Conditional expression of activated NOTCH1 in the alveolar epithelium
To model NOTCH1 activation in the lungs of adult mice we overexpressed N1ICD using a DOX-inducible system (). Transgenic mice expressing rtTA under the transcriptional control of the rat CCSP promoter (the C transgene) were used to achieve lung-specific expression. Although CCSP is expressed in Clara cells of the bronchiolar epithelium, the C transgene is transcriptionally active mainly in epithelial cells of the distal lung (11
). We bred C mice to mice carrying a transgene encoding N1ICD (sequence of human NOTCH1
encoding amino acids 1756–2556) under the transcriptional control of a tetracycline-response element (TRE) promoter (the N1 transgene) (12
Figure 1 Overexpression of N1ICD induces transient alveolar hyperplasia. (A) The C and N1 transgenes were used to direct N1ICD expression to lung epithelial cells with DOX treatment. (B) Western analysis of N1ICD expression in lung protein extracts following 0, (more ...)
A diet supplemented with DOX was used to stimulate the transactivating function of the rtTA protein and induce N1ICD expression in the resulting CN1 mice. The 110 kDa N1ICD protein fragment was induced in CN1 mouse lungs after 7 continuous days of DOX treatment and reached a maximum level of expression after 14 days (). N1ICD levels decreased after day 14 but nonetheless remained elevated (see day 30, ). We assayed the mRNA levels of a subset of known targets of the NOTCH transcriptional complex. Some responded to DOX induction, including Hes5 and Nrarp (). Notably, Hes1, a gene whose transcription is often used as a measure of NOTCH activation, was not induced. Therefore, N1ICD overexpression activated only a subset of the known NOTCH targets, presumably reflecting tissue specific differences in the transcriptional program activated by NOTCH.
N1ICD induction of alveolar hyperplasia and apoptosis
We wished to assess if there were any acute consequences associated with N1ICD overexpression. Histological examination of H&E stained sections revealed an abundance of alveolar hyperplasia after 7 days of DOX treatment (compare ). The hyperplasia was comprised of clusters of cells found in the alveolar space, reached a maximum after 14 days of DOX treatment () and then regressed (). Regression was never absolute as hyperplastic cells persisted in the lungs of older mice (compare ). Immunohistochemical staining for NOTCH1 confirmed that the hyperplastic cells overexpressed N1ICD ().
We supposed that the transient hyperplasia could result from proliferation followed by apoptosis. Immunohistochemical staining for Ki67 antigen was performed to mark cycling cells (Supplementary Figure 1A–D
). The number of cycling cells in DOX treated CN1 mice was substantially increased after 7 and 14 days of treatment, but regressed by 30 days of continuous DOX treatment (). TUNEL staining was used to detect apoptotic cells with fragmented DNA (Supplementary Figure 1E–H
). CN1 mice did not have a substantial increase in TUNEL+
cells after 7 days of continuous DOX treatment, but positive cells were abundant after 14 days and remained elevated after 30 days (). Therefore, alveolar epithelial cells proliferated in response to N1ICD overexpression, but cell death tempered the proliferative response.
Figure 2 Apoptosis contributes to the clearance of N1ICD overexpressing cells from the alveolar space. (A) Quantification of Ki67+ cells in the lungs of DOX treated CN1 mice (n≥3 mice, error bars are ± standard deviation; * p<0.01 compared (more ...)
N1ICD overexpression engages the BCL2 family of apoptotic regulators
To define mediators of the apoptosis that cleared N1ICD-induced alveolar hyperplasia, we screened protein lysates from DOX treated CN1 mice for alterations in anti- and pro-apoptotic proteins. We found that the anti-apoptotic BCL2 protein was repressed in CN1 mouse lungs after 14 days of DOX (). Induction of full-length, pro-apoptotic BCL2 family proteins BAK and BOK and the BH3-only proteins PUMA, BIK, BMF, and BIM, accompanied BCL2 repression (see Supplementary Figure 2A
for unaltered BCL2 family proteins). Concurrently, cleaved forms of CASPASES 3 and 7, and the Caspase target PARP1 accumulated. We concluded that activated NOTCH1 induced alterations in multiple BCL2 family proteins to stimulate the intrinsic apoptotic cascade.
) and Bok
) are transcriptional targets of the p53 tumor suppressor. We reasoned that p53 induction might contribute to the apoptotic clearance of N1ICD-induced alveolar hyperplasia, but expression analysis of mRNAs encoding BCL2 family proteins altered by N1ICD showed a modest but statistically significant induction of Bik
, but not Puma
(Supplementary Figure 2B
). In addition, neither p53 protein nor trp53
mRNA was induced at day 14 of DOX treatment (Supplementary Figure 3A, B
). We concluded that apoptotic signaling was engaged independent of p53 induction and that non-transcriptional regulatory mechanisms that are yet to be defined likely account for most of the observed alterations in BCL2 family proteins.
