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. Author manuscript; available in PMC: 2023 Dec 4.
Published in final edited form as: Mol Cancer Ther. 2020 Oct 21;19(12):2422–2431. doi: 10.1158/1535-7163.MCT-19-0949

ASR490, a Small Molecule, Overrides Aberrant Expression of Notch1 in Colorectal Cancer

Ashish Tyagi 1, Balaji Chandrasekaran 1, Venkatesh Kolluru 1, Becca V Baby 1, Cibi A Sripathi 1, Murali K Ankem 1, Srinivasa R Ramisetti 2, Venkat R Chirasani 2, Nikolay V Dokholyan 3, Arun K Sharma 2, Chendil Damodaran 1,*
PMCID: PMC10694926  NIHMSID: NIHMS1637235  PMID: 33087513

Abstract

Notch1 activation triggers significant oncogenic signaling that manifests as enhanced metastatic potential and tumorigenesis in colorectal cancer (CRC). Novel small molecule inhibitors, mainly plant-derived analogues, have low toxicity profiles and higher bioavailability. In this study, we have developed a small molecule, ASR490 by modifying structure of naturally occurring compound Withaferin A. ASR490 showed a growth inhibitory potential by downregulating Notch1 signaling in HCT116 and SW620 cell lines. Docking studies and thermal shift assays confirmed that ASR490 binds to Notch1 while no changes in Notch2 and Notch3 expression was seen in CRC cells. Notch1 governs epithelial-to-mesenchymal transition (EMT) signaling and is responsible for metastasis, which was abolished by ASR490 treatment. To further confirm the therapeutic potential of ASR490, we stably overexpressed Notch1 in HCT-116 cells and determined its inhibitory potential in transfected CRC (Notch1/HCT116) cells. ASR490 effectively prevented cell growth in both the vector (p=0.005) and Notch1 (p=0.05) transfectants. The downregulation of Notch1 signaling was evident, which corresponded with downregulation of mesenchymal markers including N-cadherin, Snail, and β-catenin and induction of E-cadherin in HCT-116 transfectants. Intraperitoneal administration of a 1% MTD dose of ASR490 (5 mg/kg) effectively suppressed the tumor growth in control (pCMV/HCT116) and Notch1/HCT116 in xenotransplanted mice. Additionally, downregulation of Notch1 and survival signaling in ASR-treated tumors confirmed the in vitro results. In conclusion, ASR490 appears to be a potent agent that can inhibit Notch1 signaling in CRC.

1. Introduction:

Hyper-activation of Notch1 plays a significant role in the pathogenesis of cancer [1]. Activation is triggered by binding of ligands to the receptor, which leads to protease (TACE or Kuzbanian proteases) driven sequential cleavages of the receptor followed by cleavage by γ-secretase. The cleaved Notch receptor intracellular domain (NICD) subsequently translocate to the nucleus, which induces the transcriptional activation of Notch target genes, such as HES1 [2]. Cleavage of NICD initiates a signaling cascade that has multiple interactive points with other oncogenic pathways [1, 3, 4]. Moreover, HES1 activation has been shown to promote CRC cell resistance to 5-Fu by inducing EMT [5]. Notch induction also activates several other oncogenic pathways and negatively affects pro-apoptotic pathways leading to activation of cell proliferation genes [6, 7].

In CRC, Notch1 signaling is a major pathway that governs cancer cell differentiation and proliferation [8]. Its dysregulation has been frequently associated with CRC pathogenesis, which is the second leading cause of cancer death in men and women.(CDC Colorectal stats 2019; fightcolorectalcancer.org 2019). Although recent advances in CRC treatment have resulted in dramatic reductions in CRC-related death [9],CRC-related morbidity in young adults and chemoresistance to existing therapies is a major challenge in curing patients with CRC [10].The CRC incidence rate in adults aged ≥50 years decreased by 32%, while these incidence rates increased by 22% among adults aged <50 years [11].

Although, screening at an early stage can significantly improve survival, most of the CRC patients are diagnosed at an advanced stage. Neoadjuvant therapy before surgery, which is followed by chemotherapy, are recommended for such patients. However, pharmacological therapy often is associated with toxic and harmful side effects and patients eventually develop chemoresistance [12–14]. Changes in cell signaling patterns, such as upregulation of expression or aberrant activation of several important genes such as anti-apoptotic factors (BCL-2 and BCL-XL [15]), survival signaling, and EMT signaling, have been shown to be the main causative factors of chemoresistance in CRC [5].

