-S Lee is supported by Basic Science Research Program grant 2017R1D1A3B03034076. J-W Lee is supported by Basic Science Research Program grant 2018R1C1B6001532. Y Ito is supported by the Singapore Ministry of Health’s National Medical Research Council under its Open Fund Large Collaborative Grant (LCG) Programme (MOH-OFLCG18MAY-0003), NRF Singapore, and the Singapore Ministry of Education under its Research Centres of Excellence initiative.
Y.S.L., J.Y.L., and D.M.K. generated mouse cancer models and analyzed the cancers. X.Z.C. analyzed inactivation, activation, and restoration of target genes in mouse cancer models. J.W.L. and S.H.S. analyzed
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2020; 43(10): 889-897
Published online October 31, 2020 https://doi.org/10.14348/molcells.2020.0182
Copyright © The Korean Society for Molecular and Cellular Biology.
You-Soub Lee1,3 , Ja-Yeol Lee1,3
, Soo-Hyun Song1
, Da-Mi Kim1
, Jung-Won Lee1
, Xin-Zi Chi1
, Yoshiaki Ito2
, and Suk-Chul Bae1,*
1Department of Biochemistry, School of Medicine, Institute for Tumor Research, Chungbuk National University, Cheongju 28644, Korea, 2Cancer Science Institute of Singapore, National University of Singapore, Singapore 117599, 3These authors contributed equally to this work.
Correspondence to:scbae@chungbuk.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
K-RAS is frequently mutated in human lung adenocarcinomas (ADCs), and the p53 pathway plays a central role in cellular defense against oncogenic K-RAS mutation. However, in mouse lung cancer models, oncogenic K-RAS mutation alone can induce ADCs without p53 mutation, and loss of p53 does not have a significant impact on early K-RAS–induced lung tumorigenesis. These results raise the question of how K-RAS–activated cells evade oncogene surveillance mechanisms and develop into lung ADCs. RUNX3 plays a key role at the restriction (R)-point, which governs multiple tumor suppressor pathways including the p14ARF–p53 pathway. In this study, we found that K-RAS activation in a very limited number of cells, alone or in combination with p53 inactivation, failed to induce any pathologic lesions for up to 1 year. By contrast, when Runx3 was inactivated and K-RAS was activated by the same targeting method, lung ADCs and other tumors were rapidly induced. In a urethane-induced mouse lung tumor model that recapitulates the features of K-RAS–driven human lung tumors, Runx3 was inactivated in both adenomas (ADs) and ADCs, whereas K-RAS was activated only in ADCs. Together, these results demonstrate that the R-point–associated oncogene surveillance mechanism is abrogated by Runx3 inactivation in AD cells and these cells cannot defend against K-RAS activation, resulting in the transition from AD to ADC. Therefore, K-RAS–activated lung epithelial cells do not evade oncogene surveillance mechanisms; instead, they are selected if they occur in AD cells in which Runx3 has been inactivated.
Keywords: cancer initiation, K-Ras, lung cancer, p53, Runx3
Lung adenocarcinoma (ADC) is the most frequent subtype of lung cancer. Most lung ADCs develop through stepwise progression from atypical adenomatous hyperplasia (AAH) to bronchio-alveolar carcinoma (BAC), and ultimately to multiple types of invasive ADCs (Subramanian and Govindan, 2008; Wistuba and Gazdar, 2006). Human AAH and BAC are considered to be equivalent to mouse lung adenoma (AD). Approximately 25% of human lung ADC cases harbor activating mutations in the
However, in mouse lung tumor models, oncogenic
Adenovirus carrying Cre recombinase (
To determine the order of
H&E staining was conducted according to a standard protocol. Briefly, slides were rehydrated by ethanol, xylene, and water to remove the paraffin. The nuclei were stained with hematoxylin (#S3309; DAKO, USA) for 3 min, and the cytoplasm was stained with eosin (HT110280; Sigma) for 30 s. Slides were mounted with Permount (SP15-500; Thermo Fisher Scientific, USA) after the dehydration and clearing steps.
