A phenotypic cell-binding screen identifies a novel compound

May 2, 2018 - We describe a "phenotypic cell-binding screen" by which therapeutic candidate targeting cancer cells of a particular phenotype can be is...
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A phenotypic cell-binding screen identifies a novel compound targeting triple-negative breast cancer Luxi Chen, Chao Long, Jonghae Youn, and Jiyong Lee ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.8b00026 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 3, 2018

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A phenotypic cell-binding screen identifies a novel compound targeting triple-negative breast cancer Luxi Chen†, Chao Long†, Jonghae Youn† and Jiyong Lee*† †

Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson TX, USA.

Supporting Information Placeholder ABSTRACT: We describe a “phenotypic cell-binding

screen” by which therapeutic candidate targeting cancer cells of a particular phenotype can be isolated without knowledge of drug target(s). Chemical library beads are incubated with cancer cells of the phenotype of interest in the presence of cancer cells lacking the phenotype of interest, and then the beads bound to only cancer cells of the phenotype of interest are selected as hits. We have applied this screening strategy in discovering a novel compound (LC129-8) targeting triple-negative breast cancer (TNBC). LC129-8 displayed highly specific binding to TNBC in cancer cell lines and patient-derived tumor tissues. LC129-8 exerted anti-TNBC activity by inducing apoptosis, inhibiting proliferation, reversing epithelialmesenchymal transition, downregulating cancer stem cell activity and blocking in vivo tumor growth.

Triple-negative breast cancer (TNBC), accounting for 15% to 20% of all breast cancer cases,1-2 is characterized by its lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2). TNBC reflects a substantial clinical challenge due to the absence of targeted therapeutics.3-6 Moreover, TNBC has been reported for enriched epithelial-mesenchymal transition (EMT)-associated genes and higher percentage of cancer stem cell (CSC) populations, resulting in drug resistance, metastasis and relapse.7-9 Therefore, discovering targeted therapeutic agents for TNBC is urgently needed. However, drug discovery efforts for TNBC have been challenging due to the limitation of defined therapeutic targets of TNBC. We have reasoned that TNBC carries additional characteristic surface molecule that distinguish itself from the other breast cancer subtypes, which can allow us to identify therapeutic candidates that specifically bind to TNBC over non-TNBC. Indeed, several

cell surface molecules of TNBC have been identified and evaluated as therapeutic targets of TNBC; previous studies have demonstrated the utilization of peptides or nanoparticles to target EGFR,10 11 12 CD44/neuropilin or αvβ3 integrin of TNBC. However, as we aim to target unidentified or undefined receptors of TNBC also, the screening platform to identify novel TNBC-specific therapeutic candidates should be built on the difference in phenotypes between TNBC and non-TNBC. A phenotypic cell-binding screen we describe in this study is the application of the previously reported cell-based screens employing OBOC library.13-14 However, it provides an important advance as it demonstrates hit discovery in a specific cancer cell phenotype-dependent manner without the prior knowledge of defined drug target(s). As illustrated in Figure 1a, two TNBC cell lines, MDA-MB-231 (mesenchymal stem-like subtype) and BT549 (mesenchymal subtype) were stained with a red fluorescent Qtracker655. In contrast, non-TNBC cell line MCF-7 (ER+/PR+/HER2-) was stained with a green fluorescent Qtracker565. The stained cancer cells were mixed and incubated with about 50,000 peptoid library beads (Figure S1). The resulting beads with bound cells were visualized with UV illumination. We found the following sets of beads from the screen; (1) beads with mostly red fluorescent cells: TNBC-specific ligands, (2) beads with mostly green fluorescent cells: non-TNBC specific ligands, (3) beads with both red fluorescent cells and green fluorescent cells: non-specific ligands. The co-incubated nonTNBC cells serve as competitor cancer cells that display cell surface receptors competing for ligand binding, and thus increase specificity of the isolated TNBC-specific hits. Among the 10 hits isolated, LC129-8 (Figure 1b) was chosen for evaluating its bindings to breast cancer cell lines of various subtypes. LC129-27 (Figure S2a) was a non-TNBC binder, and LC129-CON (Figure

