The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control (P < 0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function.
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Andersson ER, Sandberg R, Lendahl U (2011) Notch signaling: simplicity in design, versatility in function. Development 138(17):3593–3612
Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137(2):216–233
Arnett KL, Hass M, McArthur DG, Ilagan MX, Aster JC, Kopan R, Blacklow SC (2010) Structural and mechanistic insights into cooperative assembly of dimeric Notch transcription complexes. Nat Struct Mol Biol 17(11):1312–1317
Radtke F, Raj K (2003) The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat Rev Cancer 3(10):756–767
Ranganathan P, Weaver KL, Capobianco AJ (2011) Notch signalling in solid tumours: a little bit of everything but not all the time. Nat Rev Cancer 11(5):338–351
Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD, Sklar J (1991) TAN-1, the human homolog of the Drosophila Notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 66(4):649–661
Previs RA, Coleman RL, Harris AL, Sood AK (2015) Molecular pathways: translational and therapeutic implications of the Notch signaling pathway in cancer. Clin Cancer Res 21(5):955–961
Mazur PK, Grüner BM, Nakhai H, Sipos B, Zimber-Strobl U, Strobl LJ, Radtke F, Schmid RM, Siveke JT (2010) Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras G12D-induced skin carcinogenesis in vivo. PLoS One 5(10):e13578
Siegel R, Ma J, Zou Z, Jemal A (2014) Cancer statistics. CA Cancer J Clin 64(1):9–29
Hu C, Diévart A, Lupien M, Calvo E, Tremblay G, Jolicoeur P (2006) Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors. Am J Pathol 168(3):973–990
Harrison H, Farnie G, Howell SJ, Rock RE, Stylianou S, Brennan KR, Bundred NJ, Clarke RB (2010) Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res 70(2):709–718
Reedijk M, Odorcic S, Chang L, Zhang H, Miller N, McCready DR, Lockwood G, Egan SE (2005) High-level coexpression of JAG1 and NOTCH1 is observed in human breast cancer and is associated with poor overall survival. Cancer Res 65(18):8530–8537
Kim SH, Sehrawat A, Singh SV (2012) Notch2 activation by benzyl isothiocyanate impedes its inhibitory effect on breast cancer cell migration. Breast Cancer Res Treat 134(3):1067–1079
Sehrawat A, Sakao K, Singh SV (2014) Notch2 activation is protective against anticancer effects of zerumbone in human breast cancer cells. Breast Cancer Res Treat 146(3):543–555
Lee J, Sehrawat A, Singh SV (2012) Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells. Breast Cancer Res Treat 136(1):45–56
Chao CH, Chang CC, Wu MJ, Ko HW, Wang D, Hung MC, Yang JY, Chang CJ (2014) MicroRNA-205 signaling regulates mammary stem cell fate and tumorigenesis. J Clin Investig 124(7):3093–3106
O’Neill CF, Urs S, Cinelli C, Lincoln A, Nadeau RJ, León R, Toher J, Mouta-Bellum C, Friesel RE, Liaw L (2007) Notch2 signaling induces apoptosis and inhibits human MDA-MB-231 xenograft growth. Am J Pathol 171(3):1023–1036
Parr C, Watkins G, Jiang WG (2004) The possible correlation of Notch-1 and Notch-2 with clinical outcome and tumour clinicopathological parameters in human breast cancer. Int J Mol Med 14(5):779–786
Mishra LC, Singh BB, Dagenais S (2000) Scientific basis for the therapeutic use of Withania somnifera (Ashwagandha): a review. Altern Med Rev 5(4):334–346
Hahm ER, Lee J, Kim SH, Sehrawat A, Arlotti JA, Shiva SS, Bhargava R, Singh SV (2013) Metabolic alterations in mammary cancer prevention by withaferin A in a clinically relevant mouse model. J Natl Cancer Inst 105(15):1111–1122
Stan SD, Hahm ER, Warin R, Singh SV (2008) Withaferin A causes FOXO3a- and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo. Cancer Res 68(18):7661–7669
Singh SV, Kim SH, Sehrawat A, Arlotti JA, Hahm ER, Sakao K, Beumer JH, Jankowitz RC, Chandra-Kuntal K, Lee J, Powolny AA, Dhir R (2012) Biomarkers of phenethyl isothiocyanate-mediated mammary cancer chemoprevention in a clinically relevant mouse model. J Natl Cancer Inst 104(16):1228–1239
Powolny AA, Bommareddy A, Hahm ER, Normolle DP, Beumer JH, Nelson JB, Singh SV (2011) Chemopreventative potential of the cruciferous vegetable constituent phenethyl isothiocyanate in a mouse model of prostate cancer. J Natl Cancer Inst 103(7):571–584
Beaino W, Anderson CJ (2014) PET imaging of very late antigen-4 in melanoma: comparison of 68Ga- and 64Cu-labeled NODAGA and CB-TE1A1P-LLP2A conjugates. J Nucl Med 55(11):1856–1863
