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Notch pathway inhibition using DAPT, a γ‐secretase inhibitor (GSI), enhances the antitumor effect of cisplatin in resistant osteosarcoma

Overcoming platinum drug resistance represents a major clinical challenge in osteosarcoma (OS) treatment. The high rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug‐resistant cancer stem cells (CSCs). Notch signaling is implicated in regulating CSCs and tumor resistance to platinum. Thus, we attempt to investigate whether inhibiting of Notch pathway could sensitize cisplatin (CDDP) to CDDP‐resistant OS cells and the underlying molecular mechanisms. OS cell lines resistant to CDDP were treated with DAPT, CDDP or combination, we present evidences that DAPT enhances the cytotoxic effect of CDDP in resistant OS by inhibiting proliferation, resulting in G0/G1 cell‐cycle arrest, inducing apoptosis, and reducing motility. In addition, DAPT targeting depletes OS stem cells (OSCs), thus increasing tumor sensitivity to platinum, which indicating that a dual combination targeting both OSCs and the bulk of tumor cells are needed for tumor eradication. We also found that the combination of CDDP and DAPT exhibit additive suppression on phosphorylated AKT and ERK, contributing to the anti‐cancer effects. In animal model, this combination therapy inhibits the growth and metastasis of CDDP resistant tumor xenografts in nude mice to a greater extent than treatment with either reagent alone. Based on these results, we conclude that CDDP plus DAPT was able to sensitize CDDP‐resistant human OS cells to CDDP by downregulation of Notch signaling. CDDP and DAPT combination treatment may be effective and promising for advanced OS.

Overcoming platinum drug resistance represents a major clinical challenge in osteosarcoma (OS) treatment. The high rates and patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer stem cells (CSCs). Notch signaling is implicated in regulating CSCs and tumor resistance to platinum. Thus, we attempt to investigate whether inhibiting of Notch pathway could sensitize cisplatin (CDDP) to CDDP-resistant OS cells and the underlying molecular mechanisms. OS cell lines resistant to CDDP were treated with DAPT, CDDP or combination, we present evidences that DAPT enhances the cytotoxic effect of CDDP in resistant OS by inhibiting proliferation, resulting in G0/G1 cell-cycle arrest, inducing apoptosis, and reducing motility. In addition, DAPT targeting depletes OS stem cells (OSCs), thus increasing tumor sensitivity to platinum, which indicating that a dual combination targeting both OSCs and the bulk of tumor cells are needed for tumor eradication. We also found that the combination of CDDP and DAPT exhibit additive suppression on phosphorylated AKT and ERK, contributing to the anticancer effects. In animal model, this combination therapy inhibits the growth and metastasis of CDDP resistant tumor xenografts in nude mice to a greater extent than treatment with either reagent alone. Based on these results, we conclude that CDDP plus DAPT was able to sensitize CDDP-resistant human OS cells to CDDP by downregulation of Notch signaling. CDDP and DAPT combination treatment may be effective and promising for advanced OS.

| INTRODUCTION
Osteogenic sarcoma, also referred to osteosarcoma (OS), is the most common primary malignant bone tumor. OS affects patients of all ages but shows a substantially higher incidence in children and early adulthood, which is the leading cause of disabling for childhood and adolescence, platinum compounds-based therapies are the current global standard for neoadjuvant chemotherapy. 1,2 The platinum-based anticancer drugs, including cisplatin (CDDP) (Supplementary Figure S1A), carboplatin and oxaliplatin, are currently among the most potent and widely used chemotherapeutic agents in clinical. They are also used for treating a variety of soild cancers, such as breast, ovarian, cervical, testicular, colorectal, bladder, non-small-cell lung, and head and neck cancers. 3,4 As the leading anticancer drug, CDDP has been used for nearly four decades in standard chemotherapy regimens. 2 The effect of platinum-based anticancer chemotherapy is positively with dose, but high doses of CDDP are accompanied by severe side effects as manifested by renal impairment, ototoxicity, neurotoxicity, and vomiting on the patients during the treatment. 5 In addition, prolonged usage of CDDP in chemotherapy also induces drug resistance from intrinsic and acquired via multiple mechanisms. 6 Platinum resistance is the single most important factor after stage in determining prognosis. Development of CDDP resistance is often associated with multidrug resistant phenotype, including cancer stem cells (CSCs) and disorder of signaling pathway. 7,8 It has been postulated that within a tumor, a minor subpopulation of cells, named CSCs, drive the self-renewal and differentiation that account for the initiation, metastasis, proliferation, therapeutic resistance, and recurrence of cancer. Recently, studies have also presented that CSCs may be involved in the mechanisms of multidrug resistance. 9,10 OS stem cells (OSCs) can also evade senescence and apoptosis through the Notch pathway to resist toxicity of chemotherapy. 11 Notch signaling is one of the most important signaling cascades involved in drug resistance in tumor cells. 12 A previous study demonstrated that activation of the Notch signaling was linked to chemoresistance of urothelial carcinoma to CDDP 13

