ARTICLE

https://doi.org/10.1038/s41467-020-16936-9 OPEN NOTCH1 activation compensates BRCA1 deficiency and promotes triple-negative breast cancer formation

Kai Miao 1,2, Josh Haipeng Lei1,2, Monica Vishnu Valecha1,2, Aiping Zhang1,2, Jun Xu1,2, Lijian Wang1,2, Xueying Lyu1,2, Si Chen1,2, Zhengqiang Miao1,3, Xin Zhang1,4, Sek Man Su1,2, Fangyuan Shao1,2, Barani Kumar Rajendran1,2, Jiaolin Bao1,2, Jianming Zeng 1,2, Heng Sun1,2, Ping Chen1,2, Kaeling Tan1,3, ✉ Qiang Chen1,2, Koon Ho Wong 1,3, Xiaoling Xu 1,2,4 & Chu-Xia Deng 1,2 1234567890():,;

BRCA1 mutation carriers have a higher risk of developing triple-negative breast cancer (TNBC), which is a refractory disease due to its non-responsiveness to current clinical targeted therapies. Using the Sleeping Beauty transposon system in Brca1-deficient mice, we identified 169 putative cancer drivers, among which Notch1 is a top candidate for accelerating TNBC by promoting the epithelial-mesenchymal transition (EMT) and regulating the cell cycle. Activation of NOTCH1 suppresses mitotic catastrophe caused by BRCA1 deficiency by restoring S/G2 and G2/M cell cycle checkpoints, which may through activation of ATR-CHK1 signalling pathway. Consistently, analysis of human breast cancer tissue demonstrates NOTCH1 is highly expressed in TNBCs, and the activated form of NOTCH1 correlates posi- tively with increased phosphorylation of ATR. Additionally, we demonstrate that inhibition of the NOTCH1-ATR-CHK1 cascade together with cisplatin synergistically kills TNBC by targeting the cell cycle checkpoint, DNA damage and EMT, providing a potent clinical option for this fatal disease.

1 Cancer Center, Faculty of Health Sciences, University of Macau, Macau, SAR, China. 2 Centre for Precision Medicine Research and Training, Faculty of Health Sciences, University of Macau, Macau, SAR, China. 3 Genomics & Bioinformatics Core, Faculty of Health Sciences, University of Macau, Macau, SAR, ✉ China. 4 Transgenic and Knockout Core, Faculty of Health Sciences, University of Macau, Macau, SAR, China. email: [email protected]

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reast cancer gene 1 (BRCA1), is the first identified breast Brca1 deficiency and promotes TNBC formation by activating the cancer susceptibility gene, and is responsible for ~20–25% epithelial–mesenchymal transition (EMT) signalling pathway. B – of hereditary breast cancers and 5 10% of total breast 1 cancers . Low levels of BRCA1 expression are detected in ~30% of Results sporadic breast cancers, possibly due to hypermethylation of the SB mutagenesis enhances Brca1-mutant mammary tumour- promoter or other transcriptional regulatory mechanisms2,3. igenesis. We introduced the SB system into Brca1Co/Co;WAP-Cre Inherited mutations in the BRCA1 gene predispose carriers to mice or Brca1Co/Co;MMTV-Cre mice to generate four experi- early-onset tumourigenesis and an up to 87% cumulative lifetime mental cohorts of mice by interbreeding them with two inde- risk of developing breast cancer and/or ovarian cancer4. BRCA1- pendent SB transposase-T2Onc3 transposon mouse lines: SB; defective breast cancers are usually high grade and have poor T2Onc3-12740 and SB;T2Onc3-1277522 (Fig. 1a; Supplementary prognoses. Moreover, ~48–66% of BRCA1 mutation carriers Fig. 1a). The mouse strains were Brca1Co/Co;WAP-Cre;SB; develop triple-negative breast cancer (TNBC), a rate that is much – T2Onc3-12740 (BrWSB40, n = 188), Brca1Co/Co;WAP-Cre;SB; higher than that of non-carriers (~20%)5 7. T2Onc3-12775 (BrWSB75, n = 129), Brca1Co/Co;MMTV-Cre;SB; BRCA1iscrucialformultiplebiological processes, including T2Onc3-12740 (BrMSB40, n = 34), and Brca1Co/Co;MMTV-Cre; DNA damage repair, cell-cycle checkpoints, ubiquitination and SB;T2Onc3-12775 (BrMSB75, n = 36). Brca1Co/Co;Wap-Cre transcriptional regulation8. Studies have demonstrated that loss (BrW, n = 62) mice and Brca1Co/Co;MMTV-Cre (BrM, n = 56) of BRCA1 results in defective DNA damage repair, abnormal cen- mice without the SB transposase or transposon were used as trosome duplication, G2-M cell-cycle checkpoint defects, growth controls (Supplementary Data 1). The mice were monitored twice retardation, increased apoptosis, genetic instability and tumour- – a week for tumourigenesis, and tumours were collected when they igenesis9 11. Using mouse models, we and others have demonstrated − − reached ~1–2 cm in diameter or the mice were moribund. that complete knockout of Brca1 in the whole body (Brca1 / ) Complete necropsy was performed to assess primary and meta- causes lethality at embryonic day 7–8(E7-8)12,13; in contrast, static tumours. mammary-specific deletion of exon 11 of Brca1 (Brca1Co/Co; A total of 22/62 (35.5%) BrW mice developed mammary MMTV-Cre) results in mammary tumour formation accompanied tumours in the study period of approximately 100 weeks. The by massive genomic alterations and cellular lethality14.Thesefind- SB tumourigenesis system markedly accelerated tumourigenesis ings prompted us to hypothesise that tumourigenesis triggered by and increased tumour incidence to almost 100% and tumour Brca1 deficiency must encounter a lethal block that retards tumour burden/mouse in both the 12740 and 12775 strains (Fig. 1b, c). progression, at least in early stages. Therefore, Brca1 deficiency is a Similarly, 26/56 (46.4%) BrM mice and all (n = 70) BrMSB double-edged sword, i.e., genome instability dysregulates massive mice developed mammary tumours during the same period of tumour suppressor and oncogenic factors to promote tumourigen- time, with a faster acceleration of tumourigenesis in BrMSB75 esis, but too much DNA damage initiates a lethal block by inducing than in BrMSB40 strains (Supplementary Fig. 1b, c; Supple- apoptosis to retard tumour formation. mentary Data 1). The data showing that mammary tumour- A genetic approach by directly breeding mutant mice carrying Δ Δ igenesis was accelerated in both Brca1Co/Co;WAP-Cre and various Brca1 11/ 11 mutations has indicated that loss of function Brca1Co/Co;MMTV-Cre mice by two independent mammary- of Trp53, Atm, Chk1 or Chk2 suppresses the embryonic lethality specific SB transposition strains underscore the idea that caused by Brca1 deficiency and accelerats tumourigenesis to – secondary “hits” are required to attenuate the lethal block in varying degrees15 17. Although many oncogenic drivers for Brca1-deficient mammary epithelial cells. tumourigenesis remain elusive, because the absence of Brca1 Next, we conducted molecular subtyping for tumours from initially triggers the lethal block by inducing mitotic catastrophe these mice using Hematoxylin and eosin (H&E) and immuno- and apoptosis, researchers have hypothesised the existence of histochemistry (IHC) staining for TNBC markers (Fig. 1d). secondary “hits” to modifying some cancer drivers (oncogenes or Tumours from both the BrWSB and BrW groups demonstrated tumour suppressors) to attenuate this block, allowing Brca1- comparable diverse histology18 (Supplementary Fig. 1d). The mutant cells to overcome mitotic catastrophe and ultimately tumours of both groups also had a similar incidence of TNBC, resulting in mammary tumourigenesis18. Identifying these second i.e., 57% (85/149) and 50% (5/10), respectively (Fig. 1e), which is “hits” may benefit clinical treatment by rebooting mitotic cata- also comparable to the incidence of TNBC in human BRCA1- strophe after modifying the target gene or pathway. related breast cancers5. Furthermore we analysed 24 tumours The Sleeping Beauty (SB) DNA transposon system has been – from BrM mice, and 45.8% (11/24) were TNBC (Supplementary used to identify genes involved in multiple types of cancers19 21. Fig. 1e). These data indicate that SB-mediated insertional This system consists of a conditionally expressed transposase and mutagenesis in Brca1 mammary gland-specific knockout mice mutagenic transposon allele flanked by inverted/direct repeats. accelerates mammary tumour formation without changing the The SB transposon can be adapted to initiate mutagenesis total tumour spectrum. through random insertions to cause loss or gain of gene function while tagging potential cancer driver genes. SB transposition can also be controlled to induce mutations in a specific target tissue Identification of driver genes in Brca1-deficient tumours.To by restricting conditional expression of the SB transposase identify genes involved in promoting mammary tumourigenesis, via tissue-specific expression of Cre to avoid potential side effects. we performed high-throughput sequencing for transposon Together with high-throughput sequencing, these methods can insertion sites of 306 SB tumours from 245 mice (Supplementary rapidly identify cancer driver genes and related pathways, pro- Data 1). Common insertion sites (CISs) were identified using the viding insight into human cancers through the use of mouse TAPDANCE method, as previously described23, which led to the models. identification of over 17,626 non-redundant transposon insertion In our efforts to identify these cancer drivers, we have con- sites among the SB strains 12740 and 12775 (Supplementary ducted a functional-based driver gene screen by introducing the Data 2). Analysis of CIS distribution patterns revealed no obvious SB system (SB+;T2Onc3+)22 into the Brca1 mammary-specific bias between the different strains (i.e., 12740 or 12775 with knockout mouse model and identified 169 candidate genes cor- MMTV-Cre or WAP-Cre) for all chromosomes, including relating with tumourigenesis. Further analysis identifies Notch1 chromosomes 9 and 12, which are the original sites of as a top putative oncogene that overcomes apoptosis caused by the transposons in strains 12740 and 12775, respectively

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a b co/co 100 Brca1 10 11 12

75 BrW (n = 62) Wap-Cre Wap Cre-recombinase BrWSB40 (n = 188)

p BrWSB75 (n = 129) 50 < 0.0001 MMTV-Cre MMTV Cre-recombinase

Tumor free (%) 25 SB11 12SA 3XpASBase pA 0 0 20 40 60 80 100 120 T2Onc3 IRR SACAG SD pA En2 SA IRR Weeks

d ER PR HER2

c 100 BrWSB40 (n = 186) 90 BrWSB75 (n = 127) BrW (n = 22) 80 20 100 μm 100 μm 100 μm ER+, PR+, HER2+ % of mice 10

0 12347 Tumor number ER+, PR+

100 μm 100 μm 100 μm

e BrW control (n = 10) BrWSB (n = 149) 8.1% (12) 3.4% (5) 2.7% (4) PR+ 30% (3) 10.7% (16)

50% (5) 100 μm 100 μm 100 μm 57% (85) 8.7% (13) 20% (2) 6% (9)

3.4% (5) TNBC TNBC ER+ PR+ ER+, PR+, HER2+

ER+, PR+ ER+, HER2+ PR+, HER2+ HER2+ 100 μm 100 μm 100 μm

Fig. 1 SB mutagenesis promotes tumourigenesis in Brca1 mammary-specific knockout mice. a Overview of the engineered alleles in mutant mice. b Kaplan–Meier curve showing the mammary tumour-free rate for the indicated genotypes. BrWSB40 (n = 188) and BrWSB75 mice (n = 129) showed increased tumourigenesis compared to BrW control mice (n = 62). BrWSB40 versus BrW (p < 0.0001); BrWSB75 versus BrW (p < 0.0001); BrWSB40 versus BrWSB75 (p = 0.9984) by log-rank tests with GraphPad Prism. c Numbers of tumours per mouse in different groups. d IHC staining with antibodies against ER, PR and Her2. e TNBC incidence among different groups.