Mutations in Negative Regulatory Region (NRR) have been attributed to ligand independent activation of Notch1 and resulted in aggressive malignancies [16]. NRR is termed as an activation switch of Notch1 receptor [17]. Monoclonal antibodies (mAb) targeting NRR have shown promise by inhibiting Notch1 cleavage resulted in degradation of NICD [16, 18]. However, there is no report, to our knowledge, of compounds specifically binding to NRR and impacting Notch1 signaling. Increasing incidences of chemoresistance to existing therapies in advance CRC and importance of Notch1 signaling in maintenance of oncogenic phenotypes via uncontrolled proliferation, loss of apoptosis, and advancement to metastasis in CRC, makes it imperative to further broaden the current treatment paradigm by developing plant derived novel small molecules, which have low toxicity profile, can target Notch1 signaling and its aberrant activation and thus overcome these challenges.

We identified a small molecule, ASR490, using structure-activity relationship (SAR) studies focused on the Withaferin A analogs. ASR490 effectively inhibits CRC cell growth in both in vitro and in vivo models. Our results also suggest that ASR490 effectively suppressed Notch1 signaling, which resulted in inhibition of EMT in CRC. Targeting the multifaceted functions of Notch1 receptor, and several interlinked signaling pathways in CRC with a plant-derived potent small molecule presents a promising approach for colon cancer.

2. Materials and Methods

2.1. Synthesis of ASR490

ASR490 (Pyridine-2-carboxylic acid {17-[1-(5-hydroxymethyl-4-methyl-6-oxo-3,6-dihydro-2H-pyran-2-yl)-ethyl]-10,13-dimethyl-1-oxo-,4,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-20-oxa-cyclopropa[5,6]cyclopenta[a]phenanthren-4-yl}ester) was synthesized starting from Withaferin A (4β,5β,6β,22R)-4,27-Dihydroxy-5,6:22,26-diepoxyergosta-2,24-diene-1,26-dione) according to a synthetic strategy recently developed in our laboratory (Supplementary material Section1 and Supplementary Fig.1A) with modifications in earlier reported protocols [19, 20]. Briefly, to a stirred solution of Withaferin A (0.470 g, 1.0 mmol) and triethylamine (0.278 mL, 2.0 mmol) in CH2Cl2 (10.0 mL) at 0 °C under nitrogen atmosphere was added pyridine-2-carbonyl chloride hydrochloride (0.195 g, 1.10 mmol) and the resulting reaction mixture was stirred overnight at room temperature. After completion of the reaction as indicated by TLC, the reaction mixture was quenched with saturated NaHCO3 solution (5 mL). The organic layer was separated, followed with extraction of aqueous layer with CH2Cl2 (2 × 10 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford the crude ASR490 which was purified by silica gel column chromatography with eluent ethyl acetate: hexane (2:8) to afford the pure ASR490 (0.471 g, 82%) as a white solid The compound was characterized by NMR and MS and its purity (≥98%) was determined by HPLC. 1H NMR (600 MHz, CDCl3): δ 8.78 (d, J = 4.2 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.86 (td, J = 7.8, 1.2 Hz, 1H), 7.52−7.48 (m, 1H), 6.94 (dd, J = 6.0, 4.2 Hz, 1H), 6.22 (d, J = 10.2 Hz, 1H), 5.30−5.20 (m, 2H), 4.50−4.46 (m, 1H), 4.45−4.35 (m, 2H), 4.14 (q, J = 7.2 Hz, 1H), 3.78 (d, J = 6.0 Hz, 1H), 3.25 (1H, brs), 2.60−2.50 (m, 1H), 2.20−2.15 (m, 1H), 2.05 (2s, 6H), 2.00−1.98 (m, 1H), 1.90−1.85 (m, 1H), 1.70−1.65 (m, OH, 3H), 1.55−1.48 (m, 2H), 1.40 (s, 3H), 1.30−1.25 (m, 3H), 1.05−1.00 (m, 3H), 0.72 (s, 3H). ESI-MS m/z 576 (M+H)+.

2.2. Cell culture and supplies:

HCT116, SW-620, TCCSUP, UMUC3, HT1376, 5637, T24 and RT4 cells were purchased from ATCC (American Type Culture Collection; Manassas, VA, USA). HCT116, T24, RT4 were maintained in McCoy’s medium, TCCSUP, UMUC3, HT1376 in EMEM, SW620 in DMEM and 5637 in RPMI medium, respectively, and supplemented with 10% FBS and penicillin (100 units/mL) and streptomycin (100 units/mL; Millipore Sigma, St Louis, MI, USA) in the presence of 5% CO2 at 37°C. pCMV6-NOTCH1, vector pCMV6-Entry (NOTCH1 (NM_017617) Human ORF Clone; Origene) and NOTCH1 Human siRNA Oligo Duplex were obtained from Origene Technologies Inc. (Rockville, MD, USA). Lipofectamine 2000 reagent was used following the manufacturer’s (Cat# 11668019; ThermoFisher Scientific) instruction, transfection with overexpression vectors was performed with 500ng plasmid concentration, while the siRNA were used in 25nM concentration. Cells were allowed to be transfected for 48 hrs and later harvested or treated for further analysis. Neomycin (1 μg/mL) selection media was used to cultivate Notch1-overexpressing HCT116 clones (C1, C2, C3, C4, C5).