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Figure 1. A phenotypic cell-binding screen. (a) Illustration of the screen. Transmitted and fluorescence (DAPI channel) images of the isolated beads displaying LC129-8, LC129-27 or LC129 CON. The bead itself emits blue fluorescence. Scale bar = 150 µm. (b) Chemical structures of LC129-8, TG-LC129-8, and C-LC129-8 (C: Cys). (c) Indicated subtypes of breast cancer cell lines were incubated with biotinylated-LC129-8 (biotinylated-8, 50 µM) followed by streptavidin-conjugated Texas Red (SA-Texas Red), and fluorescent images were taken at DAPI channel. Blue fluorescence indicates nuclei. Scale bar = 100 µm. (d) Tissue microarray was incubated with biotinylated-8 (20 µM) followed by SA-Texas Red, and fluorescent images of representative tissues were shown. Scale bar = 400 µm. (e) Quantitative binding area analysis of biotinylated-8 to the indicated tissues (n = 6 for each type of tissue).

S2b) was a non-specific binder. As shown in Figure 1c, exclusive bindings of biotinylated-LC129-8 (hereafter biotinylated-8, Figure S3) to all the tested TNBC cell lines were observed while no binding was observed to non-TNBC cell lines, MCF-7 (ER+/PR+/HER2-), T47D (ER+/PR+/HER2-) and SKBR3 (ER-/PR/HER2+). Importantly, biotinylated-8 did not show any binding to MCF10A, normal mammary epithelial cells (Figure 1c). The dihydroxyphenylalanine (DOPA) moiety of the biotinylated-8 is for the future use in periodate-mediated crosslinking of binding receptors(s) for target identification studies. It is important to note that we didn’t do crosslinking reaction in evaluating the biding of biotinylated-8 described in this study. To rule out any possibility that the hydrophobic DOPA moiety contributed to the binding of biotinylated-8 to TNBC cell lines, we have examined the binding of biotinylated-8 derivative lacking DOPA moiety (biotinylated-8 wo DOPA; Figure S3). As shown in Figure S4, the biotinylated-8 wo DOPA displayed same selectivity in binding to TNBC cell lines as biotinylated-8, demonstrating the binding of biotinylated 8 was not due to the DOPA moiety. Since we have utilized cell lines for the screening and binding validation, it is possible that the binding of LC129-8 to TNBC is just cell line-dependent but not necessarily TNBC phenotype-dependent. To address this issue, we utilized tissue microarrays (TMAs) comprising patient-derived TNBC tumor tissues, non-

TNBC tumor tissues and normal tissues adjacent to tumor regions. We found that biotinylated-8 displayed extensive binding to TNBC tissues, but no binding was observed to hormone receptor (HR)-positive (ER+ and/or PR+) tissues or HER2-overexpressed tissues (Figure 1d, e). There was also low level of binding of biotinylated-8 to the normal tissues adjacent to TNBC tumors while no binding was found for adjacent normal tissues of HR-positive and HER-2 overexpressed tumors. It was reported that a small population of cancer cells in the adjacent normal tissues of TNBC tumor has some level of TNBC phenotype such as CSC activity,15 which can explain the small level of binding of biotinylated-8 to the neighboring tissue of TNBC tumor observed in our TMA analysis. Collectively, these results provide strong evidence of the high binding specificity of TNBC ligand LC129-8. Next, we examined if the observed binding specificity of LC129-8 could lead to its specific anti-cancer activity. For all the functional assays, C-LC129-8 (hereafter C-8, Figure 1b), C-LC129-27 (hereafter C27, Figure S2a) and C-LC129-CON (hereafter C-CON, Figure S2b) were used. The cysteine was added to the C-terminus of each peptoid sequence for the concentration determination of the compounds using Ellman’s reagent.16 The MTT assay results showed that C-8 caused a significant reduction in TNBC cell viability, while being much less toxic to both MCF10A

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Figure 2. LC129-8 displays TNBC-specific anti-cancer activities. (a) WB analysis of activated caspases of MDA-MB231 cells treated with C-LC129-8 (C-8), C-LC129-27 (C-27) or C-LC129-CON (C-CON) for 48 hrs. (b) WB analysis of Bax, Bak, phosphorylated Bad, and total Bad of MDA-MB-231 cells after 48 hrs treatment. (c) Quantitation of soft agar colony formation of MDA-MB-231 cells. (d) Quantitative analysis of wound healing of the MDA-MB-231 cells at the indicate times after the wound was generated. (e-f) WB analysis of selected epithelial and mesenchymal markers in MDA-MB-231 cells (e) or BT549 cells (f) after 48 hrs treatment. (g) Quantitation of WB analysis of β-catenin nuclear/cytosolic levels of MDA-MB-231 cells after 24 hrs treatment.