(Supplementary Fig. 2a–c). Therefore, all insertion sites were Activated Notch1 is an oncogenic driver for tumourigenesis. further analysed. We divided the candidate drivers into oncogenes and tumour Using a cut-off point of their appearance in at least 5% of suppressors based on the insertion patterns of the T2Onc3 tumours in the 12740 and 12775 strains, we identified 119 transposon (i.e., whether the insertions are clustered at hot-spot putative driver genes (Supplementary Data 3) from the BrWSB regions or widely distributed and in the same or opposite tran- group and 90 genes from the BrMSB group (Supplementary scriptional direction of the host gene) (Fig. 3a, Supplementary Data 4). Combining the two groups together yielded 169 distinct Data 8). Among all 169 potential cancer drivers, 33 genes, genes with 40 overlapping genes (Fig. 2a), which appeared in including Notch1, Jup and Met, showed oncogenic features 6–36% of all tumour samples (Fig. 2b). (Supplementary Fig. 3a), whereas the majority of genes, including We hypothesised that other genes, in addition to the 40 Lipc, Cntn5, Hdac4, Nrxn3 and Cntnap5c, exhibited tumour overlapping genes, might be involved in accelerating tumour- suppressor features (Supplementary Fig. 3b). igenesis. Therefore, we conducted functional enrichment Notch1 is a large transmembrane protein that is associated analysis using the KEGG and GO database, as well as gene with several human malignancies. Interaction of Notch1 with its setenrichmentanalysis(GSEA),forall169candidategenes. ligands will trigger two sequential proteolytic cleavages of Notch1 Some functional items were enriched, including ubiquitin- to release ICN1 (intracellular domain of Notch1), allowing it to be mediated proteolysis and cell adhesion-related functions, translocated to the nucleus to participate in transcriptional (Supplementary Data 5–7). We also conducted protein–protein regulation24. Although the role of Notch1 in cancer formation has interaction analysis for all 169 candidate genes. The results been well established, it is somewhat surprising that it can act as highlighted several clusters, including the ubiquitination an oncogene or a tumour suppressor in different contexts25,26. system, cytoskeleton and cell junction, gene expression regula- Thus, we decided to focus on Notch1 to illustrate the mechanism tion, and protein kinase/phosphatase modification-related underlying its oncogenic function. Of 138 insertion sites functions (Fig. 2c). identified in 108 Notch1-trapped SB tumours, 136 (98.6%) were

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a c Primary tumors

BrWSB BrMSB 40 79 50 Ubiquitination system

Cytoskeleton and cell junction

Gene expression regulation

Notch1, Jup, Lipc, Cul5, Cntnap5c, Cntn5, Hdac4, Protein kinase/phosphatase modification Nrxn3, Met, Thap1, Rtf1, Tsc22d2, Gphn, Ptprd, Arg2, Pde4d, Dmd, Gm30319, Med15, Rpl26-ps4, Supt3, Npas3, Gm40462, Gpc5, Dtna, Slc12a8, Arih1, Prep, Ptpn3, Fmnl2, Pard3b, Stag1, Magi2, Sgcz, Tbc1d22b, Fbxw4, Thsd4, Nrg3, Wwox, Kcnma1.

b BrW/MSB tumors (n=306)

36.1% Notch1 34.1% Jup 32.4% Lipc 26.4% Cul5 25.8% Cntnap5c 24.4% Cntn5 23.1% Hdac4 22.1% Nrxn3 20.4% Met 19.1% Thap1 18.7% Rtf1 18.1% Tsc22d2 17.7% Gphn 16.7% Ptprd 16.4% Arg2 15.1% Pde4d 15.1% Dmd BrWSB40 13.7% Gm30319 12.7% Med15 BrWSB75 12.7% Rpl26-ps4 BrMSB40 12.7% Supt3 BrMSB75 11.7% Npas3 11.7% Gm40462 11.7% Gpc5 11.7% Dtna 11.0% Slc12a8 11.0% Arih1 10.0% Prep 9.7% Ptpn3 9.7% Fmnl2 9.4% Pard3b 9.0% Stag1 9.0% Magi2 9.0% Sgcz 8.4% Tbc1d22b 8.4% Fbxw4 7.7% Thsd4 7.4% Nrg3 7.0% Wwox 6.0% Kcnma1

Fig. 2 SB-driven candidate gene identification and functional annotations. a Venn diagram indicating CIS genes for the BrWSB and BrMSB groups. There were 40 genes shared between the two groups. b Oncoplot of the 40 genes showing their frequency in all tumour samples. c Protein–protein interaction analysis of SB-driven candidate genes using STRING online tools. concentrated between exon 25 and exon 30 (Fig. 3b), and 73.5% Notch1 did not have a significant effect (Fig. 3e, f). In contrast, (100/136) were in the same direction as the direction of gene targeting either the N terminus or C terminus of Notch1 in expression, indicating that the transposon specifically inserted to MK1097-5R tumours did not result in any obvious effects result in overexpression of ICN1. RNA-sequencing data also (Fig. 3e, g). These data confirm that transposon insertion- demonstrated that overexpression of Notch1 starts from exons induced overexpression of ICN1 is the driver for MK1370-3R 25–27 (Fig. 3c), which is consistent with activated Notch1 protein tumours but not for MK1097-5R tumour, which may be driven expression analysis (Fig. 3d). This finding suggests an oncogenic by other factors. Consistently, we found that tumours with role for Notch1 activation in Brca1-mutant tumourigenesis Notch1 activation exhibited faster progression than did non- (Supplementary Fig. 4a). Notch1-driven tumours (Fig. 3h). In addition, we designed two shRNAs targeting endogenous We previously showed that Brca1 deficiency results in cell death Notch1 (the N terminus of Notch1, shRNA-N) or pan-Notch1, that can be repressed by p53 loss15. We suspected that Notch1 including both SB transposon-driven Notch1 and endogenous activation might also affect cell lethality to attenuate the lethal block. Notch1 (the C terminus of Notch1, shRNA-ICN) (Supplemen- To examine this possibility, we generated a Brca1 conditional tary Fig. 4a, b), and applied them to cell lines derived from knockout ES cell line (Brca1Co/−;Cre-ERT2), in which Cre-mediated Notch1-driven SB tumours (MK1370-3R) and cell lines derived recombination is controlled by 4-hydratamoxifen (4-HT) from our from non-Notch1-driven SB tumours (MK1097-5R) (Fig. 3e, Brca1fl/Exon11 ES cells27. These cells enabled us to compare the Supplementary Fig. 4c, d). Knockdown of ICN1 blocked the impact of Notch1 activation or p53 loss on the viability of Brca1- growth of MK1370-3R tumours, whereas targeting endogenous deficient cells (Supplementary Fig. 4e, f). Acute knockout of Brca1

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a 100% e Notch1 driven tumor Met driven tumor MK1370-3R MK1097-5R Jup Met 80% Notch1

Rtf1 Thap1 shRNA-Ctl shRNA-N shRNA-ICN1 shRNA-Ctl shRNA-N shRNA-ICN1 Cul5 1.0 0.84 0.08 0.34 0.06 0.08

Activating 60% Ptprd Cntn5 Lipc Full-length Notch1 Gphn Cntnap5c 250 -

Pde4d Truncating Activated Notch1 Hdac4 100 - 40% 1.0 0.84 0.08 0.34 0.06 0.08 Nrxn3 Dmd 55 - β-Actin

Sense fraction (proportion) Tsc22d2 1.0 1.0 1.1 1.0 1.0 1.1 20%

fgNotch1 driven tumor Met driven tumor p = 0.39 p = 0.0086 0% 150 p = 0.34

0 20406080100120) p = 0.94 3

) 600

p = 0.019 3 p = 0.98 Number of samples 100 400

b 50 200 1 2 3 Tumor volum (mm

Gain of function Tumor volum (mm 34 0 0 Loss of function 2 30 66

shRNA-N shRNA-N shRNA-Ctl shRNA-Ctl Exon25 shRNA-ICN1 shRNA-ICN1 Exon30

N C h 100 EGF-like repeats LNR HD TM RAM ANK TAD PEST

Notch1-ICN1 75 Notch1-driven tumor (n = 93) non-Notch1-driven tumor (n = 152) c 3.65 50 3.19 2.73 Exon1 Exon2 Exon3 Exon4 Exon5 Exon6 Exon7 Exon8 Exon9 Exon10 Exon11 Exon12 Exon13 Exon14 Exon15 Exon16 Exon17 Exon18 Exon19 Exon20 Exon21 Exon22 Exon23 Exon24 Exon25 Exon26 Exon27 Exon28 Exon29 Exon30 Exon31 Exon32 Exon33 Exon34 BrW/M (n = 118) 2.28 Tumor free (%) free Tumor MK1370-3R 1.82 25 MK1058-3L 1.37 0.91 MK1344-3R 0.45 MK1519-2L -0.00 0 MK1576-5L -0.46 -0.92 0 20 40 60 80 100 120 MK969-4R -1.37 MK1024-4LA -1.83 Weeks -2.09 MK1482-2LA -2.74 100% i 100% non-Notch1 Notch1-driven tumor d driven tumor 83.3% 80% 69.4% 62.5% 60% Brca1 knockout colony MK1097-5R MK1013-1RMK1370-3RMK1058-3L MK1344-3RMK1519-2LMK1576-5L MK969-4R MK1024-4LMK1482-2L 37.5% 1.00 1.29 6.87 3.44 5.78 7.28 2.84 0.89 2.85 3.81 40% Activated notch1 30.6% Brca1 wildtype colony 100 - 55 - 20% 16.7%