2.3. Cell proliferation and colony formation assay:

The growth inhibitory effect of ASR490 (reconstituted in 10mM DMSO) was determined by the MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Six biological replicates used for MTT assays and it was repeated twice for each experiment. Colon and bladder cancer cell lines were treated with varying concentrations of ASR490 (0–1.6 μM). . The anchorage-independent growth assay was performed and repeated in triplicate as described previously [21].

2.4. Binding Studies

The protein-ligand binding was first studied by cellular thermal shift assay (CETSA) by following previously described protocol [22]. Briefly, we treated the cells (3×106) with ASR490, incubated at different temperatures (38–55°C) to denature and precipitate proteins, performed cell lysis and centrifuged at 13000g for 10 min to collect the soluble fraction. Equal amount of cell lysate was used for ELISA with NRR antibody (Cat: NBP2–62557; Novus Biologicals). GloMelt Thermal Shift Protein Stability assay was performed as per the kit instructions (GloMelt™ Thermal Shift Protein Stability Kit; Biotium; Fermont, CA). Briefly, a qPCR reaction was setup with the purified NRR protein (Origene technologies: TP606288), the GloMelt fluorescent dye and ASR490 (10μg per reaction). A protein melt run profile was generated and Tm (melting temperature) was calculated using DNA melt curve software. To analyze protein melting, the Tm was considered at the lowest -dF/dT value (at the lowest point on the curve).

2.5. Molecular Docking studies

For molecular docking studies, the structure of NRR domain was downloaded with resolution 2 Å from the RCSB database (PDB ID: 3ETO) [23]. All bound crystal water molecules and ligands were removed prior to building missing residues through homology modeling using Modeller 9v15. Simultaneously, we built and optimized the structure of ASR490 using Marvin sketch workspace (arXiv.org). We relaxed the NRR structure using Chiron [24] and Gaia [25] for subsequent docking studies with ASR490 compound. To evaluate the extent of interaction between ASR490 and the NRR domain of Notch1 receptor, we performed molecular docking using MedusaDock [26]. H-bond interactions between Notch1-NRR domain and ASR490 compound as blue dotted lines. Notch1-NRR domain is shown as carton with α-helices in cyan, β-sheets in magenta, and loops in deep salmon color. ASR490 is shown in green licorice representation and water molecules mediating the interaction between NRR and ASR490 are shown in red spheres.

2.6. Flow cytometry analysis:

0.3X106 cells seeded in a 6 well plate and were cultured until 70–80% confluence was achieved. The cells were then treated with ASR490 for 24h. To quantify apoptosis, flow cytometry analysis of the Annexin V–FITC against Propidium Iodide (PI) assay was performed following a previously described protocol [27].. The Apoptosis detection kit was purchased from BD Pharminogen™, San Diego, CA, USA. All experiments were repeated in triplicate to achieve statistical relevance.

2.7. Cell invasion and migration assays:

The invasive capability of pCMV/HCT116 and Notch1/HCT116 was evaluated in Boyden chambers, as described in earlier studies [28]. HCT 116, SW-620, pCMV/HCT116, and Notch1/HCT116 cells were analyzed for migration capability with protocols already described in an earlier study [28]. All experiments were performed in triplicate to achieve statistical relevance.

2.8. Protein extraction and western blotting:

Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA) was used to extract total protein from pCMV/HCT116, C4, and C5 cells as well as bladder cancer cells were prepared with the according to the manufacturer’s instructions. Western blotting was performed using specific antibodies against Notch1 (Cat:3447S), Bcl-2 (Cat:2872S), E-cadherin (Cat:3195S), N-cadherin (Cat:13116S), Snail (Cat:3879S), β-catenin (Cat:8480S), NF-κB (p65), Bax (Cat:5023S), cleaved PARP (Cat:9541L) (Cell Signaling Technology; Danvers, MA, USA), and β-actin (Santa Cruz Biotechnologies, Dallas, TX, USA), HES1 (Genescript; Lot QC1851,Piscataway, NJ, USA). Actin presented in the images represent the loading control for one or more markers from same cell lysates. Chemiluminescence was used to detect the positive bands on the membrane.

