Activation of MYC induces alveolar hyperplasia and apoptosis
We created mouse models in which MYC
expression could be induced with DOX with the aim of examining the earliest stage of MYC
-induced lung tumorigenesis. Transgenic mice expressing rtTA under the control of either the human surfactant protein C (SPC) promoter (S transgene) or the rat Clara cell secretory protein (CCSP) promoter (C transgene) were used to achieve lung specific expression () (18
). Endogenous SPC is expressed in alveolar type II pneumocytes and CCSP in Clara cells of the upper airway. However, the fidelity of the CCSP expression pattern is not maintained by the C transgene (18
). Both the S and C transgenes are transcriptionally active mainly in type II cells and the main distinction between the two transgenes appears to be the level of DOX-inducible expression that can be achieved. We bred S and C mice to mice carrying a human MYC
transgene under the transcriptional control of a TRE promoter (TM transgene) (19
). A diet supplemented with DOX was used to stimulate the transactivating function of the rtTA protein and induce MYC
in the resulting STM and CTM mice.
Figure 1 Over-expression of MYC induced alveolar hyperplasia and apoptosis. (A) Schematic of transgenes used for MYC over-expression (see descriptions in text). (B) Analysis of transgenic MYC expression using whole lung RNA and a human-specific Taqman® (more ...)
Transgenic lungs were examined for MYC mRNA expression after 2, 4, and 7 days of DOX. As expected, expression of MYC was not detected in S mice (data not shown). TM and CTM mice had detectable levels of transcription from the MYC transgene ( and data not shown) but MYC protein remained below the level of detection by Western analysis (). In contrast, we observed a substantial but transient rise in MYC transgene mRNA in STM mice, with a concomitant increase in MYC protein. This coincided with the appearance of alveolar hyperplasia, which was abundant in STM mice () but absent from CTM mice (data not shown) where MYC expression was lower. Alveolar hyperplasia formed as clusters of cells that expanded the alveolar septa, reached a maximum after 4 days of DOX treatment and then resolved (). We pulse-labeled proliferating cells using BrdU. Clusters of BrdU+ cells were common in the alveolar space after 4 days of DOX treatment (), but not after 7 days (). We also detected TUNEL+ cells (). As alveolar hyperplasia resolved, TUNEL staining declined to near background levels (). We concluded that in the alveolar epithelium MYC-induced proliferation was restrained by MYC-induced apoptosis. Eradication of cells by this apoptosis could explain the transience of detectable MYC expression in response to DOX.
Mice that over-express MYC develop lung adenomas and adenocarcinomas
Reduced lifespan was observed for STM and CTM mice, both treated with DOX after weaning and in the absence of DOX, but not in a cohort of DOX-treated TM mice (). STM and CTM mice treated with DOX did fare worse than untreated mice, but decreases in survival did not reach statistical significance (Logrank; p=0.108 for STM and p=0.052 for CTM mice).
Figure 2 Mice over-expressing MYC developed lung adenomas and adenocarcinomas. (A) Kaplan-Meier plot. (B) (B) Graphical representation of lung tumor incidence in transgenic mice. The cumulative numbers of tumors up until 18 months is displayed. (C-G, I, J) H&E (more ...)
After 18 months only solid adenomas were found in 73.3% (n=15) of the DOX treated TM mice (). In contrast, STM and CTM mice developed both papillary adenomas and adenocarcinomas () with DOX increasing the penetrance of adenocarcinoma from 31.6% (n=19) to 57.9% (n=19) and 23.8% (n=21) to 56.7% (n=30) in STM and CTM mice respectively (). Tumorigenesis was rarely multi-focal and respiratory distress was observed only when an adenocarcinoma expanded to occlude an entire lobe of the lung () or the upper respiratory tract (). Metastases () were observed only in DOX treated STM (1 of 17, 5.9%) and CTM (5 of 30, 16.7%) mice.
Our findings suggest that even MYC expression below the limit of detection by Western analysis can predispose to lung tumorigenesis. Despite the initial low levels of MYC, the resulting adenomas and adenocarcinomas both had higher levels of MYC protein than normal lung samples (100% of tumors; ), and tumors from DOX-treated mice always had very high levels of MYC. Given the ability of MYC to elicit apoptosis in pulmonary epithelium, we reasoned that an anti-apoptotic function must emerge during the course of tumorigenesis elicited by activation of the MYC transgene. We sought to define events that occurred in lung tumors to allow bypass of MYC-induced apoptosis.
Figure 3 MYC transgenic tumors have activating mutations in Kras. (A) Western analysis of MYC expression in normal lung, adenomas and adenocarcinomas from transgenic mice. (B) Spectrum of mutations detected in Kras from tumors from DOX-fed MYC transgenic mice (more ...)