β-Actin % of Brca1 knockout colony 1.0 1.0 1.00.8 0.9 1.1 0.9 1.0 1.0 1.0 0.0% 0% Control P53 knockout ICN1 OV-1 ICN1 OV-2

Fig. 3 Effect of Notch1 activation on cell death and tumourigenesis. a Predicted effect of candidate genes, as indicated by their sense fraction of insertions based on the direction of the CAG promoter and the transcriptional direction of the inserted gene. Genes with a strong bias towards sense insertions are expected to be activated and might serve as oncogenic drivers. Conversely, those biased towards antisense insertions are predicted to be inactivatedor yield truncated products and serve as tumour suppressors. The dashed line in the middle indicates the equal ratio of sense and antisense insertions. b Structure of Notch1 and transposon insertion sites within the Notch1 gene. Blue arrows indicate that the promoter in the transposon is in the same orientation as the host gene, and red arrows indicate different directions. Insertion frequencies are indicated by numbers. c Expression analysis of all exons of the Notch1 gene based on RNA-seq of Notch1-driven SB tumours. d Activated Notch1 protein expression analysis based on western blotting compared with non-Notch1-driven tumours. e Western blot analysis of Notch1-driven tumours (MK1370-3R) and non-Notch1-driven tumours (MK1097-5R) after shRNA knockdown. f, g Tumour volume measured at day 30 of MK1370-3RMT (f) and MK1097-5RMT (g) after shRNA-mediated knockdown of endogenous Notch1 or pan-Notch1; n = 4 biologically independent animals. The t-test was used to determine the significance of the difference between the different sets of data. h Kaplan–Meier curve showing the mammary tumours-free rate for SB mice with Notch1-driven tumours (n = 93) and non-Notch1- driven tumours (n = 152), as well as BrW (n = 62) and BrM (n = 56) control mice. Notch1-driven tumours tended to show earlier onset compared with non-Notch1-driven tumours (p < 0.0001) by the log-rank test. i Percentage of different types of ES colonies with wild-type Brca1 and knockout after treatment with 4-HT to induce Brca1 knockout in p53 mutant, ICN1-overexpressing and parental ES cell lines. Cells were inoculated with 4-HT for 3 days (to delete Brca1), followed by disassociation and replating on feeder cells with regular medium. Single ES colonies were selected 7 days later for genotyping of their Brca1 status. Data are presented as mean values ± s.d. by 4-HT treatment resulted in no Brca1−/− ES colonies (n = 36); Overall, these data suggest that ICN serves as an oncogene to we observed 37.5% (18/48) of Brca1−/− ES colonies among p53- promote the initiation of Brca1-associated mammary tumour- knockout ES cells (Brca1Co/−;Cre-ERT2;p53−/−). In two indepen- igenesis, which may occur by suppressing the lethality effect dently derived ES cell lines overexpressing ICN1 (Brca1Co/−;Cre- caused by Brca1 deficiency. ERT2;ICN1OV), acute knockout of Brca1 after administration of 4- − − HT yielded 16.7% (6/36) and 30.6% (11/36) Brca1 / ES colonies Notch1 suppresses BRCA1 defects caused mitotic catastrophe. (Fig. 3i), indicating that activation of Notch1 suppressed, at least in To explore how ICN1 suppresses the lethality effect under Brca1- part, the lethality caused by Brca1 deficiency. deficient conditions, we used a tet-on system to induce ICN1

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a MCF10A-iICN1 cdp = 0.020

shRNA-Ctl shRNA-Brca1 p = 0.0031 p = 0.00063 (–Dox) shRNA-Ctl 30 (–Dox) shRNA-BRCA1 20 20 μm 20 μm 20 μm (+Dox) shRNA-Ctl (–) DOX 10 (+Dox) shRNA-BRCA1 Number of foci

e p = 4.5E–06 shRNA-Brca1 20 μm 20 μm 20 μm 100 p = 7.0E–07 p = 5.5E–06 (–Dox) shRNA-Ctl

(+) DOX 80 (–Dox) shRNA-BRCA1 20 μm 60 (+Dox) shRNA-Ctl Scale bar: 20 μm 40 Dox+ 20 (+Dox) shRNA-BRCA1 shRNA-Ctl shRNA-Ctl b Relative intensity 0 p = 0.0047 20 μm μ 20 μm 20 m 0.6 p = 0.094 Wash-off 0.4 i shRNA-Ctl Image DAPI, Edu Dox+ Edu 0–8 h BrdU H3-pS10 or BrdU 0.2 shRNA-Brca1 20 min 20 min shRNA-Brca1 20 μm 20 μm 20 μm 2 h 4 h 6 h 8 h 0.0 Scale bar: 20 μm DAPI/γ-H2AX Absorbance (590 nm)

(–Dox) (+Dox)

g p = 0.18 f MCF10A-iICN1 0.5Gy, 1 h 80 p = 0.031 20 μm p = 0.037 Scale bar: 20 μm DAPI/EdU/BrdU n = 20000, triplicate p = 0.0014

4 10 104 60 shRNA-Ctl shRNA-Ctl shRNA-Ctl+Dox j S-G2 progression (n > 1000) shRNA-Ctl+Dox 3 10 103 40 1.1 M phase M phase shRNA-BRCA1 cells) in G2

– 1.0 1.59% 0.74% +

2 2 shRNA-BRCA1+Dox 10 10 20 0.9 shRNA-Ctl % of control mitosis 0.8 shRNA-Ctl+Dox

101 1 10 0 and BrdU shRNA-BRCA1

+ 0.7 shRNA-BRCA1+Dox 0.6 Fraction of EdU 0 (EdU 100 10 2468 0 200 400 600 0 200 400 600 h 200 4 shRNA-Ctl 10 104 Hours post-EdU wash-off shRNA-BRCA1 shRNA-BRCA1+Dox shRNA-Ctl+Dox

103 103 150 shRNA-BRCA1 k S-M progression (n > 1000) M phase M phase shRNA-BRCA1+Dox

+ 0.015 2 1.92% 1.50% 10 102 100 shRNA-Ctl 0.010 shRNA-Ctl+Dox

101 101 50 shRNA-BRCA1

% of control mitosis 0.005

in mitosis shRNA-BRCA1+Dox h(s) 0 100 10 0 Fraction of EdU 0.000 0 200 400 600 0 200 400 600 01234 024 Hours post-EdU wash-off

Fig. 4 Notch1 activation suppresses the lethality caused by BRCA1 deficiency via cell-cycle regulation. a Overexpression of ICN1 suppressed the cell death caused by BRCA1 acute knockdown in MCF10A naïve cells. b Quantification analysis by MTT assays regarding the rescue effect of ICN1 on BRCA1 deficiency. Analysis on the 3rd day after Dox and/or lentivirus with shRNA-BRCA1 induction; n = 3 biologically independent experiments. c IF staining and foci counting d and intensity quantification e of γ-H2AX to indicate DNA damage at 48 h after BRCA1 acute knockdown with or without ICN1 overexpression; n = 17–30 independent cell measurements. f Mitotic index analysis of MCF10A cells at 1 h after 0.5 Gy gamma-irradiation treatment. A total of 20,000 cells were counted, and three repeats were used to determine the SD. g Quantification analysis of the relative M-phase portion in the different treatment groups in e; values were normalised to the no-irradiation treatment value for the individual groups; n = 3 biologically independent experiments. The p-value was determined by the t-test with one-tailed distribution. h Dynamic changes in the mitotic index at different time points after 0.5 Gy gamma-irradiation treatment; n = 3 biologically independent experiments. * indicates p < 0.05. i Pulse-chase assay to measure S-G2 and S-M progression. The yellow arrowhead indicates representative cells that progressed to G2 phase at a particular time point. j Fraction of EdU-positive cells entering G2 phase (BrdU-negative) during the chase (n = 1800). k S-M progression indicated by EdU and p-H3 double-positive cells (n = 1100). Data are presented as mean values ± s.d. overexpression in MCF10A cells by doxycycline (Dox) adminis- G2/M-phase lag (Supplementary Fig. 5j, k), even with BRCA1 tration (Supplementary Fig. 5a–c). Acute knockdown of BRCA1 knockdown. by using shRNA led to cell death, whereas Dox-induced ICN1 We previously showed that BRCA1 deficiency impairs the overexpression suppressed this lethal effect (Fig. 4a, b). Our G2/M cell-cycle checkpoint, which causes severe genomic further analysis detected markedly increased γ-H2AX and 53BP1 instability28. Thus, we investigated whether ICN1 is able to levels upon acute knockdown of BRCA1, but overexpression of restore the G2/M checkpoint, which accounts for the decreased ICN1 blocked this effect (Fig. 4c–e, Supplementary Fig. 5d, e), DNA damage. By assessing the mitotic index (MI) with ICN1 suggesting that activation of NOTCH1 abolishes the mitotic overexpression and/or BRCA1 knockdown after gamma- catastrophe caused by BRCA1 deficiency by decreasing DNA irradiation (IR), we found that ICN1 decreased the mitotic damage. The rescue effect was also observed when we treated the population in both parental and BRCA1-knockdown cells after cells with the NOTCH1 ligand Jagged1 (Supplementary Fig. 5f, g). DNA damage in both MCF10A (Fig. 4f, g), and T47D cells Moreover, the cell growth rate was decreased after ICN1 over- (Supplementary Fig. 5l, m). These data suggest a role for ICN in expression before cell confluence at day 5 (Supplementary activation of the G2/M cell-cycle checkpoint upon irradiation, Fig. 5h); and the stall in growth was maintained after replating which is sufficient to override the lethal block caused by BRCA1 the cells (Supplementary Fig. 5i). By synchronising the cell cycle deficiency. We then conducted a time course experiment after IR, with thymidine, we found that overexpression of ICN1 caused a and the data also indicated that ICN significantly reduced the