2.9. Xenograft studies:

Six to eight week old BALB/c athymic nude mice (nu/nu) (Jackson Laboratory: Bar Harbor, ME, USA) were subcutaneously injected with pCMV/HCT116 and C4 (1×106 cells).. The monitoring and measurements were performed as described previously [21].

2.10. Immunohistochemistry (IHC):

The tumor samples from the pCMV/HCT116 and Notch1/HCT116 xenografts were subjected to IHC analysis as we previously described [28]. Primary antibodies against Ki67, Notch1, HES1, and p65 were used in this study.

Statistical analysis:

The experimental data is presented as the mean ± standard deviation (SD or SEM). Unpaired Student’s t-test was used to determine the significance of the differences between different test groups. The significant differences were established at p < 0.05. Prism 6 software purchased and licensed from GraphPad Software Inc, La Jolla, CA, USA was used to perform the statistical analyses

3. Results

3.1. ASR490 specifically inhibits Notch1-mediated survival of CRC cells

To examine the therapeutic potential of ASR490 (Fig. 1A). in CRC, we assessed the cell viability of ASR490-treated HCT116 and SW620 using the MTT assay. Cell viability was significantly reduced with 24-h (HCT 116, IC50: 750 nM; p=0.007 and SW-620, IC50:1.2 μM; p=0.0008) and 48-h (HCT 116, IC50: 600 nM; p=0.005 and SW-620, IC50: 850 nM; p=0.007) treatment (Fig. 1B, C). To determine the molecular mechanism by which ASR490 inhibits the growth of CRC cells (HCT116 and SW620), we treated the cell lines with ASR490 and performed immunoblot analysis. Significant downregulation in the expression of NICD and its downstream effector HES1 protein were observed in HCT116 and SW620 cells (Fig. 1D; Supplementary Fig. 1B). Interestingly, an apparent decline in Notch1 and HES1 mRNA expression was observed (Supplementary Fig. 1C), whereas, no change in Notch2 and Notch3 expressions were seen in ASR490-treated HCT116 cells (Supplementary Fig. 1D). To confirm that ASR490 inhibits the CRC cell growth through Notch1, we silenced Notch1 expression by siRNA in HCT116 cells (Supplementary Fig.2A), then treated with vehicle or ASR490. As seen in supplementary Fig.2B, ASR490 failed to inhibit the growth of Notch1 silenced HCT116 cells as compared to scrambled transfected HCT116 cells. Bladder Cancer cells have low basal level of Notch1, we treated these cells with ASR490, which failed inhibit their growth (Supplementary Fig. 2C and 2D). These two experiments suggest that Notch1 may be a target for ASR490.

Figure 1. ASR490 specifically inhibits Notch1-mediated survival of CRC cells.

Figure 1.

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(A) Structure of ASR490. (B & C) ASR490 or vehicle were used at indicated concentrations to treat HCT 116 and SW-620 cells for 24 h and 48 h followed by the MTT assay for cell viability. (D) Immunoblot analysis of cell lysates from HCT 116 and SW620 cells treated with the IC50 concentration of ASR490 or vehicle (DMSO) for 12 and 24 h. (E) Docking study with Medusa Dock was performed with NRR domain and ASR490, the blue dotted lines (H-bond), Notch1-NRR domain with α-helices (cyan), β-sheets (magenta), loops (salmon) and ASR490 (green licorice) are represented with water molecules (red spheres). (F) Protein run melt profile with temperature plotted against first derivative of fluorescence curve (–df/dt) and the lowest curve points taken at respective melting temperature (Tm) for NRR+ASR490 and NRR+ Vehicle samples. (G) The CETSA assay was performed on ASR490-treated HCT 116 cells at the indicated temperatures followed by ELISA with NRR antibody and temperature was plotted against the absorbance (450nm) changes. (H) Analysis of ASR490-treated cells for p65/Bcl-2 expression in a time-dependent manner. Data are presented as the mean ± standard deviation (SEM/SD) of three independent experiments. Statistical significance between vehicle and treatment at each concentration was calculated with the Student’s t-test. *p < 0.05, **p < 0.01 and ***p < 0.001