and MCF-7 cells (Figure S5a). In contrast, non-TNBC ligand C-27 caused significant decrease in cell viability only in MCF-7, but not in TNBC cells (Figure S5b). To understand the underlying mode of action that cause the reduction in TNBC cell viability, we examined if C-8 induced apoptosis. It was found that C-8 increased the levels of cleaved caspase-8, -9 and -3 in TNBC cells compared to C-CON-treated or C-27treated cells (Figure 2a). Importantly, C-8 did not cause induction of caspases activation in MCF10A and MCF-7 cells, while C-27 was able to induce caspases activation of MCF-7 (Figure S6). It is important to note that, as shown in Figure S7, the cysteine of C-8 didn’t contribute or affect the activity of LC129-8 in activating caspases. Next, effects of C-8 on pro-apoptotic Bcl-2 family proteins expressions were examined. We have found that only C-8, but not C-27, induced significant upregulation of the pro-apoptotic Bcl-2 family proteins Bax and Bak in TNBC cells. Also, only C-8 led to dephosphorylation of Bad in TNBC cells (Figure 2b). Moreover, result from the soft-agar colony-forming assay showed that C-8 suppressed proliferation capabilities of only TNBC cells but not of MCF-7 cells (Figure 2c, Figure S8). In contrast, C-27 inhibited colony formation of MCF-7 but not of MDA-MB-231 cells (Figure S8). Collectively, these results suggested that LC129-8 displays anti-cancer activities in TNBC cells specifically, and the phenotypic cell-binding screen was able to afford ligands with high specificity in bio-

logical activity toward to cancer cells of a particular phenotype. TNBC has been suggested for its higher metastatic potential than the other subtypes of breast cancer.17 To evaluate potential anti-metastasis activity of LC129-8, we have performed a wound-healing assay on MDA-MB-231 cells and found that C-8 effectively inhibited MDA-MB-231 cell migration (Figure 2d). We examined if anti-metastatic activity of C-8 was due to its inhibition of EMT. EMT in tumor promotes cancer cell dissemination, migration and tumor invasion, leading to metastatic progression of cancer.18-20 Recent studies suggest targeting EMT can be a promising strategy in inhibiting TNBC metastasis.21-23 During EMT, it is observed the loss of epithelial cell marker such as E-cadherin, and the upregulation of the mesenchymal markers such as N-cadherin, and vimentin.18-20 Western blot analysis showed that C-8 induced downregulation of the mesenchymal markers by 20 - 33% in MDA-MB-231 cells, and by 50% in BT549 cells (Figure 2e, f). On the other hand, C-8 was able to induce increased expression of the epithelial marker E-cadherin in TNBC cells by at least 3.5 folds (Figure 2e, f). C-8 also led to a significant decrease in nuclear localization of β-catenin in TNBC cells (Figure 2g), suggesting that C-8 could attenuate mesenchymal phenotype and, at the same time, promote epithelial phenotype in TNBC cells by preventing the nuclear translocation of β-catenin. These experimental results strongly suggest that LC129-8 can inhibit cancer metastasis by reversing EMT.

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Next, in vivo study using mouse xenograft indicated that tumors from the C-CON-treated mice continuously grew, while C-8-treated mice didn’t show any significant tumor growth (Figure 3a). This result suggests that the C-8 can efficiently suppress TNBC tumor growth in vivo. To understand how tumor growth inhibition was achieved by C-8, we investigated if C-8 altered the phenotype of TNBC by characterizing the extracted xenograft tumor cells. Soft-agar colonyforming assay result showed that C-8-treated tumor cells displayed a significant reduction in the colonyforming capability, by approximately 50% of the CCON-treated tumor cells (Figure 3b). In addition, C-8treated tumor cells expressed more than 2.5-fold higher level of E-cadherin than the C-CON-treated tumor cells. Moreover, they expressed 50% or less levels of the mesenchymal markers, such as Ncadherin, vimentin and slug, than the C-CON-treated tumor cells (Figure S9a). C-8-treated tumor cells also showed 20% to 60% lower expressions of the CSC markers (c-Myc, KLF4 and Nanog) than the C-CONtreated tumor cells (Figure S9b). We also examined if these changes of TNBC phenotype can lead to attenuation of chemotherapy resistance of TNBC. We found that C-8-treated tumor