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a shRNA-Ctl shRNA-BRCA1 bc CHK1 knockdown p < 0.01 p < 0.01 Dox (h) 0 6 8 10 12 16 24 0 6 8 10 12 16 24 1.0 250 0.8 BRCA1 Control shRNA1 shRNA2 shRNA3 1.00 1.03 1.56 1.38 0.94 1.59 0.94 0.44 0.25 0.19 0.41 0.35 0.14 0.09 0.6 Dox(h) 08 16 0 8 16 0 8 160 8 16 0.4 ICN1 100 0.2 1.00 4.69 7.51 7.46 3.79 1.69 2.03 0.97 5.12 7.58 6.95 7.17 8.79 6.66 55 p-Chk1 1.0 1.8 2.4 1.2 1.1 1.2 1.3 1.3 1.2 1.3 1.6 1.2 0.0 55 p-CHK1-317 % of control mitosis 1.00 1.88 2.18 1.57 2.63 1.40 2.31 1.95 2.67 3.83 3.98 6.04 3.56 6.52 t-Chk1 (–Dox) (+Dox) 55 1.0 2.0 2.8 0.1 0.1 0.7 0.6 0.3 0.8 0.7 0.8 1.2 55 55 p-CHK1-345 β shRNA-Ctl shRNA-CHK1_2 1.00 0.98 1.25 1.71 1.82 4.20 3.29 1.95 1.94 2.15 2.92 3.29 5.48 5.69 -actin 1.0 1.1 0.9 1.0 1.1 0.9 0.8 0.6 0.8 0.9 0.8 0.9 shRNA-CHK1_1 shRNA-CHK1_3 t-CHK1 55 1.00 0.96 1.01 1.16 0.97 0.97 0.98 0.99 0.86 0.97 0.98 0.99 0.99 1.04 d p = 0.009 p = 0.022 p-ATR-428 p = 0.011 p = 0.003 250 1.00 0.91 0.98 0.93 0.96 0.85 0.88 0.57 0.69 0.67 0.83 0.92 1.22 1.59 0.25 (–Dox) shRNA-Ctl 0.20 p-ATR-1989 p = 0.64 p = 0.027 1.00 1.69 2.00 2.20 5.08 2.31 2.10 1.73 1.81 3.26 p = 0.016 250 3.03 7.81 1.84 4.19 0.15 p = 0.012 (–Dox) shRNA-BRCA1 t-ATR 0.10 (+Dox) shRNA-Ctl 250 1.00 0.90 0.82 0.86 0.85 0.78 0.67 0.75 0.96 0.91 0.86 0.88 0.92 0.79 0.05 (+Dox) shRNA-BRCA1 p-ATM 250 1.00 0.74 0.92 0.96 0.95 0.98 0.97 0.85 0.76 0.81 0.98 1.02 1.12 1.15 0.00 t-ATM 1.00 0.94 1.02 1.06 0.96 0.98 0.99 0.95 0.96 0.91 0.99 1.01 1.02 1.04 250 shRNA-Ctl 55 β-Actin shRNA-CHK1_1 shRNA-CHK1_2 shRNA-CHK1_3 1.00 0.98 1.01 1.01 0.99 0.96 0.96 0.99 0.99 0.97 0.99 0.96 0.94 0.96

g p = 0.009 p = 0.012 p = 0.007 p = 0.028 e ATR knockdown f 0.25 p < 0.05 p < 0.05 1.0 0.20 p = 0.68 p = 0.046 (–Dox) shRNA-Ctl p = 0.034 p = 0.0015 Control shRNA1 shRNA2 shRNA3 0.8 0.15 (–Dox) shRNA-BRCA1 Dox(h) 08 16 08 16 0 8 16 0 8 16 0.6 0.10 (+Dox) shRNA-Ctl 0.4 ATR 0.05 (+Dox) shRNA-BRCA1 250 1.0 1.0 1.1 0.3 0.3 0.3 0.8 0.8 0.8 0.1 0.1 0.1 0.2 p-Chk1 0.00 55 0.0 Absorbance (590 nm) 1.0 1.9 4.2 2.0 1.6 1.7 1.6 1.4 1.2 1.3 1.2 1.2 % of control mitosis (–Dox) (+Dox) 55 t-Chk1 1.0 2.0 3.1 1.3 1.5 2.3 1.3 1.4 1.9 1.0 1.1 2.3 shRNA-Ctl shRNA-ATR_2 shRNA-Ctl 55 β-actin shRNA-ATR_1 shRNA-ATR_2 shRNA-ATR_3 1.0 1.1 0.9 1.2 1.0 0.8 0.9 0.8 0.9 1.0 1.1 0.8 shRNA-ATR_1 shRNA-ATR_3

h ij0.18 IP: R = 0.54 , p = 4e–08 R = 0.3 , p = 0.0099 Input IgG 0.13 ICN1 ATR 0.16 ICN1 100 0.11 ATR ATR 0.14 ATR 250 0.09 0.12

0.08 0.09 0.10 0.11 0.12 0.05 0.07 0.09 Notch1 Notch1

Fig. 5 Notch1 activation rescues the lethal effect caused by BRCA1 deficiency through the ATR–CHK1 axis. a Western blotting analysis of cell-cycle checkpoint proteins at different time points after Dox administration. b Western blotting analysis of CHK1 phosphorylation after ICN1 induction after knockdown of CHK1. c MI assay of MCF10A cells when CHK1 was knocked down with/without ICN1 overexpression. Cells were collected at 1 h after 0.5 Gy irradiation treatment; n = 3 biologically independent experiments. d MTT assay evaluating the rescue effect of Brca1 acute knockdown on the 3rd day when CHK1 was knocked down with shRNA; n = 3 biologically independent experiments. e Western blotting analysis of CHK1 phosphorylation after ICN1 induction following ATR knockdown. f MI assay of MCF10A cells when ATR was knocked down with/without ICN1 overexpression. Cells were collected at 1 h after 0.5 Gy irradiation treatment; n = 3 biologically independent experiments. g MTT assay evaluating the rescue effect of Brca1 acute knockdown on the 3rd day after ATR knockdown with shRNA; n = 3 biologically independent experiments. h IP analysis indicated that ICN1 can directly bind with ATR. i, j Immunohistochemistry staining of a human TNBC patient tissue microarray for target proteins. The scatter plots indicate a positive correlation between ICN1 and p-ATR (n = 90 for i, n = 72 for j); R and p-values were obtained based on Spearman’s rank correlation coefficient R’s and probability (p) value calculator. Data are presented as mean values ± s.d. population of MI in BRCA1 WT and knockdown cells at multiple restore the S/G2 or G2/M checkpoint to suppress apoptosis time points (Fig. 4h, Supplementary Fig. 5n). caused by BRCA1 defects. Next, we specifically evaluated S-G2 and S-M progression using The CHK1 kinase acts downstream of the ATR/ATM kinase, quantitative image-based cytometry combined with a pulse-chase which is essential for embryonic development and tumour assay (Fig. 4i). The fraction of EdU-positive cells that progressed suppression29,30. To determine whether CHK1 phosphorylation to G2 phase (BrdU-negative) during the chase indicated that is mediated by ATR or ATM after ICN1 overexpression, we BRCA1 deficiency resulted in premature entry into mitosis and examined the expression levels of these two kinases and found that hyperactivation of NOTCH1 restored cell-cycle regulation that ATR phosphorylation was moderately increased but that (Fig. 4j). Meanwhile, ICN1 also markedly delayed S-M progres- ATM phosphorylation was not (Fig. 5a). In addition, ATR sion (Fig. 4k). knockdown blocked ICN1-triggered CHK1 phosphorylation To provide further evidence, we performed western blotting (Fig. 5e), increased MI (Fig. 5f), and, similar to CHK1 knock- for key proteins involved in cell-cycle checkpoints, and the down, abolished the ability of ICN1 to suppress the apoptosis results showed increased CHK1 phosphorylation at Ser317 and caused by acute knockdown of BRCA1 (Fig. 5g). However, such Ser345 along with ICN1 induction in both parental and effects were not found when ATM was inhibited (Supplementary BRCA1-knockdown cells to varying degrees (Fig. 5a). To Fig. 6a). These observations indicate that ICN1 regulates CHK1 investigate the function of p-CHK1, we knocked down CHK1 phosphorylation through ATR but not ATM. Immunofluorescent with shRNA (Fig. 5b) and found that it increased MI (Fig. 5c) staining results showed that ICN1 induction markedly increased and abolished the suppressive effect of ICN1 on the lethality p-ATR as well as p-CHK1 localisation to the nucleus (Supple- caused by acute knockdown of BRCA1 (Fig. 5d). These data mentary Fig. 6d, e). Furthermore, immunoprecipitation analysis suggest that activated NOTCH1 functions through p-CHK1 to demonstrated physical binding between ICN1 and ATR (Fig. 5h),