Next, to analyze whether ASR490 binds directly to Notch1, we performed molecular docking studies. The CASTp predictions confirmed binding sites of ASR490 in Negative Regulatory Region (NRR) of Notch1 (Fig. 1E). The catalytic pocket in NRR is lined by the residues: Lys-1462, Cys-1464, Asp-1479, Cys-1480, Leu-1482, Asn-1483, Ala-1708, Gly-1711, Leu-1713, Asn-1714, Ile-1715, Tyr-1717, Lys-1718, Ile-1719, and Glu-1720. The estimated binding energy between the NRR domain and ASR490 was −52.55 kcal/mol which signifies strong interaction between ASR490 and NRR domain. The residue-wise interaction analysis estimated three hydrogen-bond interactions between ASR490 and NRR residues Asn-1483, Glu-1673, and Gly-1664 mediated by water molecules (Fig. 1E). To further confirm the binding at protein level we performed Protein Thermal Shift differential scanning fluorimetry assay with purified NRR protein (Fig.1F). The results suggest that an increased stabilization of NRR protein in presence of ASR490 than with vehicle (DMSO). Further, traditional CETSA was performed and NRR specific antibody was used in ELISA as detection method. The absorbance profile (495 nm for TMB substrate) of ASR490-treated HCT116 cells confirmed that ASR490 binds directly to NRR (Fig. 1G). To further confirm that the inhibition of Notch1 activation alters the expression of key genes that regulate cancer cell survival signaling in CRC cells, we analyzed the effect of ASR490 treatment on pro-survival genes. As shown in Fig. 1H, ASR490 treatment significantly inhibited p65 and Bcl-2 expression in CRC cells.

3.2. Notch1 inhibition resulted in EMT downregulation in CRC cells

To examine whether Inhibition of Notch1 signaling facilitates induction of pro-apoptotic signaling, we performed apoptotic assays in ASR490 treated CRC cells. Induction of apoptosis in ASR490-treated HCT116 (19.9%, p=0.01; 24 h) as well as SW-620 (9.57%, p=0.011; 24 h) cells in FACS analysis showed significant apoptotic cell death (Fig. 2A, B). A time-dependent up-regulation of the pro-apoptotic markers Bax and cleaved PARP expression was observed in ASR490-treated HCT116 and SW620 cells (Fig. 2C, D). More importantly, inhibition of the migratory (25.18%, p=0.05; HCT116 and 32.36%, p=0.032; SW620) capability of CRC cells was observed in response to ASR490 treatment for 24 h (Fig. 2E, F). Additionally, the time-dependent increase in the E-cadherin (an epithelial marker) and a significant decrease in mesenchymal markers N-cadherin and β-catenin expression were observed in CRC cells treated with ASR490 for both 12 and 24 h (Fig. 2G, H).

Figure 2. Inhibition of Notch1-mediated oncogenic signaling in CRC cells.

Figure 2.

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(A& B) HCT116 as well as SW620 cells treated with IC50 concentration of ASR490 or vehicle, were stained with Annexin V-FITC and PI. Values, mean ± SEM. *P < 0.05; **P < 0.01 (Student t test). (C& D) Cell lysates from ASR490-treated HCT 116 and SW620 cells were analyzed for cleaved PARP and Bax expression. (E & F) Migration assay were performed for HCT 116 and SW620 cells that are treated with ASR490 and vehicle. Values, mean ± SEM. *P < 0.05; **P < 0.01 (Student t test). (G & H) Immunoblotting analysis of ASR490-treated and vehicle-treated HCT 116 and SW620 cell lysates for E-cadherin, N-cadherin, and β-catenin expression.

3.3. ASR490 overcame Notch1 overexpression and inhibited the growth of Notch1/HCT116 transfectants

To assess the proliferative attribute of Notch1 in CRC, we first generated Notch1 expressing stable HCT116 cell lines i.e. clones C1, C2, C3, C4, and C5 (Fig. 3A). C4 and C5 were used for further studies as they expressed higher Notch1 compared to other clones. pCMV/HCT116 and Notch1/HCT116 clones C4 and C5 were assessed for cellular growth,. Notch1 transfectants showed a significantly higher growth compared to the control (pCMV/HCT116) cells (C4: 42%, p=0.0226; and C5: 25.8%, p=0.0236; Fig. 3B). ASR490 treatment significantly inhibited cell growth in both C4 (IC50: 800 nM; p=0.0016) and C5 (IC50: 1.1 μM; p=0.0028) transfectants, showing the ability of ASR490 to override Notch1 mediated overgrowth of CRC cells (Fig. 3B). To understand the effect of Notch1 in increasing the tumorigenic capability of CRC cells, we performed a colony forming assay. The colony forming ability in Notch1 transfectants increased significantly (C4 −42%; p=0.0238 and C5 −32.4%; p=0.0238) compared to vector-transfected HCT116 cells. However, ASR490 treatment significantly reduced the colony forming ability of pCMV/HCT116 (22.3%; p=0.0169), C4 (38%; p=0.0406), and C5 (26.66%; p=0.0127) cells (Fig. 3C). Immunoblot and densitometry analysis of ASR490-treated transformants (C4 and C5) demonstrated inhibition of Notch1 and HES1 expression (Fig. 3D, E). Additionally, p65 and BCl2 expression (survival markers) was downregulated (Fig. 4A, B). Next, we observed increase in expression of pro-apoptotic genes such as cleaved-PARP and Bax (Fig. 4C) along with induction of apoptosis in ASR490-treated pCMV/HCT116 (25.8%, p=0.0165) as well as C4 (13.4%, p=0.0102) and C5 (13.2%, p = 0.0112) cells during FACS analysis (Fig. 4D).