and inhibition of
Notch signaling with gamma-secretase inhibitors (GSIs) could target the leukemia-initiating cells, sensitize acute myeloid leukemia to chemotherapy, and be synergistic with some antineoplastic agents. 14 Notch genes encode transmembrane receptors that are highly conserved from invertebrates to mammals. These receptors interact with ligands expressed by adjacent cells to regulate cell fate specification, proliferation, differentiation, and survival. 15 The Notch system in vertebrates is comprised of four receptors (Notch1-4) and at least five ligands from the families Delta/Serrate/LAG-2 (DSL): Deltalike (Dll)-1, Dll-3, Dll-4, Jagged-1, and Jagged-2. 15 In ovarian cancer patients who received CDDP treatment, the activity of Notch signaling in tumor tissue correlates with drug resistance and poor prognosis. 16 Also, in a mouse model, the actived Notch pathway promotes acquired resistance to CDDP in serially passaged OS xenografts. 8 Similar drug resistance to Doxorubicin, Methotrexate, and Cyclophosphamide were reported in OS cells and lymphoblastic leukemia cells, both due to intracellular Notch signaling. 17,18 Additionally, treating mice with a Notch inhibitor restores CDDP sensitivity. 8 Most interestingly, other groups found that inhibition of Notch signaling result in downregulation of phosphor-AKT (p-AKT) and phosphor-ERK (p-ERK) expression, 19 this indicates that Notch signaling occurs upstream of AKT and ERK signaling, and Notch may positively regulate p-AKT and p-ERK expression directly or indirectly.
The undesirable properties of the CDDP have triggered intense research interest to design a new pattern for drug application, such as combination therapy. In this study, we sought to identify therapeutic agents to enhance the sensitivity of CDDP in resistant OS. Here, we reported that the activity of CDDP can be pharmacologically enhanced by Figure S1B) and 143B were established by serial desensitization of these three cell lines for 9 months and partly according previous studies, 8,12,20 and these resistant OS cell lines are named U2OS/R, MG63/R, and 143B/R , respectively. In parallel, parental U2OS, MG-63 and 143B cells were exposed to DMSO (vehicle solution) in the same dose-escalation manner. Generation of parental cells and resistant OS cells and growth conditions were described previously. 8

| Cell viability
Cell viability was assessed as described previously. 8

| Synergy analysis
The synergistic effect of CDDP and DAPT was determined based on the combination index (CI) using CalcuSyn, version 2.1 (Biosoft®). The CI indicated synergism at less than 1.0, antagonism at greater than 1.0, and an additive effect at 1.0. 21 2.4 | Reverse transcription quantitative real-time PCR Quantitative real-time polymerase chain reaction (qRT-RCR) was done according standard techniques, as described previously. 22 The gene specific primers used are listed in Supplementary Table S1.

| Western-blot analysis
For western blot analysis, proteins were extracted with Protein Lysis Buffer (Roche, Rotkreuz, Switzerland). And detail protocol for western blotting is described previously 8 and manufacturer's instructions.