NATURE COMMUNICATIONS | (2020) 11:3256 | https://doi.org/10.1038/s41467-020-16936-9 | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16936-9 indicating that NOTCH1 activation modulates p-ATR localisa- In conclusion, our results indicate that NOTCH1 may be an tion to regulate the ATR–CHK1 pathway; these findings may oncogenic driver for BRCA1-related TNBC and basal-type need further validation. Moreover, ATR–CHK1 activation by tumours. ICN1 was also detected in T47D cells, as well as in MCF10A cells treated with the NOTCH1 ligand Jagged1 (Supplementary Fig. 6b, NOTCH1 activation stimulates EMT to promote TNBC for- c), suggesting a general feature of NOTCH1 to regulate the cell mation. To further explore the related mechanism, we conducted cycle in breast cancer cells. gene expression analysis of Notch1-driven TNBC tumours To further confirm the correlation between ICN1 and p-ATR, compared with non-Notch1-driven TNBC tumours (Fig. 7a). we used two sets of human TNBC patient tissue microarrays to Gene set enrichment analysis (GSEA) demonstrated that genes conduct immunohistochemistry staining (Supplementary Fig. 6f). upregulated in the EMT process were highly expressed in The results showed that the p-ATR protein level correlated Notch1-driven tumours (Fig. 7b, Supplementary Data 9). In significantly with the NOTCH1 activation level (Fig. 5i, j); contrast, genes downregulated during EMT revealed low levels of conversely, other proteins did not show a consistent correlation expression in Notch1-driven tumours (Fig. 7c, Supplementary with NOTCH1 (Supplementary Fig. 6g, h). Data 9). These results indicate that EMT-related genes correlated Taken together, these data reveal that BRCA1 deficiency strictly with Notch1 activation in TNBC tumours. It has been impairs the S/G2 and G2/M cell-cycle checkpoints, which reported that Notch1 signalling regulates the EMT in human triggers the lethal block, but that ICN1 overexpression restores breast cancer33,34, and that expression of EMT-related markers in these checkpoints and suppresses the mitotic catastrophe TNBCs can serve as a signature of a certain subgroup of caused by BRCA1 deficiency through a non-canonical target: TNBC35,36. Therefore, we studied whether NOTCH1 promotes the ATR–CHK1 axis. EMT transition, leading to TNBC formation in BRCA1-defective conditions. By using a tet-on system to induce ICN1 overexpression in NOTCH1 correlates with increased human TNBC formation. different mammary cell lines, we found that activated NOTCH1 BRCA1 mutation increases the formation of malignant TNBC in upregulated Fibronectin 1, Vimentin and Slug to varying degrees humans5. To identify the driver genes responsible for BRCA1- in MCF10A, T47D and MCF7 cells (Fig. 7d, e, Supplementary deficient TNBC formation, we used comparative coefficient Fig. 9c, d). Moreover, we induced Notch1 expression in ATR or analysis to select candidates correlating closely with TNBC CHK1 knockdown cells and then assessed their EMT status. As incidence. First, we converted 169 candidate genes to human shown in the Supplementary Fig. 9e, f, Dox-mediated Notch1 genes based on mouse-human orthologues. Then, we traced the induction increased expression of Fibronectin; in contrast, expression data and corresponding pathology information for knockdown of ATR or CHK1 did not affect expression. These each gene from the human clinical databases TCGA31 and data suggest that the function of Notch1 in EMT stimulation is METABRIC32 and separated the patients into defined equal independent of ATR or CHK1. Human BRCA1-mutant breast cohorts based on the expression level of candidate genes. Finally, cancer samples also showed high levels of mesenchymal markers the TNBC percentage was calculated for each cohort to determine (Fig. 7f). Furthermore, a meta-analysis of BRCA1-related cancer the correlation between gene expression and TNBC incidence revealed that all basal-like tumours belong to the TNBC subtype, (Fig. 6a, Supplementary Fig. 7). much higher than the average level (Supplementary Fig. 8g, h). Fifteen genes in our 169-gene pool showed good correlations These data suggest the EMT might be the driver of BRCA1- with TNBC patients in both TCGA and METABRIC databases mutant cell transdifferentiation. Therefore, we conclude that (Fig. 6b). NOTCH1, ARHGAP21, FMNL2 and TCF20 correlated NOTCH1 hyperactivation promotes EMT, which might induce positively with TNBC incidence, i.e., patients with high expres- the formation of TNBC and basal-like breast cancer. sion of these genes tended to develop TNBC. Expression pattern analysis of the NOTCH1 gene indicated that TNBC and basal- type tumours exhibited significantly increased levels of NOTCH1, Inhibition of ATR–CHK1 enhances the tumouricidal activity. regardless of whether BRCA1 was mutated (Fig. 6c). TNBC and Cisplatin preferentially kills BRCA1-deficient breast cancers basal tumour incidence markedly increased with increasing compared to BRCA1-proficient cancers; however, the inhibition NOTCH1 expression (Fig. 6d). In 54 BRCA1-deficient breast efficiency is always compromised when cancers gain various cancers in this cohort, increased expression of NOTCH1 also additional alterations37,38.Furthermore,ahighdosageof correlated with TNBC and the basal-type. Analysis of protein cisplatin can cause serious nephrotoxicity39,40. Our previous levels in BRCA1-deficient tumours indicated that a high protein data indicate that cisplatin inhibits tumour metastasis by expression level of NOTCH1 correlated with TNBC incidence blocking EMT, though prolonged treatment may result in drug (Supplementary Fig. 8a, b). Moreover, under BRCA1-defective resistance41. conditions, NOTCH1 tended to display an enhanced ability to TNBC is the most difficult type of breast cancer to treat due to promote TNBC and basal-type breast cancer (Supplementary its unresponsiveness to current clinical targeted therapies, high Fig. 8c, d). A Kaplan–Meier survival curve indicated high rate of recurrence and poor prognosis42. A high expression level expression levels of NOTCH1 to be associated with worse of Notch1 correlates closely with TNBC incidence and activates outcomes in TNBC patients (Supplementary Fig. 8e, f). ATR–CHK1 signalling to compensate for the DNA damage repair To investigate whether Notch1 activation in our SB tumours deficiency and benefit tumourigenesis, and cisplatin causes DNA also contributes to TNBC formation, we conducted IHC staining crosslinking, leading to DNA double strand breaks (DSBs). for ER, PR and Her2, The data demonstrated that 74.24% (49/66) Accordingly, we hypothesised that ATR or CHK1 inhibitor of ICN1-driven SB tumours were TNBC (Supplementary Fig. 9a), combined with cisplatin might have a synthetic lethal effect on which was much higher than the average percentage of BrWSB TNBC samples. To investigate this, we first assessed the effect on (57%) and BrW control (50%) tumours (Fig. 1e). Moreover, we the HCC1937 cell line, and the results indicated that a low dosage observed that 62.5% (20/32) of ICN1-driven SB tumours were of an ATR inhibitor (AZD6738 or VE821) combined with the basal-type, compared with 40% (8/20) of randomly selected different concentrations of cisplatin effectively killed HCC1937 non-ICN1-driven SB tumours (Supplementary Fig. 9b), suggest- cells (Fig. 8a, b). Notably, combined treatment with cisplatin and ing that ICN1 also enhances basal-type tumour formation. an ATM inhibitor (KU-60019) failed to show a synergistic effect

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a • Rank the samples based on individual gene expression. Count percentage of TNBC TCGA • Equally separate the in each cohort for each genes database All patients samples into several cohort 169 candidates Human gene Gene expression & from mouse orthologs Pathology information SB tumors

METABRIC Brca1 mutant • Rank the samples based on Count percentage of TNBC database individual gene expression. • Equally separate the in each cohort for each genes samples into several cohort

b METABRIC-BRCA1 mutant patients c p = 2.53889E–65 p = 4.85266E–06 p = 1.65916E–53 p = 0.00015523

6 METABRIC−BRCA1 mutant patients FBXO7, TSC22D2, DDX60, DLG3, FBXO9, RUVBL2 5 4 2 TNBC 15 NOTCH1, ARHGAP21, FMNL2, TCF20, ANKS1B, 2 Non-TNBC ATP6V1D, CNTNAP2, Basal TCGA–All patients METABRIC−All patients 0 31 16 CORO2A, CPD, ESR1, Non-Basal 20 FUT8, MYO6, NEDD4L, NFIA, THSD4 −2 NOTCH1 expression level

METABRIC-all patients All patientsBRCA1 All patients BRCA1 TCGA-all patients mutant patients mutant patients

d NOTCH1 expression Status PAM50 Study Status 4 TNBC Er−/Her2−/Pr+ 2 ER+/Her2− ER+/Her2+ All patients 0 Er−/Her2+ Er+/Her−/Pr− Expression −2 Er−/Her+/Pr+ Er+/Her+/Pr− PAM50 Basal Status LumA PAM50 Her2 Study LumB 3 Normal Claudin-low 2 Study Metabric 1 TCGA2015

0 Expression

BRCA1 mutant patients −1

Fig. 6 Correlation of NOTCH1 expression with human TNBC and basal-type breast cancer. a Flowchart illustrating the strategy of TNBC correlation analysis for candidate genes. b Venn diagram showing TNBC-correlated genes obtained from TCGA and METABRIC databases. Genes labelled with red correlated positively with TNBC incidence, and genes highlighted with blue correlated negatively. c Distribution of NOTCH1 expression levels in TNBC vs. non-TNBC and basal vs. non-basal human patients. The t-test was used to determine the significance of differences between the different sets of data. d Correlation between NOTCH1 expression level and tumour subtype. Tumours were ranked based on NOTCH1 expression level, and tumour subtypes are labelled on the top. Data were obtained from TCGA (n = 403) and METABRIC (n = 1898) databases. in killing cancer cells, which is consistent with our previous Notably, combined with ATRi, cisplatin at a low dose (1.5 mg/kg) finding that ICN1 regulates downstream effects through ATR significantly inhibited tumour progression. According to IHC but not ATM. Moreover, the CHK1 inhibitor CCT245737 or staining, double treatment induced massive apoptosis/death LY2603618 also increased sensitivity to cisplatin (Fig. 8a, b). Next, compared with high-dosage single treatment of ATRi or cisplatin we tested four additional BRCA1-mutant TNBC cell lines and (Fig. 8e, f). found that the ATR or CHK1 inhibitor markedly enhanced In conclusion, cisplatin-mediated inhibition of the NOTCH1 killing but that the ATM inhibitor showed the opposite effect non-canonical target ATR–CHK1 cascade can synergistically kill (Fig. 8c). These data verify our model that ICN1 can, through the BRCA1-related TNBC both in vitro and in vivo. non-canonical target ATR or CHK1, regulate the cell cycle and promote TNBC formation. Discussion Furthermore, we conducted in vivo treatment of TM00091 Tumours initiate from normal cells through a long “evolution” PDX tumours, which carry two BRCA1 point mutations (Q356R process, encountering various blocks, including immune check- and C61G), with NOTCH1 hyperactivation (Supplementary points, telomere limitation, contact inhibition and genome Fig. 10a). Because a high dose of cisplatin markedly decreased instability-induced apoptosis, among others. Our previous studies mouse body weight (Supplementary Fig. 10b) indicating a toxic indicated that the progression of Brca1-related tumourigenesis effect, we administered cisplatin at a low dose. The results showed occurs through a long latency that is accompanied by initial that AZD6738 single treatment did not affect tumour growth, cellular lethality12,14, suggesting the existence of such blocks. In even at high doses. A low dosage of AZD6738 markedly increased the present study, SB-mediated mutagenesis revealed several the killing effect of cisplatin (Fig. 8d, Supplementary Fig. 10c). findings that advance our understanding of this situation. We

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a b c Notch1-driven non-Notch1-driven Enrichment plot: HOLLERN_EMT_BREAST_TUMOR_UP Enrichment plot: HOLLERN_EMT_BREAST_TUMOR_DN 0.6 0.0 0.5 –0.1 0.4 –0.2 0.3 –0.3 0.2 –0.4

0.1 –0.5 Enrichment score (ES) Enrichment score (ES) 0.0 –0.6

‘Notch1’ (positively correlated) ‘Notch1’ (positively correlated) 1.0 1.0 0.5 0.5 0.0 Zero cross at 16163 0.0 Zero cross at 16163 –0.5 –0.5 –1.0 –1.0 –1.5 ‘Non-Notch1’ (negatively correlated) –1.5 ‘Non-Notch1’ (negatively correlated) 0 5,000 10,000 15,000 20,000 25,000 30,000 0 5,000 10,000 15,000 20,000 25,000 30,000 Ranked list metric (Signal2Noise) Rank in ordered dataset Ranked list metric (Signal2Noise) Rank in ordered dataset

