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. 2006 Jun 13;103(24):9262-7.
doi: 10.1073/pnas.0603371103. Epub 2006 Jun 2.

Myc is a Notch1 transcriptional target and a requisite for Notch1-induced mammary tumorigenesis in mice

Apostolos Klinakis  1 , Matthias Szabolcs, Katerina Politi, Hippokratis Kiaris, Spyros Artavanis-Tsakonas, Argiris Efstratiadis
Affiliations

Myc is a Notch1 transcriptional target and a requisite for Notch1-induced mammary tumorigenesis in mice

Apostolos Klinakis et al. Proc Natl Acad Sci U S A. .

Abstract

To explore the potential involvement of aberrant Notch1 signaling in breast cancer pathogenesis, we have used a transgenic mouse model. In these animals, mouse mammary tumor virus LTR-driven expression of the constitutively active intracellular domain of the Notch1 receptor (N1(IC)) causes development of lactation-dependent mammary tumors that regress upon gland involution but progress to nonregressing, invasive adenocarcinomas in subsequent pregnancies. Up-regulation of Myc in these tumors prompted a genetic investigation of a potential Notch1/Myc functional relationship in breast carcinogenesis. Conditional ablation of Myc in the mammary epithelium prevented the induction of regressing N1(IC) neoplasms and also reduced the incidence of nonregressing carcinomas, which developed with significantly increased latency. Molecular analyses revealed that both the mouse and human Myc genes are direct transcriptional targets of N1(IC) acting through its downstream Cbf1 transcriptional effector. Consistent with this mechanistic link, Notch1 and Myc expression is positively correlated by immunostaining in 38% of examined human breast carcinomas.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Molecular characterization of N1IC-induced mammary tumors. (A) Northern blot analysis of total cell RNA from WT mammary glands of virgin (V), pregnant (P), lactating (L), and postinvolutional (PI) female mice and from regressing (R) and nonregressing (NR) N1IC-induced mammary tumors (two specimens of each class). The same membrane was sequentially hybridized (without stripping), with specific probes detecting the indicated RNA species. The relative Myc levels in the controls were approximately V 1.0, P 2.0, L 1.0, and PI 1.5. There was an ≈7-fold increase in Myc expression in regressing tumors relative to the amount in normal lactating glands. A similar Myc increase (≈7.5-fold on average) was detected in nonregressing carcinomas compared with corresponding control postinvolutional glands. Expression of the transgenic N1IC resulted in up-regulation of the endogenous Notch1 mRNA (row mN1, lanes 5–8). The level of mN1 transcripts in the control specimens was below detection limits by Northern blot analysis under our conditions. Hybridization to 18S rRNA (loading control) was performed to normalize the data for quantitation by using a PhosphorImager (Molecular Dynamics). (B) Northern blot analysis shows that the level of Myc mRNA expression in MMTV-Myc-induced carcinomas (≈45-fold greater than normal) is approximately six times higher than that found in nonregressing N1IC tumors. The transcript derived from the MMTV-Myc transgenic construct (14) is longer than the endogenous Myc mRNA. Lanes 1 and 2 are the same as Myc lanes 7 and 8 in A (different exposure times). (C) Southern blot analysis of EcoRI-digested DNA from a lactating mammary gland of a female mouse with an MMTV-N1IC/Mycfl/fl/Wapcre genotype after a second pregnancy, to assess the level of Cre-mediated DNA excision (lane 2) from the “floxed” (fl) Myc locus (generation of Δ allele). The control DNA (floxed allele; lane 1) was prepared from a nonregressing tumor of an animal with the same genotype. (D) Comparative Northern blot analysis of Myc mRNA expression levels at 2 weeks postpartum between N1IC-induced regressing tumors developed in females carrying MMTV-N1IC in functional Myc background (R; lanes 1 and 2; same as Myc lanes 5 and 6 in A) and tumor-free lactating glands of MMTV-N1IC/Mycfl/fl/Wapcre mice, in which Cre-mediated recombination (r) at the Myc locus had occurred. RNA was extracted from glands after the first pregnancy (lanes 3 and 4) or after a second pregnancy (lane 5).
Fig. 2.
Fig. 2.
Histopathological analysis and immunophenotyping of mammary tumors. (A) Comparison of sections of lactating mammary glands (hematoxylin/eosin staining). (Magnification: ×20.) The specimens were from animals carrying the Notch1IC transgene, in which the Myc gene was either conditionally ablated (a and b) or functional (control) (c). The glands were isolated at 2 weeks postpartum after a first (a) or a second (b and c) pregnancy. After a first pregnancy, only miniscule and rare solid intraalveolar lesions occupying <1% of the surface area were observed in experimental animals (Inset). (Magnification: ×400.) After a second pregnancy, however, several small papillary lesions were detected (b, outlined). Although they are morphologically the same as those found in controls (c, outlined), the lesions of experimental mice were fewer and occupied collectively 4.2% of the alveolar surface area vs. 14.5% in controls (area of each lesion 0.21 ± 0.16 and 0.78 ± 0.45 mm2, respectively; P < 10−6). (B) Immunohistochemical staining (brown reaction product) for Notch1IC and Myc of WT lactating glands, and regressing and nonregressing tumors from mice with an MMTV-N1IC/Mycfl/fl/Wapcre genotype. (Magnification: ×400.) In contrast to the positive Notch1 immunoreactivity, normal lactating glands lack Myc