N1ICD-induced lung adenomas
We reasoned that over time genetic and/or epigenetic defects might accumulate in the lungs of CN1 mice, enabling cells to evade intrinsic tumor suppressive functions, such as apoptosis. Therefore, we monitored DOX-fed CN1 mice at different time-points for signs of tumorigenesis. Beginning as early as 8 months of age we observed that N1ICD overexpressing cells displayed an altered pattern of growth. Instead of growing as cell clusters (as in ), some N1ICD+ cells expanded laterally along the alveolar walls in a bronchioalveolar pattern (). Small adenomas could be found in each of the 8-month old CN1 mice (n=4 mice). These early tumors had bronchioalveolar patterning () and appeared to have formed from the coalescence of N1ICD+ cells ().
Figure 3 Adenomas develop in the lungs of DOX treated CN1 mice. (A–D) Sections from CN1 mice maintained on DOX for 8 months. (A) H&E stained section showing organized growth along the alveolar walls. (B) Immunohistochemical staining for NOTCH1 (more ...)
Eventually DOX-fed CN1 mice developed multiple adenomas with papillary histology () and succumbed to their tumor burden at an average age of 15.1±2.2 months (n=11 mice). We did not find adenocarcinomas (size ≥ 5mm) in any of these mice. Only rare adenomas were found in CN1 mice not fed a DOX diet (average = 0.38±0.72 per mouse, n=16 mice).
We investigated the expression of markers of differentiated lung epithelial lineages in alveolar hyperplasia and the adenomas that arose in DOX fed CN1 mice. Alveolar hyperplasia in CN1 mice treated with DOX for 7 days was TTF-1+
(Supplementary Figure 4A–C
), suggesting it may have arisen from type II pneumocytes where TTF-1 and SPC are co-expressed. Adenomas from CN1 mice were TTF-1+
with scattered SPC+
cells (). Therefore, both the expression of SPC and the anatomical location in which N1ICD+
cells first arise suggest that N1ICD+
adenomas may derive from type II cells.
The ARF/MDM2/p53 network in N1ICD-induced adenomas
We wondered what other factors contributed to the formation of lung adenomas in DOX treated CN1 mice. For example, in T-ALL, N1ICD suppresses the ARF/MDM2/p53 network (12
). However, examination of the ARF/MDM2/p53 network in the lungs of CN1 mice demonstrated that p19ARF
was induced, not repressed, at both the protein and mRNA level when N1ICD was overexpressed (Supplementary Figure 3A
). In addition, Mdm2
was transcriptionally repressed in adenomas (Supplementary Figure 5B
). Both findings are consistent with stabilization, not degradation, of p53, although no p53 accumulation was observed (Supplementary Figure 3A
). Our data suggests that in the lung N1ICD does not inhibit p53 through repression of p19ARF
, a finding that contrasts with findings in T-ALL (12
MYC and MYCL1 are induced in adenomas
We also examined the hypothesis that long-term formation of adenomas in CN1 mice could be mediated, at least in part, by MYC activity. Transcription from MYC
is induced by activated NOTCH1 in T-ALL (17
) and in mammary tumors (19
). We measured expression of MYC, MYCL1 and MYCN in lung protein extracts from CN1 mice treated with DOX. MYC and to a lesser extent MYCL1 were induced in the lungs of mice treated with DOX for 7 and 14 days (). The expression of both proteins was undetectable by day 30 of DOX treatment when hyperplasia had mostly resolved. However, expression of MYC and MYCL1 reemerged in adenomas from CN1 mice receiving long-term treatment with DOX ().
Figure 4 MYC and MYCL1 are upregulated in N1ICD-induced adenomas. (A) Western analysis of MYC, MYCL1 and MYCN expression in the lungs of CN1 mice treated with DOX for 0, 7, 14 and 30 days and in adenomas from CN1 mice continuously fed a DOX diet until sacrifice (more ...)
Next we measured the mRNA level of the Myc genes in the lungs and adenomas of CN1 mice. Although MYC and MYCL1 protein could be easily detected after just 7 days of DOX treatment, no changes in the transcription of any of the Myc genes was detected (). Adenomas, however, had upregulated transcription of both Myc and MycL1, but not MycN, a result that mirrored changes observed at the protein level. Therefore, transcriptional induction of both Myc and Mycl1 could account for the presence of MYC and MYCL1 in adenomas, but altered post-transcriptional regulation likely underlies the upregulation of MYC and MYCL1 protein in N1ICD-induced alveolar hyperplasia. In any case, we concluded that MYC and MYCL1 expression reemerged in adenomas, presumably in response to selection that favored tumorigenesis.
MYC and N1ICD cooperate in lung tumorigenesis
To test whether sustained MYC activity could facilitate the formation of N1ICD-induced adenomas, we crossed CN1 mice to mice carrying a DOX-regulatable MYC
) (the M transgene) and created compound CN1M mice (schematic in ). We found that mice overexpressing both N1ICD and MYC had dramatically decreased survival () compared to mice overexpressing either N1ICD (CN1 mice) or MYC (CM mice) alone.