Enrichment profile Hits Ranking metric scores Enrichment profile Hits Ranking metric scores

d MCF10A_iICN1 f shRNA-Ctl shRNA-BRCA1 Dox (h) 0 6 8 10 12 16 24 0 6 8 10 12 16 24

130 E-cadherin Status 1.00 0.80 0.67 0.54 0.49 0.56 0.56 0.51 0.50 0.60 0.62 0.69 0.82 1.23 CDH1 TNBC 100 β-catenin CTNNB1 Er−/Her2−/Pr+ 1.00 1.04 1.00 0.79 1.12 1.27 0.92 0.70 0.77 0.89 0.77 0.75 0.62 0.83 ER+/Her2− ER+/Her2+ N-cadherin OCLN 130 1.00 1.34 0.67 1.12 0.99 1.12 1.50 1.25 1.10 1.03 1.10 1.04 1.10 1.21 Er−/Her2+ LAMB1 Er+/Her−/Pr− 55 Vimentin Er+/Her+/Pr− 1.00 1.34 0.67 1.12 0.99 1.12 1.50 1.25 1.10 1.03 1.10 1.04 1.10 1.21 CDH2 PAM50 Fibronectin VIM Basal 250 1.00 1.12 2.07 2.03 2.16 2.15 2.57 0.73 0.89 0.32 1.58 1.51 1.56 1.45 LumA 35 FN1 Her2 Slug LumB 25 1.00 1.35 1.23 1.45 1.11 1.57 1.83 0.85 0.94 1.02 0.92 1.09 1.28 2.57 Normal 55 SNAI1 Claudin−low β-actin 1.00 1.03 1.00 1.05 0.79 0.97 0.94 0.85 0.74 0.99 0.92 1.03 1.98 1.07 SNAI2 Study Metabric CLDN1 TCGA2015 T47D_iICN1 e ZEB1 shRNA-Ctl shRNA-BRCA1 TWIST1 Dox (h) 0 6 8 10 12 16 24 0 6 8 10 12 16 24 250 KRT5 BRCA1 Marker 1.00 0.77 0.86 0.82 0.86 0.94 0.97 0.59 0.52 0.58 0.56 0.73 0.63 0.57 KRT6A TNBC Epithelial_Marker ICN1 110 KRT14 Mesenchymal_Marker 1.00 1.32 2.13 2.31 3.03 8.93 10.01 0.55 1.71 2.38 2.28 5.22 8.92 3.41 Luminal_cytokeratins KRT17 130 E-cadherin Basal_cytokeratins 1.00 0.86 0.73 0.71 0.75 0.77 0.70 0.58 0.52 0.52 0.58 0.63 0.72 0.91 KRT8 Expression 4 130 1.00 2.45 2.90 2.62 2.80 3.82 3.15 2.00 1.71 1.59 1.26 1.69 1.75 1.08 N-cadherin KRT18 2 0 KRT19 –2 55 Vimentin –4 1.00 0.87 0.92 0.85 0.43 0.75 0.67 0.47 0.79 0.86 0.72 0.66 0.47 0.40 ESR1

Fibronectin PGR 250 1.00 0.91 1.07 0.61 0.48 0.86 0.79 0.88 1.17 1.81 1.92 1.94 1.96 1.99 55 ERBB2 β-Actin 1.00 0.95 0.96 0.97 0.93 0.97 0.97 0.90 0.94 0.99 0.99 0.93 0.98 0.97

Fig. 7 Notch1 stimulates BRCA1-related TNBC progression by promoting EMT. a Gene expression analysis of Notch1-driven TNBC SB tumours (n = 24) compared with non-Notch1-driven TNBC SB tumours (n = 4). b GSEA analysis reveals that upregulated genes in Notch1-driven tumours are enriched in the EMT_breast_tumour_up term. c GSEA analysis indicates that downregulated genes in Notch1-driven tumours are enriched in the EMT_breast_tumour_dn term. d Western blotting analysis of EMT markers after induction of ICN1 in MCF10A and MCF10A-BRCA1-knockdown cells. e Western blotting analysis of EMT markers after induction of ICN1 in T47D and T47D-BRCA1-knockdown cells. f Meta-analysis of BRCA1-mutant human breast tumours for EMT markers, basal markers, luminal markers and TNBC markers. demonstrated that a defective G2/M cell-cycle checkpoint caused creates an environment for mutations leading to tumourigenesis by Brca1 deficiency serves as one such block, as restoration of this if p53 is mutated14. We further showed that loss of p53 enables checkpoint by Notch1 activation suppressed lethality and allowed Brca1-deficient cells to survive despite the presence of widespread Brca1-mutant cells to grow. Further analysis revealed several genetic instability due to inactivation of apoptosis mediated by previously unknown functions of Notch1 that may be necessary p5315. Notably, we found that activation of Notch1 also enables in accelerating Brca1-related TNBC tumourigenesis. Brca1-deficient cells to survive through a distinct mechanism, The Notch signalling pathway has an important role in several namely, partial restoration of the G2/M cell-cycle checkpoint biological processes. Deregulated Notch signalling is associated possibly through the ATR–CHK1 cascade, which is known to with several human malignancies, such as carcinomas of the lung, have an essential role in activating G2/M29. ATR also activates colon, head and neck, breast, pancreas and kidney, either as an intra-S and S/G2 cell-cycle checkpoints45,46, which prompted us oncogene or a tumour suppressor26,43,44. In the present study, our to examine these checkpoints. Our data indicate that Notch1 data clearly demonstrate that expression of ICN1, representing activation does not have an obvious role in the intra-S checkpoint Notch1 activation, promotes Brca1-associated mammary tumour but that it does activate the S/G2 cell-cycle checkpoint. Activation formation. One of the major abnormalities induced by Brca1 of these checkpoints delays cell-cycle progression, as reflected by deficiency is a defective G2/M cell-cycle checkpoint28, which may reduced cell proliferation and attenuation of the lethal block serve as a double-edged sword that, on the one hand, causes caused by BRCA1 deficiency. Clinical sample analysis demon- genetic instability leading to cell death and, on the other hand, strated that NOTCH1 activation correlated positively with the

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a b p = 6.5E–07 e Ki67 Cleaved caspase-3 100% p = 5.7E–07 Control p = 4.9E–06 80% CCT245737_0.5 μM p = 2.0E–06 LY2603618_0.5 μM 80% p = 0.057 60% μ Ctl AZD6738_0.5 M 60% VE821_0.5 μM 40%

40% μ μ KU60019-0.5 μM 50 m 50 m

Viability (% control) 20% 20%

0% Viability (% control) 0% 024681012 M M M M M μ μ μ μ μ 5 mg/kg Cisplatin (μM) Control Cisplatin-high

50 μm 50 μm VE821_0.5 KU60019-0.5 AZD6738_0.5 c d CCT245737_0.5LY2603618_0.5 800 MDA-MB-436MDA-MB-468Sum149HCC1937TM00091 Ctl

Cisplatin ) AZD6738-high_10 mg/kg

1.09 3 CCT245737 1.04 600 AZD6738-low_0.5 mg/kg 1.5 mg/kg 0.99 Cisplatin-low

M LY2603618 0.93 μ Cisplatin-low & AZD6738-low AZD6738 0.88 50 μm 50 μm 0.83

0.5 VE821 400 Cisplatin-low_1.5 mg/kg 0.77 KU60019 0.72 Cisplatin-high_5 mg/kg CCT245737 0.67 0.62 200 LY2603618 0.56 ns M 0.51 Tumor volum (mm

μ AZD6738 0.46 1 VE821 0.41 0.35 0 KU60019 Cisplatin-low

1481114 & AZD6738-low 50 μm 50 μm f p = 2.8E–05 Days p = 0.0010 p = 5.1E–05 p = 5.9E–05 p = 0.077 g 1000 BRCA1 defective NOTCH1 hyperactivation 800 600 Cisplatin 0.5 mg/kg

400 Genome instability AZD6738-low ATR EMT 50 μm 50 μm 200 ATR/CHK 1 inhibitor (per 1000 cells)

No. of dead cells 0 Mitotic catastrophe CHK1 Luminal Ctl transdifferentiation 10 mg/kg Apoptosis p53 Tumorigenesis TNBC tumor AZD6738-high

μ 50 m 50 μm Cisplatin-high_5Cisplatin-low_1.5 mg/kg mg/kg AZD6738-high_10AZD6738-low_0.5 mg/kg mg/kg Cisplatin-low & AZD6738-low

Fig. 8 Combined drug treatment of TNBC tumours. a Effect of combined treatment of cisplatin with ATR, ATM or CHK1 inhibitors on HCC1937 cells; n = 4 biologically independent experiments. b Quantification analysis of combined drug treatment in HCC1937 cells. Cisplatin (2.5 µM), ATR, ATM or CHK1 inhibitor (0.5 µM); n = 3 biologically independent experiments. c Killing effect on TNBC cells treated with cisplatin combined with different inhibitors. The colour in the heatmap represents the relative values of the areas under the curves. d In vivo effect of cisplatin combined with ATR inhibitor (AZD6738) on tumours in the PDX model (TM00091), which carries the BRCA1 mutation and highly expresses NOTCH1. Six mice were used for each treatment group. e IHC staining of Ki67 and cleaved caspase-3 in PDX tumours after drug treatment. Five tumours from different mice were examined for each treatment group. f Quantification analysis of dead cells (arrowhead), including cleaved caspase-3-positive cells (arrow) in each treatment group. Six to ten areas from different tumours were counted. g Illustration of the ICN1 function for TNBC formation in BRCA1-defective conditions. The t-test was used to determine the significance of differences between the different sets of data. Data are presented as mean values ± s.d. * indicates p < 0.05. phosphorylation level of ATR plus the capacity of direct binding originate from luminal cells, which are largely ERα positive between NOTCH1 and ATR; therefore, we speculate that initially49. Our previous study analysing tumours at different NOTCH1 rescues BRCA1-defective lethality through a non- stages also showed that Brca1−/− mammary tumours are initially canonical target, the ATR–CHK1 signalling pathway. Conse- ERα positive and gradually become negative as they grow50; quently, we conclude that NOTCH1 has an oncogenic function in nonetheless, it remains unclear how ERα is lost. EMT-related the presence of BRCA1 loss. Nonetheless, we should note that markers in TNBC might comprise a signature of a certain sub- under some other conditions, e.g., in tumours with intact DNA group of TNBC35,36,51, and the EMT programme has an damage repair systems or cell-cycle checkpoints, activation of important role in basal breast cancer52,53. Thus, in light of our ATR–CHK1 signalling would inhibit cell-cycle progression and finding that activation of Notch1 can enhance expression of EMT elicit tumour suppressor functions to block tumour development. signature genes, including Fibronectin and Slug, we believe that This may help to explain the dual functions of NOTCH1 as a Notch1 may drive TNBC formation under BRCA1-defective tumour suppressor and an oncogene under different circum- conditions by inducing expression of EMT signature genes, stances26. Obviously, further studies are needed to support this thereby promoting the transdifferentiation of luminal cells into hypothesis. the basal-type and at the same time strongly enhancing TNBC Previous studies have revealed that activation of NOTCH1 progression. This mechanism may also explain why basal-like correlates with cancer stem cell maintenance and expansion47 tumours largely overlap with TNBC in both human and mouse and that high NOTCH1 expression may serve as a prognostic BRCA1-related breast tumours. marker in patients with TNBC48, yet the underlying mechanism Analysis of the human breast cancer database revealed a close remains elusive. Our study demonstrates that activation of correlation between high expression levels of NOTCH1 and Notch1 promotes TNBC formation. BRCA1-deficient cancers TNBC incidence, especially BRCA1-related TNBC. Thus, it is