Figure 3. ASR490 overcomes Notch1 overexpression and inhibits the growth of HCT/Notch1 transfectants.

Figure 3.

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(A) Western blot analysis of Notch1 basal expression in HCT116 (pCMV-transfected and Notch1-transfected) cells. (B) For assessment of cell viability with MTT assay, IC50 concentration of ASR490 or Vehicle (DMSO) was used to treat HCT116 stable transfectants C4 and C5 for 24h. One way ANOVA with multiple comparison test was used to calculate the statistical significance between different experimental groups. *p < 0.05 and **p < 0.01. (C) Colony-forming assay on pCMV/HCT116 (vector transfected) and HCT116 stable transfectants C4 and C5 treated with ASR490 or vehicle (DMSO) were performed. All experiments were performed in triplicate. One way ANOVA with multiple comparison test was used to calculate the statistical significance between different experimental groups. *p < 0.05 and **p < 0.01. (D) Immunoblot analysis of ASR490-treated pCMV/HCT, C4, and C5 cells for Notch1 and HES1 expression in a time-dependent manner (12 and 24h). (E) Densitometry analysis was performed with ImageJ software for the immunoblots. Values, mean ± SEM. *P < 0.05; **P < 0.01 (Student t test).

Figure 4. Suppression of Notch1-mediated survival and induction of apoptosis in Notch1 transfectants.

Figure 4.

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(A) pCMV/HCT116 and stable clones (C4 and C5) were treated with the indicated concentration of ASR490 or vehicle (DMSO) for the indicated time points and total protein lysates were analyzed for the pro-survival markers NF-κB (p65) and Bcl-2. (B) Densitometry analysis was performed with ImageJ software for the immunoblots. Values, mean ± SEM. Statistical significance between vehicle and treatment at each concentration was calculated with the Student’s t-test. *p < 0.05. (C) Total protein lysates from ASR490-treated pCMV/HCT 116, C4, and C5 cells were analyzed for expression of the pro-apoptotic markers cleaved PARP and Bax. (D) FACS analysis was performed (Annexin V-FITC and PI staining) in non-transfected and Notch1-overexpressing HCT116 transfectants that were treated with the IC50 concentration of ASR490 or vehicle (DMSO). Values, mean ± SEM. *P < 0.05; **P < 0.01 (Student t test).

3.4. ASR490 overcame Notch1-induced EMT and decreased tumorigenicity of CRC cells

Next, we determined whether Notch1 overexpression influences EMT signaling in Notch1/HCT116 cells. The invasive capability in C4 and C5 cells increased by 65.5% (p=0.0316) and 63.5% (p=0.0253), respectively (Fig. 5A). Similarly, we observed a 29% (p=0.036) and 28.68% (p=0.0474) increase in the migratory capability of C4 and C5 CRC cells, respectively, (Fig. 5B) compared to pCMV/HCT116 cells. To analyze whether ASR490 treatment can inhibit the enhanced migratory and invasive capability of C4 and C5 cells, both transfectants were treated with the respective IC50 doses of ASR490 for 24 h. A significant decline in the migratory potential of pCMV/HCT116 (25.11%; p=0.0073) and Notch1 transfectants (C4 −49.3%; p=0.0031 and C5 −44%; p=0.0130) and the invasive capacity of pCMV/HCT116 (40%; p=0.0047) and both C4 (60.75%; p=0.0305) and C5 cells (65.5%; p=0.0301) was observed (Fig. 5A, B). Next we analyzed ASR490-treated pCMV/HCT116, C4, and C5 cells for expression of genes that regulate EMT. EMT markers such as N-cadherin, and MMP-9 were significantly downregulated, whilethe epithelial marker E-cadherin expression upregulated, which are hallmarks of EMT (Fig. 5C, D). Notch1 plays an active role in the EMT process, and the results collectively indicate that ASR490 can overcome Notch1-induced EMT signaling in CRC cells.