| Animal experiment
To determine the tumorigenicity of resistant model in vivo, 4-week-old male BALB/c-nu/nu nude mice were obtained from Center for Animal Experiment of Wuhan University and housed in laminar flow cabinets under specific pathogen-free conditions with food and water ad libitum.

| Statistics
Statistical analyses were performed using the SPSS 16.0 statistical software package. P value was calculated using chi-square in contingency table. All data are expressed as the mean ± SD of at least three independent experiments. The Student's t-test was used to compare the means of two groups. Where more than three means were compared, one-way ANOVA followed by multiple comparisons among the means was used. Enhanced expression of NICD1 in OS versus ANTTs was determined by the Mann-Whitney U Test. Spearman's correlation tests were used to analyze the association between Hes1 and Oct4 mRNA expression in OS tissues. The Kaplan-Meier method was used to calculate the survival curve, and Log-Rank test to determine statistical significance. Statistical significance is indicated by *, where P < 0.05, **, where P < 0.01, and ***, where P < 0.001.   23 In the present study, we investigated NICD1 (Notch1 intracellular domain, NICD1) expression in OS and the adjacent non-tumor tissues (ANTTs) using immunohistochemistry (IHC). We detected NICD1 expression which localized to the cytoplasm and nucleus in a total of 43 OS and 36 normal specimens. Figures 1A and 1B and Table 1 showed very low NICD1 expression in the ANTTs, but higher expression in the OS tissues. Intriguingly, NICD1 was gradually upregulation in the ANTTs (19.44%) and in OS chemosensitivity (72.41%) and chemoresistance (100.00%), and the strong positive rates of NICD1 also followed in that order ( Figure 1B and Table 1 Figures 1C and 1D). The mRNA levels of Notch target genes (Hes1, Hes5, and HeyL) also elevated in tumor samples compared with ANTTs ( Figure 1E). It is noteworthy that the whether NICD1 protein expression levels or Notch downstream target genes mRNA transcription levels exhibit features that their were higher in chemotherapyresistant group than in chemotherapy-sensitive group ( Figure 1A-E).

Additional information is described in Supplementary Materials
These results indicate that NICD1 is upregulated in OS tissues and plays an important role in OS tumorigenesis and chemoresistance. In view of the fact that tumor resistance to chemotherapy is closely related to stem cell-like properties, 25,26 we examined the expression of stem cell related gene Oct4 in normal tissues, drug-sensitive, and drugresistant OS tissues. qRT-PCR analysis showed a gradually increasing mRNA levels of Oct4 in ANTTs, chemosensitive, and chemoresistant OS tissues. And the difference was statistically significant ( Figure 1F).
Spearman's correlation analysis disclosed a positive correlation between Hes1 expression and that of Oct4 ( Figure 1G). Collectively, the Notch signaling pathway was activated in resistant osteosarcoma and the characteristics of stem cell-like properties were enhanced, and there was a positive correlation between them, it hints that combination of CDDP and Notch inhibitor may have a better therapeutic effect in resistant OS, so we performed a further study in vitro and in vivo.

| Anti-proliferative activity of CDDP and DAPT in resistant OS cells
Initially, to study the phenomenon of drug-resistance, relapse and metastasis following CDDP therapy, we generated in vitro chemo-  Figure S2A and Table S2). The expression of MDR1 (multi-drug resistance gene1) and its corresponding expression protein P-gp (P-glycoprotein) in resistant OS were significantly increased, which fully demonstrated the generation of drug resistance. 24 The resistance index of these three resistant OS cell lines increased by 2.56-, 2.03-, and 2.26-folds, respectively, according to the 24 h IC50 (half-inhibitory concentration) ( Figure 2C and Supplementary Figure S2A and Table S2).

| DAPT synergistically increased CDDP-mediated cytotoxicity in resistant OS
The treatment of OS with CDDP is linked with its concentrationlimiting toxicity. 25 Therefore, we aimed to improve the cytotoxic  Table S4).