Tumors induced by MYC over-expression harbor activating Kras mutations
We immunostained lung tumors from STM and CTM mice for proteins expressed in lung epithelial lineages. Tumors from both genotypes resembled tumors elicited by mutant RAS (23
) in being TTF-1+
(Supplementary Figure 1
). This suggested that the pathogenesis of tumors in STM and CTM mice might also involve activated RAS. We assayed RAS activity and found elevated activity in all the MYC
transgenic tumors (Supplementary Figure 2
). Sequencing of Ras
genes revealed activating Kras
mutations in 100% of the tumors (). Since adenomas harbored Kras
mutation (4/4 adenomas; 7/7 adenocarcinomas; 0/4 control lungs), we presume that the mutations occurred before the transition from adenoma to adenocarcinoma. We concluded that mutant RAS and MYC cooperated in lung tumorigenesis in our MYC
Over-expression of MYC impedes tumorigenesis by activated RAS
It is widely held that RAS blocks MYC-induced apoptosis through the PI3K/AKT pathway, while RAS-induced senescence is overcome by MYC-induced proliferation (26
). Therefore, we reasoned that signaling downstream of activated KRAS might be responsible for abrogation of MYC-induced apoptosis in lung tumorigenesis.
In an initial effort to explore this possibility, we treated mice with the DNA-alkylating agent N-methyl-N-nitrosourea (MNU) to jump-start tumorigenesis with a synchronous wave of mutagenesis in Kras
). As expected, all the lung tumors from MNU-treated mice harbored activating Kras
mutation (data not shown). The survival of TM mice was unaffected by MNU ( and data not shown for DOX-free mice), as these mice only developed a few adenomas ( and data not shown). In contrast, treatment of STM mice with MNU substantially accelerated their usual rate of demise (; compare to ) as adenocarcinomas developed in both the DOX treated and untreated groups. STM mice fed a DOX diet fared better than untreated mice (; Logrank, p
) and had a significantly lower number of tumors compared to mutagenized STM mice kept on a DOX-free diet (; T-test, p
). Therefore, MYC
induction hindered, rather than aided MNU-induced tumorigenesis. These results present an apparent paradox when compared to those in , where activation of the MYC
transgene exacerbated tumorigenesis. We attribute the paradox to the different time frames of tumorigenesis in the two sets of experiments. The relatively rapid course of tumorigenesis in response to MNU might reduce the likelihood that an anti-apoptotic event could occur in time to protect developing tumor cells from the pro-apoptotic effect of MYC.
Figure 4 Activation of the MYC transgene inhibits tumorigenesis induced by activated RAS. (A) Kaplan-Meier plot. Mice were administered 50 mg/kg MNU intraperitoneally and monitored for up to 6 months. (B) Lesions visible on the pleural surface were quantified (more ...)
We further explored the interaction between MYC and activated RAS through retroviral transduction of alveolar hyperplasia (). In brief, we gave STM mice DOX for one day prior to tracheal instillation of concentrated retrovirus encoding mutant RAS. DOX induced the MYC transgene and the proliferation of alveolar cells, a prerequisite for retroviral infection of the otherwise quiescent alveolar epithelium. Transduction was not successful in DOX-treated CTM mice (data not shown), in which the level of MYC induction was below the threshold required to induce hyperplasia (see above).
Our initial proof-of-concept experiments utilized retrovirus expressing activated human HRAS
downstream of an internal ribosomal entry site so that cells expressing activated RAS would be marked. We found that activated forms of both Kras
induced identical tumor phenotypes in this assay. As with NMU-treated animals, transduction with activated RAS accelerated the demise of STM mice (). Continuous DOX administration increased survival (Logrank, p
) and 2/5 mice remained tumor-free (Supplementary Figure 3A
). In contrast, mice treated with DOX only for the 24 hours prior to transduction developed multiple adenomas and adenocarcinomas () and adenomas could be detected as early as one month following transduction (). Only solitary transduced cells were found in mice treated with DOX for one month (n=4 mice) (). At least some of these cells maintained their tumorigenic potential, however, because removal of DOX after one month resulted in tumors (Supplementary Figure 3B
) and intermediate survival (). The data present the same paradox in comparison with as observed with MNU. We presume that the same explanation applies.
Our results suggest that the anti-tumor effects of MYC are not mitigated by activated RAS. This proved to be the case both in the MNU mutagenesis experiment, where activated Kras was expressed from its endogenous promoter, and when activated RAS was ectopically expressed using retrovirus.