NATURE COMMUNICATIONS | (2020) 11:3256 | https://doi.org/10.1038/s41467-020-16936-9 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16936-9 important to develop an effective therapy for this group of respectively. Because of the several transgenic strains used in this study, the tumours. We recently found that cisplatin, which kills BRCA1- resulting cohorts of mice were of a mixed genetic background, including C57BL/6J, deficient cancers with high efficiency by causing DNA cross- 129SVE and FVB. Mice without the SB transposase or transposon served as a 54 negative control to compare the function of the transposon on tumour formation linking , also blocks tumour metastasis through inhibition of under Brca1-defective conditions. Animals in all experimental groups were EMT41. Given our finding that NOTCH1 promotes BRCA1- examined twice a week for tumourigenesis. Complete necropsies were performed to deficient tumour growth by activating ATR–CHK1 and inducing assess the primary tumour and metastasis in other organs. Only female mice were – EMT, we inhibited the ATR/CHK1 pathway in combination used for the experiment, and all the mice were pregnant once at 2 4 months of age to activate expression of WAP-Cre or MMTV-Cre. We named the tumour based on with a low dose of cisplatin to effectively kill TNBC in our mouse the mouse eartag number and the tumour location information; for example, 55 model and PDX model . This approach might avoid the non- MK1370-3R means that the tumour was collected from the third right mammary specific cytotoxic side effects commonly associated with high gland of mouse MK1370. doses of cisplatin, thus providing a potent clinical option for TNBC treatment. Histology and IHC. Routine haematoxylin and eosin staining was performed on PARP1 inhibitors are used for the treatment of BRCA1- 6-μm sections of formalin-fixed, paraffin-embedded (FFPE) tissues. IHC for ERα deficient cancers, as inhibition of PARP1 generates more single- (1:50, Santa Cruz, sc-542), PR (1:50, Santa Cruz, sc-538), HER2 (1:50, Santa Cruz, sc-284), cleaved caspase-3 (1:500, CST, 9664) and Ki67 (1:200, Abcam, ab16667) strand DNA damage, which results in DSBs and consequently was performed to characterise tumour histopathology using a Histostain-Plus IHC leads to a synthetic lethality with BRCA1 deficiency. Our strategy kit (Thermo, 859043, Lot: 1954379A) after antigen retrieval (pH 6.0), quenching of of blocking ATR–CHK1 is likely to target the cell cycle, i.e., it endogenous peroxidase and overnight incubation with the primary antibody. targets different aspects of PARP1. Our data indicate that inhi- bition of ATR or CHK1 works with cisplatin, which causes DNA CIS identification and annotation. A sequence library of SB transposon insertion inter-strand crosslink, leading to DSBs. As the effect of cisplatin sites was constructed using Splinkerette-PCR58 followed by a second round of PCR shares some similarities with that of PARP1 inhibitors, we suspect with SB Illumina adaptors (Supplementary Data 10). Samples were sequenced – using the Illumina HiSeq X-Ten platform with paired-end 150-bp reads. The that blocking ATR CHK1 might also involve PARP1 function, resulting reads were filtered and trimmed using cutadapt version 1.16 for con- which should be investigated in the near future. taminant sequences, including Illumina adaptor sequences, splinkerette linker Of note, alteration of many genes involved in some other systems sequences and transposon sequences. Reads without a valid adaptor and trans- and pathways can accelerate tumourigenesis, including the ubi- poson sequence were excluded from further analysis. Reads <20 bp after trimming were discarded. Clean reads were aligned to the mouse reference genome mm10, quitination system, cytoskeleton and cell junction, gene expression allowing 3 mismatches, using Bowtie2 version 2.3.4 followed by TAPDANCE regulation, and protein kinase/phosphatase modification-related analysis23. The aligned locations identified as CISs were automatically annotated functions. As Brca1 is an E3 ligase56, enrichment of ubiquitin- based on mouse reference genes (mm10.gtf). CISs located in the gene body plus related genes may have a compensatory role in Brca1 deficiency. 3 kb upstream with p-values < 0.05 were analysed further. CIS genes were then annotated with the criteria of gene location plus the Cytoskeleton rearrangement is involved in the EMT and tumour upstream 3 kb. Although local hopping of the SB transposon always increased metastasis, whereas tight junctions are a specialised structure of within the chromosome where the transposon concatamer was located, the epithelial and endothelial cell membranes and have an important insertion patterns among the SB strains 12740 or 12775 did not show bias with role in maintaining most apical junctional complexes57.Indeed, regard to chromosome, regardless of which Cre (MMTV-Cre or WAP-Cre) was components of all enriched pathways among our candidate genes used (Supplementary Fig. 2a). Therefore, we assessed all insertion sites for candidate gene identification, including those in Chr9 or Chr12, which are the are known to participate in cell proliferation, transformation and original insertion sites for the T2Onc3 transposon in strains 12740 or 12775, metastasis suppression, though their specificroleinBrca1-related respectively. However, to avoid the bias of local hopping, we chose genes identified tumourigenesis remains elusive. Regardless, these data demonstrate in more than 5% of the samples in both 12740 and 12775 as strict criteria to filter that these mutations can activate multiple oncogenic pathways to out fake genes. fi CIS genes were considered oncogenic drivers if all insertions were located in a overcome the lethal block of Brca1 de ciency in mammary epi- specific region and the CAG promoter of the transposon was in the same thelial cells to promote the initiation and progression of cancer orientation as the gene’s ORF, which will drive target gene overexpression from the formation. insertion site. Conversely, genes were considered tumour suppressors in the event In summary, our study revealed that activation of Notch1 has of an unbiased insertion site and no bias orientation of the CAG promoter, various important consequences, including cell-cycle check- whereby the polyadenylation signal of the transposon most likely disrupts the gene. point activation and promotion of transdifferentiation by inducing expression of EMT signature genes. By stimulating Transcriptome sequencing and analysis. RNA-seq libraries were constructed – using an Illumina TruSeq RNA library Prep Kit and sequenced with the Illumina ATR CHK1 signalling, activation of the G2/M and S/G2 cell- HiSeq X-Ten platform to generate a minimum of 25 million paired-end 150-bp cycle checkpoints, at least in part, overcomes the lethal block reads. Sequencing reads were aligned to the mouse reference genome mm10 and caused by Brca1 deficiency through suppression of the mitotic processed using HISAT2 version 2.1.059. Differential expression analysis was catastrophe and promotes tumour initiation (Fig. 8g). However, conducted using DESeq2 version 1.22.260. For exon-level expression, reads from each exon of the target gene were counted followed by normalisation with total activation of Notch1 also promotes TNBC formation by acti- reads and length of individual exons to avoid bias due to different exon sizes. vating the EMT signalling pathway. The combination of these PAM50 subtype assignment was conducted using Genefu (2.18.1)61. effects enhances tumourigenesis; conversely, treatment with – cisplatin, which inhibits the EMT, and ATR CHK1 inhibitors Functional enrichment analysis. Gene function annotation enrichment analysis that target the cell-cycle checkpoint display good synergy in was performed with DAVID Bioinformatics v6.8 using the Gene Ontology and inhibiting TNBC, thus providing a potent clinical option for KEGG pathway data sets62,63. GSEA (GSEA v3.0)64 was also used for gene this fatal disease. expression difference analysis. Analysis of driver gene molecular interactions among CIS genes was conducted using STRING v11.0 online tools65.

Methods Cross-species TNBC correlation analysis. Gene expression data for human Mice. All mouse experiments were performed under the ethical guidelines of the patient samples were downloaded from TCGA31 and METABRIC32 databases by University of Macau (animal protocol number: UMAEC-037-2015). Mice were using R studio (version 3.5.1) with cgdsr packages. Clinical pathology information housed in a Specific-pathogen-free (SPF) facility at 23–25 °C on a 12-h light/dark was also retrieved. We obtained 1898 and 403 patients from the METABRIC and cycle. The following mouse strains were used in this study. (1) Brca1 conditional TCGA databases, respectively. Among them, 37 BRCA1-mutant samples were knockout (Brca1Co/Co) mice, in which deletion of exon 11 of Brca1 is controlled by obtained from the former. For each gene, we ranked the samples based on two mammary tissue-specific Cre transgenes (WAP-Cre or MMTV-Cre)14. (2) Two expression levels. The samples were separated equally into 10 (for all patients) or 6 strains with a conditionally expressed SB11 transposase that includes a floxed (for BRCA1-mutant patients) cohorts. Finally, we counted the TNBC proportion of transcriptional stop cassette to be activated by Cre. (3) Independent transgenic lines each single cohort to determine the correlation between the gene expression level of T2onc3 (12740 and 12775)22, located on chromosome 9 and chromosome 12, and TNBC morbidity for each individual gene.