Figure 3. LC129-8 inhibits TNBC tumor growth in vivo and modulates TNBC phenotype. (a) Tumor growth of mice treated with C-8 or C-CON (n = 4 for each group). (b) Quantitation of soft-agar colony formation of the cells extracted from C-8 or C-CON-treated tumors. (c) Cell viability of the C-8 or C-CON- treated tumors cells with increasing doses of cisplatin. (d) WB analysis to evaluate the level of cleaved PARP of C-8 or C-CONtreated tumor cells upon treatment with cisplatin for 24 hrs.

C-CON-treated tumor (Figure 3c). Particularly, 10 µM cisplatin caused 40% more cell death in C-8-treated tumor cells when compared with C-CON-treated tumor cells. Additionally, 1 µM cisplatin was shown to induce 5.5-fold increase in PARP cleavage from C-8treated tumor cells (Figure 3d), suggesting C-8 treated tumor cells became more apoptotic. These findings demonstrated that prolonged exposure of TNBC tumors to LC129-8 could change the phenotype of TNBC, in terms of suppressed tumor growth, reduced mesenchymal and CSC properties, lowered capability of malignant transformation, as well as increased sensitivity towards chemotherapy. In summary, we have demonstrated a phenotypic cell-binding screen by which a therapeutic candidate specifically targeting TNBC over non-TNBC was isolated. Exclusive binding to TNBC by LC129-8 was validated not only in cancer cell lines but also in patientderived tumor tissues. Importantly, LC129-8 was found to display TNBC-specific anti-tumor activity in vitro and in vivo. We are currently in the process of characterizing the pharmacokinetic and pharmacodynamic properties of LC129-8. Preliminary study showed excellent stability of LC129-8 in serum (Figure S10); this was expected as LC129-8 is a peptoid which is known to be protease-resistant.24 The significance of this study is that the specific binder of TNBC was shown to display anti-TNBC activities including anti-proliferative activity. The elucidation of the exact mode of action of LC129-8 requires identification of cell surface target(s) of LC129-8, which is currently underway. Possible cell surface receptor targets of LC129-8 include Notch receptors. Previous studies suggested that activated Notch pathway plays essential roles in cell proliferation and tumor growth of triple-negative breast cancer25-28. Particularly, it has been proven that Notch-1 receptor is overexpressed in TNBC with high Ki-67 proliferation marker expression and poor prognosis.29-31 We are currently investigating whether LC129-8 interacts with the Notch-1 receptor, as well as the mechanism in which LC129-8 modulates Notch-1 activity in TNBC. We understand that phenotypic screens32 with functional readouts have been used to identify therapeutic candidates of cells of a particular phenotype without the knowledge of drug targets. However, our phenotypic cell-binding screen is advantageous as it can provide not only therapeutic candidates but also specific binders of cells of a particular phenotype, thus enables theranostic applications. We anticipate versatile applications of the phenotypic-cell binding screen to identify therapeutic candidates of various cancerrelated phenotypes.

cells were more sensitive to cisplatin than cells from

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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Experimental section, supporting figures (PDF)

AUTHOR INFORMATION Corresponding Author

*[email protected] ORCID

Jiyong Lee: 0000-0002-4455-3358 Notes The authors declare no competing financial interest.