MCL1 over-expression allows the progression of MYC over-expressing tumors
We reasoned that MYC
transgenic tumors must harbor additional tumorigenic changes and took a candidate gene approach to search for anti-apoptotic events that could account for the ability of transgenic tumors to over-express MYC. Caspases, members of the inhibitor of apoptosis protein (IAP) family and members of the BCL2 protein family were measured by Western blotting of lysate from tumors and adjacent normal tissue (data not shown and see legend to ). This approach yielded one candidate, MCL1, an anti-apoptotic member of the BCL2 family of proteins (28
). MCL1 was over-expressed in one STM and one CTM adenocarcinoma (2/13 in total) ( and data not shown). This suggested that MCL1 over-expression could be an adaptive trait acquired by some tumors to offset MYC-induced apoptosis. Other, unknown anti-apoptotic events presumably fulfilled a similar role in transgenic tumors not over-expressing MCL1.
Figure 5 MCL1 acts as an oncoprotein when co-expressed with MYC. (A) Of the anti-apoptotic BCL2 family proteins screened by Western analysis of lysates from lungs and tumors of DOX treated MYC transgenic mice (BCL2, BCL2L1/BCL-XL, BCL2L2/BCLW, BCL2A1/BFL1, MCL1), (more ...)
In order to validate that MCL1 cooperated with MYC, we utilized retroviral transduction to introduce Mcl1
alone, or both KrasG12V
together into STM mice (). Retrovirus expressing only Mcl1
had no tumorigenic effect in STM mice (n=8, data not shown). Like mice transduced with HRASG12V
retrovirus (), both KrasG12V
transduced mice had a high tumor burden when placed on a DOX-free diet (Supplementary Figure 4A, B
). There was no significant difference in their survival (Supplementary Figure 4C
). In contrast, continuous induction of the MYC
transgene (Supplementary Figure 4D
) reduced the tumor burden in KrasG12V
transduced mice () but not in KrasG12V
transduced mice (), and the former had significantly better survival relative to the latter (; Logrank, p
tumors from DOX-fed STM mice had a mixed morphology of papillary adenocarcinoma interspersed with areas of large cell undifferentiated carcinoma () that lacked the expression of differentiation markers (Supplementary Figure 5
). This contrasts with the strictly papillary growth in the tumors of the original CTM and STM mice (see ). In human NSCLC, lack of differentiation is correlated with poor prognosis (29
). It appears that the KrasG12V
tumors from mice given DOX to induce MYC
may mimic aggressive human NSCLCs.
Presumably, the effects of MCL1 stemmed from its ability to protect against MYC-induced apoptosis, as MCL1 over-expression did not alter either expression of MYC (Supplementary Figure 4D
) or RAS activity (Supplementary Figure 4E
). We concluded that MCL1 acted as an oncogene, but its tumorigenic potential was manifest only when MYC was over-expressed.
Combined high MCL1 and MYC expression is a poor prognostic indicator in NSCLC
To explore whether the interaction between MCL1 and MYC has biological significance in human NSCLC, we evaluated their expression by immunohistochemistry. Compared to normal lung tissue, MYC and MCL1 were each over-expressed in a subset of tumors (Supplementary Table 1
, Supplementary Figure 6
). High expression of MYC and MCL1 was detected in 30.5% and 49.1% of the tumors, respectively. Of the MYC over-expressing tumors, 72.2% also over-expressed MCL1 (, ). Therefore, MYC and MCL1 over-expression overlapped significantly (22% of all the NSCLCs examined).
Demographic characteristics of human NSCLC cases used in survival analysis
Figure 6 High MCL1 expression correlates with poor prognosis in human NSCLC co-expressing MYC. (A) Percentage of MYC positive and MYC negative human NSCLC that co-stained for high versus low levels of MCL1. MYCpos tumors are those with an immunoreactive score (more ...)
A trend was observed for both MYC () and MCL1 (Supplementary Table 2
) to be highly expressed in NSCLCs that were less differentiated, a finding that prompted us to examine the correlation of combined expression with prognosis. Among the 59 NSCLCs with full follow-up (demographics in ), neither MYC nor MCL1 expression alone was prognostic (data not shown). However, two completely different outcomes were observed when tumors expressing MCL1 were stratified by MYC expression. Whereas high expression of MCL1 was marginally associated with better survival when MYC was not expressed (p
, ), it was significantly associated with poorer overall survival when MYC was expressed (p
, ). The data suggest that high MCL1 and MYC expression may cooperate during the genesis of a subset of human NSCLCs and combined expression has implications for patient survival. A likely explanation for our findings is that MCL1 facilitates NSCLC progression by impeding MYC’s pro-apoptotic function.