Figure 5. ASR490 overcomes Notch1-induced EMT and decreases tumorigenicity of CRC cells.

Figure 5.

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(A) A trans-well invasion assay was performed for pCMV/HCT and Notch1 overexpressing HCT116 transfectants (C4 and C5) that were treated with either ASR490 or vehicle (DMSO). (B) Migration assays were performed in vector-transfected and Notch1-overexpressing cells (C4 and C5) that were treated with ASR490 or vehicle. Analysis was carried out with Image J software and values are presented as mean ± SEM. *P < 0.05; **P < 0.01 (Student t test). (C) Cell lysates from ASR490-treated and vehicle-treated pCMV/HCT, C4, and C5 cells were subjected to western blot analysis using E-cadherin, N-cadherin, Snail, β-catenin, MMP-9, and Snail antibodies. (D) Densitometry analysis was performed with ImageJ software for the immunoblots. Values plotted are mean ± SEM. Statistical significance between vehicle and treatment at each concentration was calculated by the Student’s t-test. *p < 0.05 and **p < 0.01

3.5. ASR490 overcomes Notch1 induced tumor growth in xenotransplanted mice

Earlier we reported that CRC xenografts with overexpression of AKT (Notch1 and AKT signaling are interlinked) are significantly aggressive compared to control-transfected CRC xenografts [21]. To determine anti-tumor potential of ASR490, pCMV/HCT116 and Notch1/HCT116 (clone 4; C4) cells were subcutaneously injected into nu/nu mice. The maximum tolerated dose (MTD) was checked for ASr490 in nu/nu mice and ASR490 was found to be safe till 500mg/kg dose. Nocth1/HCT116 tumors showed rapid and aggressive growth compared to pCMV/HCT116 tumors (Figure 6A). On the other hand, significant tumor growth inhibition was noted in both the ASR490 treated (5 mg/kg of mouse body weight for 4 weeks) pCMV/HCT116 and Notch1/HCT116 (C4) xenografts.

Figure 6. ASR490 reduces Notch1-mediated tumor burden in xenografts.

Figure 6.

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pCMV/HCT116 and C4 (1×106) were injected subcutaneously into separate flanks of the mice (n=6–8). ASR490 (5mg/kg) or 1% DMSO (Vehicle) (100μl volume) was administered intraperitoneally thrice a week. (A) Weekly thrice the tumor volume (mm3) was measured in both ASR490 and vehicle treated mice.. Mean tumor volumes ± SEM are shown. *p < 0.05 and **p < 0.01 by two-tailed Student t test. (B) IHC analysis of Ki-67, Notch1, HES1, and NFκB (p65) (C) Protein isolated from tissue samples taken from HCT/Notch1 xenografts was subjected to immunoblot analysis with Notch1 and HES1 antibodies.

We then assessed the survival (p65, Notch1, HES1) and proliferative (Ki67) in both pCMV/HCT116 and Notch1/HCT116 (C4) tumors. Notch1 and HES1 expression was higher in Notch1/HCT116 tumors compared to pCMV/HCT116 tumors (Fig. 6B). ASR490 treatment resulted in significant reduction of Ki67 expression (cellular proliferation) in pCMV/HCT116, as well as Notch1-overexpressing Notch1/HCT116 (C4) tumors (Fig. 6B). In addition, ASR490 treatment significantly reduced the expression of the pro-survival marker pAKT (ser473) and p65 in all tumors (Fig. 6B). Next, to assess the effect of ASR490 treatment on Notch1 signaling in xenografts, we analyzed Notch1 and HES1 mRNA and protein expression in pCMV/HCT116 and Notch1/HCT116 (C4) tissues. Consistent with the immunohistochemistry results, an inhibition in both Notch1 and HES1 transcripts and protein levels were observed (Fig. 6C).

4. Discussion

In the current study, we have demonstrated that aberrant Notch1 overexpression causes CRC cells to grow rapidly and demonstrate aggressive migratory behavior and our newly identified small molecule, ASR490, overrides aberrant overexpression of Notch1 in in vitro and in vivo CRC models to achieve antiproliferative and antitumorigenic effects.

Modern treatment concepts in CRC are multimodal and use interdisciplinary approaches, including the use of adjuvant, neo-adjuvant chemotherapy, radiotherapy, and immunotherapy, are followed based on the CRC stage and localization [29]. However, it is well understood that the need for optimization of adjuvant therapies [30] and increasing instances of resistance in neo-adjuvant therapies [31] warrant identification of new therapeutic targets and support the search for new compounds with low toxicity profiles and better bioavailability.