REFERENCES (1) Bauer, K. R.; Brown, M.; Cress, R. D.; Parise, C. A.; Caggiano, V. Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype: a population-based study from the California cancer Registry. Cancer 2007, 109, 1721. (2) Rakha, E. A.; Elsheikh, S. E.; Aleskandarany, M. A.; Habashi, H. O.; Green, A. R.; Powe, D. G.; El-Sayed, M. E.; Benhasouna, A.; Brunet, J. S.; Akslen, L. A.; Evans, A. J.; Blamey, R.; Reis-Filho, J. S.; Foulkes, W. D.; Ellis, I. O. Triple-negative breast cancer: distinguishing between basal and nonbasal subtypes. Clin. Cancer Res. 2009, 15, 2302. (3) Guarneri, V.; Dieci, M. V.; Conte, P. Relapsed triple-negative breast cancer: challenges and treatment strategies. Drugs 2013, 73, 1257. (4) Andreopoulou, E.; Kelly, C. M.; McDaid, H. M. Therapeutic Advances and New Directions for Triple-Negative Breast Cancer. Breast Care (Basel) 2017, 12, 21. (5) Zhang, J. F.; Liu, J.; Wang, Y.; Zhang, B. Novel therapeutic strategies for patients with triple-negative breast cancer. Onco Targets Ther. 2016, 9, 6519. (6) Kalimutho, M.; Parsons, K.; Mittal, D.; López, J. A.; Srihari, S.; Khanna, K. K. Targeted Therapies for Triple-Negative Breast Cancer: Combating a Stubborn Disease. Trends in pharmacological sciences 2015, 36, 822. (7) Dai, X.; Li, T.; Bai, Z.; Yang, Y.; Liu, X.; Zhan, J.; Shi, B. Breast cancer intrinsic subtype classification, clinical use and future trends. American journal of cancer research 2015, 5, 2929. (8) Lehmann, B. D.; Bauer, J. A.; Chen, X.; Sanders, M. E.; Chakravarthy, A. B.; Shyr, Y.; Pietenpol, J. A. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. The Journal of clinical investigation 2011, 121, 2750. (9) Ma F, L. H.; Wang, H.; Shi, X.; Fan, Y.; Ding, X.; Lin, C.; Zhan, Q.; Qian, H.; Xu, B. Enriched CD44+/CD24− population drives the aggressive phenotypes presented in triple-negative breast cancer (TNBC). Cancer Letters 2014, 353, 153. (10) Corkery, B.; Crown, J.; Clynes, M.; O'Donovan, N. Epidermal growth factor receptor as a potential therapeutic target in triplenegative breast cancer. Ann. Oncol. 2009, 20, 862. (11) Liang, D. S.; Zhang, W. J.; Wang, A. T.; Su, H. T.; Zhong, H. J.; Qi, X. R. Treating metastatic triple negative breast cancer with CD44/neuropilin dual molecular targets of multifunctional nanoparticles. Biomaterials 2017, 137, 23. (12) Jin, G.; He, R.; Liu, Q.; Dong, Y.; Lin, M.; Li, W.; Xu, F. Theranostics of Triple-Negative Breast Cancer Based on Conjugated Polymer Nanoparticles. ACS Appl. Mater. Interfaces 2018, 10, 10634.