Moura MB, Hahm ER, Van Houten B, Singh SV (2014) The use of seahorse extracellular flux analyzer in mechanistic studies of naturally occurring cancer chemopreventive agents. In: Bode AM, Dong Z (eds) Cancer prevention: dietary factors and pharmacology, methods in pharmacology and toxicology. Springer Science + Business Media, New York, pp 173–187
Lahat G, Zhu QS, Huang KL, Wang S, Bolshakov S, Liu J, Torres K, Langley RR, Lazar AJ, Hung MC, Lev D (2010) Vimentin is a novel anti-cancer therapeutic target; Insights from in vitro and in vivo mice xenograft studies. PLoS One 5(4):e10105
Zang S, Chen F, Dai J, Guo D, Tse W, Qu X, Ma D, Ji C (2010) RNAi-mediated knockdown of Notch-1 leads to cell growth inhibition and enhanced chemosensitivity in human breast cancer. Oncol Rep 23(4):893–899
Simmons MJ, Serra R, Hermance N, Kelliher MA (2012) NOTCH1 inhibition in vivo results in mammary tumor regression and reduced mammary tumorsphere-forming activity in vitro. Breast Cancer Res 14(5):R126
Yuan X, Zhang M, Wu H, Xu H, Han N, Chu Q, Yu S, Chen Y, Wu K (2015) Expression of Notch1 correlates with breast cancer progression and prognosis. PLoS One 10(6):e0131689
Hurvitz S, Mead M (2016) Triple-negative breast cancer: advancements in characterization and treatment approach. Curr Opin Obstet Gynecol 28(1):59–69
Yadav BS, Chanana P, Jhamb S (2015) Biomarkers in triple negative breast cancer: a review. World J Clin Oncol 6(6):252–263
Chavez KJ, Garimella SV, Lipkowitz S (2010) Triple negative breast cancer cell lines: one tool in the search for better treatment of triple negative breast cancer. Breast Dis 32(1–2):35–48
Barnabas N, Cohen D (2013) Phenotypic and molecular characterization of MCF10DCIS and SUM breast cancer cell lines. Int J Breast Cancer 2013:872743
Vyas AR, Singh SV (2014) Molecular targets and mechanisms of cancer prevention and treatment by withaferin A, a naturally occurring steroidal lactone. AAPS J 16(1):1–10
Zou J, Han Z, Zhou L, Cai C, Luo H, Huang Y, Liang Y, He H, Jiang F, Wang C, Zhong W (2015) Elevated expression of IMPDH2 is associated with progression of kidney and bladder cancer. Med Oncol 32(1):373
Zhou L, Xia D, Zhu J, Chen Y, Chen G, Mo R, Zeng Y, Dai Q, He H, Liang Y, Jiang F, Zhong W (2014) Enhanced expression of IMPDH2 promotes metastasis and advanced tumor progression in patients with prostate cancer. Clin Transl Oncol 16(10):906–913
Ramos FS, Serino LT, Carvalho CM, Lima RS, Urban CA, Cavalli IJ, Ribeiro EM (2015) PDIA3 and PDIA6 gene expression as an aggressiveness marker in primary ductal breast cancer. Genet Mol Res 14(2):6960–6967
Dethlefsen C, Højfeldt G, Hojman P (2013) The role of intratumoral and systemic IL-6 in breast cancer. Breast Cancer Res Treat 138(3):657–664
Todorović-Raković N, Milovanović J (2013) Interleukin-8 in breast cancer progression. J Interferon Cytokine Res 33(10):563–570
Divella R, Daniele A, Savino E, Palma F, Bellizzi A, Giotta F, Simone G, Lioce M, Quaranta M, Paradiso A, Mazzocca A (2013) Circulating levels of transforming growth factor-βeta (TGF-β) and chemokine (C-X-C motif) ligand-1 (CXCL1) as predictors of distant seeding of circulating tumor cells in patients with metastatic breast cancer. Anticancer Res 33(4):1491–1497
Lin S, Gan Z, Han K, Yao Y, Min D (2015) Interleukin-6 as a prognostic marker for breast cancer: a meta-analysis. Tumori 101(5):535–541
Hartman ZC, Poage GM, den Hollander P, Tsimelzon A, Hill J, Panupinthu N, Zhang Y, Mazumdar A, Hilsenbeck SG, Mills GB, Brown PH (2013) Growth of triple-negative breast cancer cells relies upon coordinate autocrine expression of the proinflammatory cytokines IL-6 and IL-8. Cancer Res 73(11):3470–3480
Freund A, Chauveau C, Brouillet JP, Lucas A, Lacroix M, Licznar A, Vignon F, Lazennec G (2003) IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells. Oncogene 22(2):256–265
This investigation was supported by the National Cancer Institute, National Institutes of Health grant award RO1 CA142604-06 (to SVS). This research project used Animal Facility, In Vivo Imaging Facility, Tissue and Research Pathology Facility, and Proteomics Facility supported in part by a grant from the National Cancer Institute at the National Institutes of Health (P30 CA047904).
Conflict of interest
The authors do not have any conflict of interest.
Use of mice and their care was in accordance by the Institutional Animal Care and Use Committee.
Su-Hyeong Kim and Eun-Ryeong Hahm have contributed equally to this work.
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Kim, S., Hahm, E., Arlotti, J.A. et al. Withaferin A inhibits in vivo growth of breast cancer cells accelerated by Notch2 knockdown. Breast Cancer Res Treat 157, 41–54 (2016). https://doi.org/10.1007/s10549-016-3795-y
- Breast cancer
- Withaferin A