For histological analysis, lungs were inflated with 4% paraformaldehyde or formalin (3.7% formaldehyde) and fixed for 36 h. Fixed paraffin sections were rehydrated, subjected to antigen retrieval, and blocked in Tris-buffered saline (0.1% Triton X-100 containing 1% bovine serum albumin) or protein-free blocking solution (DAKO), and sequentially incubated with specific primary antibodies and biotinylated (DAKO) or Alexa Fluor-conjugated secondary antibodies (Invitrogen, USA). Images were produced with a conventional microscope mounted with a DP71 digital camera (Olympus, Japan), an LSM 710 T-PMT confocal microscope (Carl Zeiss, Germany), and an AXIO Zoom.V16 and ApoTome.2 (Carl Zeiss). Images were processed with equivalent parameters using the ZEN Light Edition software (Carl Zeiss).
Standard exome-capture libraries were generated from 1 µg input DNA using the Agilent SureSelect Target Enrichment protocol for Illumina paired-end sequencing library (ver. B.3, June 2015). Probe sets were SureSelect Human All-Exon V6 or SureSelect Mouse All-Exon (Agilent, USA). DNA was quantitated using PicoGreen, and DNA quality was assessed by agarose gel electrophoresis. One microgram of DNA from each cell line was diluted in EB buffer and sheared to a target peak size of 150 to 200 bp using the Covaris LE220 focused ultrasonicator (Covaris, USA). The 8-microTUBE Strips were loaded into the tube holder of the ultrasonicator, and the DNA was sheared using the following settings: mode, frequency sweeping; duty cycle, 10%; intensity, 5; cycles per burst, 200; duration, 60 s × 6 cycles; temperature, 4°C to 7°C. The fragmented DNA was repaired, an ‘A’ was ligated to the 3′ end, and Agilent adapters were ligated to the fragments. Once ligation was assessed, the adapter ligated product was amplified by polymerase chain reaction (PCR). The final purified product was quantified using TapeStation DNA ScreenTape D1000 (Agilent). For exome capture, 250 ng of DNA library was mixed with hybridization buffers, blocking mixes, RNase block, and 5 µl of SureSelect All-Exon capture library according to the standard Agilent SureSelect Target Enrichment protocol. Hybridization to the capture baits was conducted for 24 h at 65°C in a PCR machine, with the thermal cycler lid heated to 105°C. The captured DNA was washed and amplified. The final purified product was quantified by qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and quantified again using the TapeStation DNA ScreenTape D1000 (Agilent). Finally, the DNA was sequenced on the HiSeq 2500 platform (Illumina, USA).
To determine whether the oncogenic
Currently, all available genetically engineered
All
To determine whether the same thing would happen when
Similar to what we observed with
Overall, these findings indicate that
Leaky activation of Cretm/ERT1 occurs in a wide range of tissues (Kemp et al., 2004). Indeed, tumors in
Notably, thymic lymphoma cells infiltrated into the lung along the broncho-vascular bundle in some
Mouse lung tumors induced by urethane, a tobacco carcinogen, recapitulate the natural history of smoking-associated,
Although
To confirm the existence of this defense mechanism and identify the critical genes involved, we need to understand why tumor development in animal models requires activation of oncogenes in a large number of cells. For example,
The Arf–p53 pathway is the primary defense against oncogenic
Targeted therapies that inhibit activated oncogenes have yielded clinical responses but eventually lead to cancer recurrence with secondary oncogene activation in almost all malignancies (Janne et al., 2009; Podsypanina et al., 2008). Therefore, it would be of great therapeutic value to understand why cancers recur after the failure of therapies that target activated oncogenes. Our results explain why cancers recur: inhibition of an activated oncogene causes the cancer to regress, but the regressed cells remain cancer-prone because their oncogene surveillance mechanisms are inactivated.
S-C Bae is supported by a Creative Research Grant (2014R1A3A2030690) through the National Research Foundation (NRF) of Korea. Y-S Lee is supported by Basic Science Research Program grant 2017R1D1A3B03034076. J-W Lee is supported by Basic Science Research Program grant 2018R1C1B6001532. Y Ito is supported by the Singapore Ministry of Health’s National Medical Research Council under its Open Fund Large Collaborative Grant (LCG) Programme (MOH-OFLCG18MAY-0003), NRF Singapore, and the Singapore Ministry of Education under its Research Centres of Excellence initiative.
Y.S.L., J.Y.L., and D.M.K. generated mouse cancer models and analyzed the cancers. X.Z.C. analyzed inactivation, activation, and restoration of target genes in mouse cancer models. J.W.L. and S.H.S. analyzed
The authors have no potential conflicts of interest to disclose.
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