(13) Aina, O. H.; Liu, R.; Sutcliffe, J. L.; Marik, J.; Pan, C. X.; Lam, K. S. From combinatorial chemistry to cancer-targeting peptides. Mol. Pharm. 2007, 4, 631. (14) Udugamasooriya, D. G.; Dineen, S. P.; Brekken, R. A.; Kodadek, T. A peptoid "antibody surrogate" that antagonizes VEGF receptor 2 activity. Journal of the American Chemical Society 2008, 130, 5744. (15) Atkinson, R. L.; Yang, W. T.; Rosen, D. G.; Landis, M. D.; Wong, H.; Lewis, M. T.; Creighton, C. J.; Sexton, K. R.; Hilsenbeck, S. G.; Sahin, A. A.; Brewster, A. M.; Woodward, W. A.; Chang, J. C. Cancer stem cell markers are enriched in normal tissue adjacent to triple negative breast cancer and inversely correlated with DNA repair deficiency. Breast Cancer Res. 2013, 15, R77. (16) Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82, 70. (17) Lehmann, B. D.; Bauer, J. A.; Chen, X.; Sanders, M. E.; Chakravarthy, A. B.; Shyr, Y.; Pietenpol, J. A. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J. Clin. Invest. 2011, 121, 2750. (18) Guarino, M.; Rubino, B.; Ballabio, G. The role of epithelialmesenchymal transition in cancer pathology. Pathology 2007, 39, 305. (19) Schmalhofer, O.; Brabletz, S.; Brabletz, T. E-cadherin, betacatenin, and ZEB1 in malignant progression of cancer. Cancer Metastasis Rev. 2009, 28, 151. (20) Thiery, J. P.; Sleeman, J. P. Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. Cell Biol. 2006, 7, 131. (21) Granados-Principal, S.; Liu, Y.; Guevara, M. L.; Blanco, E.; Choi, D. S.; Qian, W.; Patel, T.; Rodriguez, A. A.; Cusimano, J.; Weiss, H. L.; Zhao, H.; Landis, M. D.; Dave, B.; Gross, S. S.; Chang, J. C. Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer. Breast Cancer Res. 2015, 17, 25. (22) Zhang, X.; Liu, X.; Luo, J.; Xiao, W.; Ye, X.; Chen, M.; Li, Y.; Zhang, G. J. Notch3 inhibits epithelial-mesenchymal transition by activating Kibra-mediated Hippo/YAP signaling in breast cancer epithelial cells. Oncogenesis 2016, 5, e269. (23) Wang, Y.; Xu, X.; Zhao, P.; Tong, B.; Wei, Z.; Dai, Y. Escin Ia suppresses the metastasis of triple-negative breast cancer by inhibiting epithelial-mesenchymal transition via down-regulating LOXL2 expression. Oncotarget 2016, 7, 23684. (24) Zuckermann, R. N.; Kodadek, T. Peptoids as potential therapeutics. Curr. Opin. Mol. Ther. 2009, 11, 299. (25) Guo, H.; Lu, Y.; Wang, J.; Liu, X.; Keller, E. T.; Liu, Q.; Zhou, Q.; Zhang, J. Targeting the Notch signaling pathway in cancer therapeutics. Nat. Rev. Mol. Cell Biol. 2014, 5, 473. (26) Han, J.; Hendzel, M. J.; Allalunis-Turner, J. Notch signaling as a therapeutic target for breast cancer treatment? Breast Cancer Res. 2011, 13, 210. (27) Yuan, X.; Wu, H.; Xu, H.; Xiong, H.; Chu, Q.; Yu, S.; Wu, G. S.; Wu, K. Notch signaling: an emerging therapeutic target for cancer treatment. Cancer Lett. 2015, 369, 20. (28) Yamaguchi, N.; Oyama, T.; Ito, E.; Satoh, H.; Azuma, S.; Hayashi, M.; Shimizu, K.; Honma, R.; Yanagisawa, Y.; Nishikawa, A.; Kawamura, M.; Imai, J.; Ohwada, S.; Tatsuta, K.; Inoue, J.; Semba, K.; Watanabe, S. NOTCH3 signaling pathway plays crucial roles in the proliferation of ErbB2-negative human breast cancer cells. Cancer Res. 2008, 68, 1881. (29) Zhu, H.; Bhaijee, F.; Ishaq, N.; Pepper, D. J.; Backus, K.; Brown, A. S.; Zhou, X.; Miele, L. Correlation of Notch1, pAKT and nuclear NF-kappaB expression in triple negative breast cancer. Am. J. Cancer Res. 2013, 3, 230. (30) Zhong, Y.; Shen, S.; Zhou, Y.; Mao, F.; Lin, Y.; Guan, J.; Xu, Y.; Zhang, S.; Liu, X.; Sun, Q. NOTCH1 is a poor prognostic factor for breast cancer and is associated with breast cancer stem cells. Onco Targets Ther. 2016, 9, 6865. (31) Qiu, M.; Peng, Q.; Jiang, I.; Carroll, C.; Han, G.; Rymer, I.; Lippincott, J.; Zachwieja, J.; Gajiwala, K.; Kraynov, E.; Thibault, S.;

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Stone, D.; Gao, Y.; Sofia, S.; Gallo, J.; Li, G.; Yang, J.; Li, K.; Wei, P. Specific inhibition of Notch1 signaling enhances the antitumor efficacy of chemotherapy in triple negative breast cancer through reduction of cancer stem cells. Cancer Lett. 2013, 328, 261. (32) Moffat, J. G.; Vincent, F.; Lee, J. A.; Eder, J.; Prunotto, M. Opportunities and challenges in phenotypic drug discovery: an industry perspective. Nat. Rev. Drug Discov. 2017, 16, 531.

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