immunostaining. However, there is highly positive Myc immunodetection in nonneoplastic epithelial cells of transgenic mice expressing N1IC (d Inset, same genotype as in Ac). In the regressing tumor (same as in Ab) there is strong nuclear staining for Notch1 in the neoplasm (b, arrow) and also in the nonneoplastic tissue (b, arrowheads). In contrast to a mosaic pattern of Myc immunoreactivity in the regressing tumor (e, arrows), the adjacent nonneoplastic tissue is Myc-negative (e, arrowheads). The Notch1IC-induced nonregressing carcinoma shows strong labeling for both Notch1 (c) and Myc (f). (C) Morphological patterns of nonregressing tumors developing in transgenic animals carrying MMTV LTR-driven Notch1IC (a) or Myc (b) or in bitransgenic mice expressing both oncogenes (c). (Magnification: ×400.) The histological architecture of Notch1IC-induced tumors is mostly papillary (a, arrows), whereas the Myc tumors are composed of small glandular elements and nests (b, arrowheads). The carcinomas of bitransgenic animals exhibit a hybrid architecture of interspersed small glandular elements (c, arrowhead) and large solid papillary structures (c, arrow). At the cellular level (Insets), the Myc tumors consist of large cells with large irregular nuclei and exhibit many apoptotic bodies (b Inset, arrow), whereas the Notch1 tumors have smaller cells with uniform nuclei and no apoptotic bodies (a Inset). (Magnification: ×1,000.) The cellular features of the cancers in bitransgenic mice resemble those of the Myc tumors, including the presence of apoptotic bodies (c Inset, arrow). (D) An example of a human breast cancer (serial sections) exhibiting strong nuclear immunoreactivity for both NotchIC (a) and Myc (b). (Magnification: ×400.) Histologically, this carcinoma has a growth pattern of small, solid, irregular nests (c, hematoxylin/eosin staining). (Scale bars: A, 1,000 μm; Aa Inset, B, C, and D, 50 μm; C Insets, 20 μm.)
Fig. 3.
Fig. 3.
Kaplan–Meier tumor-free mouse survival curves. The survival of mice (from the date of birth until detection of palpable N1IC-induced nonregressing tumors) is compared by a standard rank test between controls carrying MMTV-N1IC and possessing functional Myc (dark green) and females with MMTV-N1IC/MycDfl/Dfl/Wapcre genotype (red) in which the majority of the mammary epithelium has become “null” for Myc expression by Cre-mediated recombination. Also shown for further comparisons are survival curves of MMTV-Myc transgenic (blue) and MMTV-N1IC/MMTV-Myc bitransgenic (light green) animals.
Fig. 4.
Fig. 4.
Analysis of Cbf1-binding sites in the mouse Myc promoter. (A) Shown is a diagram of the mouse Myc gene 5′ flanking region upstream from the first exon transcribed by activation of the most frequently used promoter P2. The positions of putative Cbf1-binding sites (sites A–C) and of the PCR primers used in ChIP experiments are indicated. (B) ChIP assays. In the examples shown, chromatin from a nonregressing N1IC-induced tumor was immunoprecipitated with antibodies against Notch1 (aN1) or Cbf1 (aCbf1) or with control rabbit IgG and analyzed by PCR using primers specific for the indicated promoters. The Myc promoter was analyzed for sites A and B. Hes1 and Hey1 were examined as positive controls; Cdc2a served as a negative control. “Input” corresponds to products generated by PCR (still in exponential phase; 25 cycles) by using DNA extracted from nonimmunoprecipitated chromatin (10% of the amount used in experimental samples) as a template. −, no antibody was added to the reaction mixture. (C) EMSA was performed by using a recombinant Cbf1 protein fragment and the indicated labeled oligonucleotide probes. Myc oligoB and oligoC span, respectively, the corresponding Cbf1 sites shown in A. An oligonucleotide representing two known Cbf1-binding sites of the Hes1 promoter was used as a positive control. (We attribute the shift of two bands to the presence of these two sites.) Mutated versions of Myc oligonucleotides B (Bmut) and C (Cmut) and an unrelated loxP oligonucleotide served as negative controls. In the competition assays, unlabeled Hes1, Bmut, and Cmut oligonucleotides were added to the binding mixture at a 50-fold molar excess.
Fig. 5.
Fig. 5.
Luciferase reporter assays. (A) Alignment of a region of the human and mouse Myc promoter sequences containing a recognition site for Cbf1 binding (site A). The start sites of promoters P1 and P2 are indicated. Segments of the plasmid constructs used as reporters are shown (for definition of binding sites, see Fig. 4A). In the diagrams, the putative Cbf1-binding sites (closed ovals) are intact, whereas, in the other constructs, one or more sites have been mutagenized (open ovals), as indicated. Constructs in which shorter segments of the mouse and human Myc promoters were used in association with a minimal Junb gene promoter (hatched rectangle) are also shown. (B) Results of reporter assays. 293T cells were cotransfected with Renilla luciferase plasmid and an effector plasmid expressing Cbf1-VP16 or a control plasmid (empty pcDNA3 vector; mock) in combination with one of the indicated reporter plasmids shown in A containing either intact or mutated Cbf1-binding sites. After normalization to Renilla luciferase activity, firefly luciferase activity relative to that of the control plasmid was calculated for each of the reporters. These relative values (mean ± SEM), measured in at least three independent experiments, are represented by the bars in the bar chart.
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