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Western blotting. Cultured cells were homogenised in RIPA buffer supplemented Millipore, 05-636, Lot: 3076468) and 53BP1 (1:200, Santa Cruz, sc-22760, Lot: with phosphatase and protease inhibitor cocktails (Sigma). After measurement and I1813) immunofluorescence staining are same with MI analysis. Secondary anti- normalisation of the protein concentration, whole-cell lysates were loaded onto bodies for this experiments are: F(ab′)2-Goat anti-Rabbit IgG (H + L) Cross- polyacrylamide gels for electrophoresis. Proteins were electrotransferred onto Adsorbed Secondary Antibody, Alexa Fluor 488 (1:1000, Thermo, A-11070, Lot: PVDF membranes (Bio-Rad), blocked with 5% BSA in TBS-T buffer for 1 h at 1494754), F(ab′)2-Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary room temperature and incubated with the primary antibody overnight at 4 °C. The Antibody, Alexa Fluor 594 (1:1000, Thermo, A11072, Lot: 1431810), F(ab′)2-Goat blots were incubated with the corresponding secondary antibody (Cell Signaling, anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 CST), and immunoreactive bands were developed with the ECL substrate (Milli- (1:1000, Thermo, A-11020, Lot: 1454439), F(ab′)2-Goat anti-Mouse IgG (H + L) pore). The primary antibodies used in this study were as follows: BRCA1 (1:200, Cross-Adsorbed Secondary Antibody and Alexa Fluor 488 (1:1000, Thermo, A- Santa Cruz, sc-642), NOTCH1 (1:1000, CST 4380), E-cadherin (1:1000, CST 3195), 11017, Lot: 1557766) β-catenin (1:1000, Abcam, ab32572), N-cadherin (1:1000, Abcam, ab76057), Vimentin (1:1000, CST 5741), Fibronectin (1:1000, Abcam, ab2431), Slug (1:1000, Cell viability analysis. Cell viability was assessed using different approaches, CST 9585), β-actin (1:4000, Sigma, A5316), ATM (1:1000, Abcam ab78), pATM including the MTT assay and the Alamar Blue assay. For the MTT assay, MTT dye (1:1000, Abcam, ab81292), ATR (1:1000, CST 2790), p-ATR-1989 (1:1000, Abcam, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue, ab227851), p-ATR-428 (1:1000, CST 2853), CHK1 (1:200, Santa Cruz, sc-56288), Sigma-Aldrich) was added (final concentration: 0.5 mg/ml) to cultured cells for 4 h. p-CHK1-317 (1:1000,CST 12302) and p-CHK1-345 (1:1000, CST 2348). The sec- The formazan crystals were solubilised with DMSO, and absorbance was measured ondary antibodies used in this study were as follows: Anti-rabbit IgG, HRP-linked at 570 nm using a plate spectrophotometer. The Alamar Blue assay was used for Antibody (1:5000, CST 7074, Lot:28), Anti-mouse IgG and HRP-linked Antibody cell drug treatment analysis. Briefly, culture medium containing 0.02% Alamar Blue (1:5000, CST 7076, Lot:32). was added to cultured cells, and absorbance at 590 nm under 560 nm excitation was measured 2 h of incubation at 37 °C. Quantitative RT-PCR. Total RNA was extracted using TRIzol reagent (Thermo) by following their routine procedure. cDNA was synthesised with the QuantiTect Allograft/xenograft studies. Tumours or cultured cells were dissociated into Reverse Transcription kit (Qiagen, 205313). Quantitative PCR was performed single cells and resuspended in 50% Matrigel (Corning, 356234) for inoculation. using FastStart Universal SYBR Green Master (Rox) mix (Sigma) on a QuantStudio NSG or nude mice were anaesthetised with tribromoethanol, and a small 7 Flex Real-Time PCR System (Thermo). The primer sequences used are listed in abdominal incision was made. Mammary fat pads were exposed gently by forceps, Supplementary Data 10. and 1 million cells were injected using a microlitre syringe with a 27-gauge needle. The mice were maintained in an SPF facility for further drug administration. 3 Vector construction. shRNA plasmids were constructed by inserting annealed The mice began receiving drug treatment when the tumours reached ~200 mm . targeting oligos into the pLKO.1 backbone (Addgene, 8453) after digestion with Tumour volume was calculated as V = (W2 × L)/270. AZD6738 was administered AgeI and EcoRI (NEB, R0552 and R0101). ICN1 cDNA was cloned from the Puro- by oral gavage at 10 or 0.5 mg/kg every 3 days together with cisplatin, which was iNotch1IC plasmid (Addgene, 75338)66 for mice and EF.hICN1.CMV.GFP plasmid administered by intraperitoneal injection at 1.5 or 5 mg/kg. (Addgene, 17623)67 for humans and then inserted into the tet-on plasmid 68 (Addgene, 80921) . An HA tag was also added to the N terminus of ICN1. Statistics and reproducibility. Microsoft Excel, GraphPad Prism (version 6) and fi Routine PCR and Sanger sequencing were performed to con rm successful R (version 3.5.1) software were used for statistical calculations. Specific statistical insertion into the vector. The oligonucleotide sequences used for shRNA knock- tests, sample number and other information are indicated in the main text or figure down are listed in Supplementary Data 10. legends. The t-test and two-sided statistical analysis approach was used to deter- mine the significance of the difference between the different sets of data if there was fi Cell culture. Mouse primary mammary tumour cells were derived from SB no speci c indication. tumours after dissociation with digestion buffer, which contained DMEM/F12 All experiments were conducted at least three times independently, and similar medium (Thermo), 5% FBS, 10 ng/ml EGF (Thermo, 13247-051), 0.5 mg/ml results were adopted for further analysis to guarantee reproducibility. hydrocortisone (Sigma, H0888), 20 ng/ml cholera toxin (Sigma, C-3012), 5 μg/ml insulin (Sigma, 350-020), 300 U/ml collagenase III (Worthington, S4M7602S) and Reporting summary. Further information on research design is available in hyaluronidase (Sigma, H3506)10. MCF10A cells were cultured in DMEM/F12 the Nature Research Reporting Summary linked to this article. medium containing 5% horse serum (Thermo), 20 ng/ml EGF, 0.5 mg/ml hydro- cortisone, 100 ng/ml cholera toxin, 10 μg/ml insulin and pen/strep (Thermo). T47D, MCF7, MDA-MB-231, MDA-MB-436, MDA-MB-468, Sum149 and Data availability HCC1937 cells were cultured in DMEM supplemented with 10% FBS, glutamine The RNA sequence data have been deposited in the Sequence Read Archive (SRA) (Thermo), insulin and pen/strep. The human primary breast cancer cell line database under the accession code PRJNA529536. The data from TCGA and TM00091 was derived from a PDX model (Jax, TM00091)55, which carries a METABRIC referenced in the study are available in a public repository from the BRCA1 mutation with high expression of NOTCH1, by using the same approach cBioPortal website (https://www.cbioportal.org/). All the other data supporting the used for the mouse primary cells. All cells were routinely tested to exclude findings of this study can be found within the supplementary files. A reporting summary mycoplasma contamination. for this article is available as a Supplementary Information file. Source data for all the plots are provided as a Source Data file. Cell-cycle synchronisation. The double thymidine block approach was applied for cell-cycle synchronisation as previously reported69. Briefly, cells were blocked using Received: 11 June 2019; Accepted: 2 June 2020; 2 mM thymidine for 18 h when they reached 25–30% confluence. After 9 h of culture in regular medium, 15 h of thymidine incubation was performed for the second round of blocking. The cells were then released and collected at different time points for cell-cycle flow cytometry analysis.

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14 NATURE COMMUNICATIONS | (2020) 11:3256 | https://doi.org/10.1038/s41467-020-16936-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16936-9 ARTICLE

66. Zhu, Z. R. et al. Genome editing of lineage determinants in human pluripotent Competing interests stem cells reveals mechanisms of pancreatic development and diabetes. Cell The authors declare no competing interests. Stem Cell 18, 755–768 (2016). 67. Yu, X. B. et al. HES1 inhibits cycling of hematopoietic progenitor cells via Additional information DNA binding. Stem Cells 24, 876–888 (2006). 68. Barger, C. J., Branick, C., Chee, L. & Karpf, A. R. Pan-cancer analyses reveal Supplementary information is available for this paper at https://doi.org/10.1038/s41467- genomic features of FOXM1 overexpression in cancer. Cancers 11, 251 (2019). 020-16936-9. 69. Whitfield, M. L. et al. Identification of genes periodically expressed in the human cell cycle and their expression in tumors. Mol. Biol. Cell 13, 1977–2000 (2002). Correspondence and requests for materials should be addressed to C.-X.D. 70. Faustino-Rocha, A. et al. Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab Anim. 42, 217–224 (2013). Peer review information Nature Communications thanks Branden Moriarity, Marcel van Vugt and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Acknowledgements We thank members of the C. Deng and X. Xu laboratories for helpful advice and Reprints and permission information is available at http://www.nature.com/reprints discussion, the Animal Research Core for providing the animal housing, and the ’ Information and Communication Technology Office (ICTO) for providing the HPC for Publisher s note Springer Nature remains neutral with regard to jurisdictional claims in fi data processing. This work was supported by the Chair Professor Grant granted to C.D., published maps and institutional af liations. multi-year research grant (MYRG) 2016-00139-FHS to C.D. and 2018-00186-FHS to K.M. by the University of Macau, Macau SAR, China; the Macao Science and Technology Development Fund (FDCT) Grant 094/2015/A3, 0011/2019/AKP, 0034/2019/AGJ and Open Access This article is licensed under a Creative Commons 0048/2019/A1 to C.D.; 029/2017/A1 and 0101/2018/A3 to X.X., and 111/2017/A to K.M.; Attribution 4.0 International License, which permits use, sharing, and the National Natural Science Foundation of China grant 81672603 to Q.C. adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Author contributions Commons license, and indicate if changes were made. The images or other third party ’ C.D. and K.M. designed this research; K.M., H.L., J.X., S.C., X.Z., S.M.S. and F.S. per- material in this article are included in the article s Creative Commons license, unless formed experiments; H.L., L.W. and S.M.S. performed the histopathological analysis and indicated otherwise in a credit line to the material. If material is not included in the ’ immunohistochemical staining; J.X., J.B., H.S. and Q.C. assisted with the cell-cycle article s Creative Commons license and your intended use is not permitted by statutory experiments and data analysis; M.V.V., A.Z., X.L., B.K.R. and J.Z. identified the CISs, regulation or exceeds the permitted use, you will need to obtain permission directly from analysed the RNA-sequencing data and performed other bioinformatics analyses; K.M., the copyright holder. To view a copy of this license, visit http://creativecommons.org/ F.S. and P.C. performed the drug treatment experiments; Z.M. and K.H.W. initiated the licenses/by/4.0/. bioinformatics analysis for identifying CISs; K.T. was responsible for sequencing the SB libraries; K.H.W. and K.M. supervised the bioinformatics analysis; C.D., X.X. and Q.C. © The Author(s) 2020 supervised the experiments; and K.M. and C.D. wrote the manuscript.

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