Abstract 4851: NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells
Authors: Leontovich Alexey , Salisbury Jeffrey , Haddox Candace L. , Mills Lisa , Haddad Tufia , McCubrey James , Goetz Matthew , Ingle James , Galanis Evanthia , D'Assoro Antonino B. 
Published: July 2017

Background: NOTCH oncogenic signaling induces epithelial-to-mesenchymal transition (EMT) and

tumor stemness that play a critical role in driving tumor progression. Following interaction of NOTCH

receptors (NOTCH 1, 2, 3, and 4) with their ligands (DELTA-LIKE 1, 3, 4 and JAGGED 1, 2), γ-

secretase complex performs an intra-membrane cleavage releasing the NOTCH intracellular domain

(NNICD). NNICD translocates to the nucleus and induces the expression of HEY1 and HES1

transcription factors that in turn orchestrate NOTCH-mediated EMT and stemness reprogramming.

Materials and Methods: Parental MCF-7 and unique metastatic MCF-7RAF-1 xenografts with

constitutively active MAPK signaling (D’Assoro et al., Oncogene 2014: 33:599-610) were used to define

the extent to which increased expression of NOTCH signaling was linked to distant metastases. SKY

analysis was performed to assess the grade of chromosomal instability (CIN) in tumor xenografts.

Stemness activity was assessed by culturing breast cancer cells under non-adherent conditions to

generate mammospheres. Expression of CD24 luminal marker was characterized by FACS analysis

and immunofluorescence. Global transcriptome analysis was performed using Affymetrix® microarray.

Highly invasive MDA-MB 231 breast cancer cells were used to perform a tail vein cancer cell-seeding

assay. The LY411575 γ-secretase inhibitor (Pan-NOTCH inhibitor) was used to block NOTCH signaling

in MDA-MB 231 cells.

Results: Metastasis Initiating Cells (MICs) isolated from the lungs of nude mice carrying MCF-7RAF-1

xenografts exhibited a higher degree of genomic stability than primary tumor xenografts that was

characterized by the absence of centrosome amplification and non-clonal chromosomal abnormalities.

MICs also showed higher mammosphere forming capacity that was linked to lower expression of luminal

marker CD24. Significantly, global transcriptome analysis showed that expression of NOTCH3 and

HES1 stemness genes was increased in MICs. To corroborate the role of NOTCH signaling in inducing

metastatic seeding, MDA-MB 231 breast cancer cells were pre-treated with 1 µM of LY411575 After 72

hours viable cells were injected into the tail vein of immune-compromised mice to develop lung

metastases. While animals injected with untreated MDA-MB 231 cells developed lung metastases,

those injected with the LY411575-treated MDA-MB 231 cells failed to develop lung metastatic lesions.

Conclusions: These preliminary findings demonstrate the role of NOTCH stemness signaling in

promoting metastatic seeding of breast cancer cells. Since NOTCH signaling is a “druggable target”,

these results also provide the rationale to develop novel NOTCH-targeted therapies to inhibit tumor

stemness and early onset of distant metastases.

Citation Format: Alexey Leontovich, Jeffrey Salisbury, Candace L. Haddox, Lisa Mills, Tufia Haddad, James McCubrey, Matthew Goetz, James Ingle, Evanthia Galanis, Antonino B. D'Assoro. NOTCH signaling promotes metastatic seeding and growth of human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4851. doi:10.1158/1538-7445.AM2017-4851