The recent progress in determination of the crystal structure of the NRR has improved the understanding of mechanisms that are responsible the self-inhibitory effects of HD domain on the processing and activation of NOTCH receptors [32]. Recently, mAbs have been used to target NRR region in-order to stabilize the region and prevent ligand-independent activation and wild type Notch1 activation and thus decrease in NICD expression [16, 18]. In our results, molecular docking studies suggest ASR490 binds to NRR region of Notch1. Further, the CETSA and GloMelt protein thermal shift assay performed with NRR specific antibody of Notch1 on the purified NRR protein, and those results further confirmed that ASR binds to NRR of Notch1 and downregulated Notch1 expression. Similarly, Notch3 antibodies against NRR domain have shown to inhibit expressions of NICD and HES1, whereas the anti-LBD antibody failed to achieve that [18]. It is possible that ASR490 may elicit the similar response as anti-NRR antibodies although the exact mechanism needs to be elucidated in detail. In addition to downregulation of NICD expression, we have also seen inhibition of Notch1 gene expression. However, the exact mechanism by which ASR490 inhibits Notch1 gene expression is yet to be elucidated.

Notch1 activation is associated with early development of cancer [33] and activation of its downstream events such as overexpression of HES1 has been linked with CRC progression [34] and metastasis [35]. Silencing Notch1 activity through lentiviral-encoding Notch-1-siRNA and Notch1 inhibitors such as DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) have demonstrated capability to induce apoptosis in CRC cells [36], proving that Notch1 can be an effective target for CRC management. However, the current landscape of inhibitors, particularly gamma secretase inhibitors (GSIs) such as LY-411,575 or DAPT [37] can have unintended biological implications because of broad substrate profile of gamma secretase [38, 39]. Natural compounds such as Butein [40] and more recently compounds isolated from Nerium indicum [41] have been reported as inhibitors of Notch1. Keeping in mind the low toxicity profiles of compounds derived from natural sources and high bioavailability [42], results from our study showing the detrimental effect on Notch1 signaling by ASR490 derived from a natural compound are encouraging.

Notch1-mediated survival has been shown to be a primary driver of cell proliferation and tumor recurrence in vivo [43]. Moreover, its aberrant activation has been found to be responsible for uncontrolled cellular growth in several cancer types [44]. Inhibition of its expression and downstream signaling has resulted in induction of apoptosis and thus growth arrest in HT29 cells [36]. A tripeptide of GSIs category inhibited the proliferation of MDA-MB231 cells [45], whereas natural compounds such as genistein [46] induce apoptosis in cancer cells by downregulating survival signaling, particularly, NF-κB expression. Similar alteration in survival as well as apoptotic signaling was seen in ASR490-treated CRC cells in our study. The Notch1 overexpressing transfectants mimicking aberrant overexpression conditions also showed downregulation of pro-apoptotic and pro-survival markers, indicating that uncontrolled growth of CRC cells in the case of Notch1 activation can be managed by ASR490 treatment.

Notch1 signaling is also recognized as a major regulator of EMT in several cancer types [47–49] including colon cancer [50]. Activation of Notch1 signaling accelerates EMT by positively regulating Snail, a slug family protein, and repressing E-cadherin function. This in turn affects the progression of tumors in cancer cells [51]. Additionally, elevated HES1 expression has been correlated with several neoplastic conditions (15–18). Its interaction with multiple signaling pathways has been attributed to its contribution towards promotion of cell metastasis by evading tumor cell differentiation. Alleviation of Notch1-induced EMT may well be a direct result of the inhibition of Notch1/HES1/NFκB-p65 signaling. The ability of ASR490 to overcome Notch1 signaling and inhibit tumorigenic capacity was shown in our preclinical models of CRC. Our studies suggest that ASR490 is safe up to a dose of 500 mg/kg, which is 100 times more than the dose used in our efficacy studies indicating a high therapeutic index.

In summary, our results suggest that ASR490 a potent small molecule overcome Notch1-mediated pro-survival signaling and EMT, which resulted in growth inhibition in preclinical models of CRC. Additional studies may require optimizing the therapeutic efficiency of ASR490 that might lead to clinical settings.

Supplementary Material

1

Acknowledgement:

We acknowledge support from the NIH/NCI-1R01CA185972 to CD, 1R35 GM134864 to NVD and the Passan Foundation. The current projectwas also supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1 TR002014. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Footnotes

Disclosure Statement: The authors declare no conflict of interest.

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Supplementary Materials

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NIHMS1637235-supplement-1.pdf (512KB, pdf)

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