Figure 5 N1ICD and MYC co-expression cooperates to induce lung adenocarcinoma. (A) Schematic of transgenes used for coincident overexpression of N1ICD and MYC. (B) Kaplan-Meier plot of DOX treated CM (n=34), CN1 (n=11) and CN1M (n=14) mice monitored for 18 months (more ...)
Tumorigenesis was always multi-focal in DOX fed CN1 mice (35.6±17.5 tumors visible on the pleural surface, n=11; ), but the tumors in CN1 mice were always adenomas, never adenocarcinoma. In DOX fed CN1M mice we also observed multi-focal tumorigenesis (). However, not only were there more tumors in CN1M mice (113.3 ± 45.4 tumors visible on the pleural surface, n=10) but large adenocarcinomas, as well as adenomas, were found in each mouse. A significant proportion of CN1M mice harbored gross metastases (4/13, 30.8%) (). Metastatic cells were observed in enlarged lymph nodes, the liver and lining the walls of the thoracic cavity. TTF-1 staining confirmed the lung origin of distant metastases ().
We previously reported that a high percentage of CM mice succumb to lung adenocarcinomas (10
). Usually, a single adenocarcinoma grew to occlude the parenchymal space () before symptoms of respiratory distress were observed (1.3 ± 1.1 tumors per mouse, n=31). The data suggest that the combination of activated NOTCH1 and MYC induced a more aggressive lung tumor phenotype compared to mice expressing activated NOTCH1 or MYC alone.
N1ICD substitutes for activated RAS in lung tumors overexpressing MYC
Western analysis of tumor protein lysates suggested that even more of the 110 kDa N1ICD was present in CN1M adenocarcinomas than in CN1 adenomas (). MYC overexpression may therefore stabilize N1ICD, although a mechanism by which this might occur is not apparent. We concluded that both N1ICD and MYC were overexpressed in CN1M adenocarcinomas.
Figure 6 Increased tumor cell cycling correlates with enhanced tumorigenesis in CN1M mice. (A) Western analysis of NOTCH1 and MYC expression in lungs and tumors from CN1 and CN1M transgenic mice. (B) Taqman® analysis comparing Hes5 expression in the lungs (more ...)
Like the levels of N1ICD, Hes5
induction was highest in CN1M adenocarcinomas (), although the difference in expression compared to CN1 adenomas was not statistically significant. Other Hes
genes were transcriptionally repressed in CN1M adenocarcinomas compared to CN1 adenomas (Supplementary Figure 6
). This reinforces the supposition that Hes5
is a target of N1ICD in the lung epithelium, while other Hes
basic helix-loop-helix genes may not be.
Previously, we demonstrated that lung tumors from CM mice harbor mutations in Kras
that cooperate with MYC in tumorigenesis (10
). Crosstalk between NOTCH1 and RAS has been suggested to be important for transformation (20
) and in some mouse tumor models activated NOTCH1 cooperates with activated KRAS (22
). We hypothesized that cooperativity between N1ICD and MYC in lung tumorigenesis may require RAS activation and carried out a RAS activity assay on protein lysates of tumors from CN1, CN1M and CM mice (). No increase in RAS activity was seen in CN1 adenomas or CN1M adenocarcinomas. This suggests not only that RAS activation is not required for the formation of N1ICD-induced adenomas, but also that N1ICD can substitute for mutation of Kras
in a cooperation with MYC that produces lung adenocarcinoma.
N1ICD and MYC have a synergistic effect on tumor cell proliferation
Acute overexpression of MYC
induces apoptosis in a variety of tissues (24
), including the lung (10
). Compensatory genetic and/or epigenetic events are thought to counter the pro-apoptotic effects of MYC during tumorigenesis (25
). Given that overexpression of either N1ICD or MYC can elicit an anti-tumor response in the form of apoptosis, we wondered how cooperativity was achieved during lung tumorigenesis.
Tumors from CN1, CN1M and CM mice were immunostained for phospho-H3S10, a marker of cells in the mitotic phase of the cell cycle (Supplementary Figure 7A–C
). We observed a significant increase in mitotic cells in CN1M adenocarcinomas compared to both CN1 adenomas (3.7 fold) and CM adenocarcinomas (3.3 fold) (). We observed similar changes with immunostaining for Ki67 antigen (data not shown), which stains cells in all phases of the cell cycle. Therefore, the combination of N1ICD and MYC expression had a synergistic effect on tumor cell cycling.
We also observed an increase in TUNEL staining in adenocarcinomas from CN1M mice compared to CN1 adenomas (3.2 fold) and CM adenocarcinomas (2.8 fold) (, Supplementary Figure 7D–F
). Therefore, the combination of N1ICD and MYC expression also augmented the induction of apoptosis. However, since the fold increase in phospho-H3S10+
cells was higher than the increase in TUNEL+
cells, the pro-proliferative effect of co-expressing activated NOTCH1 and MYC outcompeted apoptosis induction. Thus, synergistic activation of tumor cell cycling contributed to the cooperative effect of N1ICD and MYC on lung tumorigenesis.