| Inhibition of Notch signaling pathway decreases the CSCs phenotype in resistant OS cells
We have confirmed that CDDP-resistant OS cells could enrich CSCs previously. 8 And in this study we also showed that the expression of

| CDDP combined with DAPT inhibits Notch signaling, p-AKT, and p-ERK activation
As the combination of CDDP and DAPT showed the greater inhibition of cell proliferation, induction apoptosis and reduction mobility as synthetic way, insight was gained with regard to the mechanisms.   Figure 5L).
We also determine whether AKT and ERK activities were affected by combination treatment. As shown in Figure Figure 6E. In addition, tumor metastasis were found in the lungs according to the HE staining sections ( Figure 6F), and we found that combined CDDP and DAPT compared to the vehicle and either agent alone, regardless of the number or volume of metastases were significantly less or smaller (Figures 6F and 6G).
In addition, apoptosis related mRNA and protein levels were detected from xenografts mice of tumor sections. We tested the expressions of Bax, Bcl-2, Caspase-3, and Caspase-9, respectively.
qRT-PCR analysis demonstrated alterations in the levels of expression of Bax (proapoptotic) and Bcl-2 (antiapoptotic), resulting in a significant increase in Bax after treatment of the resistant xenografts with CDDP plus DAPT, and the Bcl-2 shown opposite result ( Figure 6H). The relative mRNA expression of Caspase-3 and Caspase-9 of mice tumor tissues were highest in CDDP + DAPT group followed by CDDP alone, DAPT alone, and vehicle in that order ( Figure 6H). Subsequently, Western blotting were performed and the results were similar to qRT-PCR as shown in Figure 6I. We also examined expression levels of NICD1, p-AKT, p-ERK, and TUNEL in sectioned mice tumors after CDDP and DAPT alone or in combination administration ( Figure 6K). TUNEL expression significantly increased in combination treatment, whereas NICD1 expression decreased by ≈45% ( Figure 6L). In addition, combination treatment resulted in significant downregulation of p-AKT and p-ERK compared to the control group ( Figures 6K and 6L). However, both the expression p-AKT and p-ERK decreased in the CDDP treatment group, which indicated that CDDP could inhibit the activities of PI3K/AKT and MAPK signaling pathway ( Figures 6K and 6L).
To further confirm the results mentioned above, Notch, PI3K/ AKT, and MAPK signaling pathway in resistant xenografts mice tumor tissues were analysis. Consistent with the in vitro and above data, there was a reduction of NICD1, p-AKT, and p-ERK proteina levels after combined treatment compared with the vehicle (Figures 6M and   6N). Thus, all these results indicated that CDDP in combination with DAPT treatment could markedly inhibit 143B chemoresistant xenograft growth in vivo and improves nude mice survival.

| DISCUSSION
Chemotherapy resistance of OS is a difficult problem in clinical. Herein we demonstrate that Notch signaling plays a key role for OS stem cell maintain and underlies mechanism for resistance to chemotherapy.
We present evidence of human specimen that Notch signaling is much greater in the chemoresistance OS and drives a CSCs phenotype. We DAI ET AL. | 13 demonstrate that Notch pathway is critical for these OSCs and thus targeting the Notch signaling pathway may be an effective therapeutic strategy.
Defects in apoptosis are common phenomena in many types of cancer as well as critical steps in tumorigenesis and resistance to therapy. Thus, traditional cancer therapy mainly targets the cell apoptosis machinery or enhances apoptosis. 30 CDDP is a platinum based, alkylating-like drug that crosslinks DNA to induce apoptosis. It is used to treat many types of solid cancer, such as ovary, testicular, bladder, lung, head and neck, thoracic, and colorectal cancers. 3,4 Although CDDP has potent antitumor effects in various cancer models, it is limited in clinical practice due to its toxicities and intrinsic or acquired drug resistance. 25,31 Many research teams have attempted to find ways to improve its efficacy and reduce its toxicity, such as changing its structure or assessing combination therapy with other drugs. 4 Researches have shown that combination therapy based on the synergistic effect between drugs owning multiple drug targets has been used to overcome drug resistance. 16,19,32,33 Recently many research groups have reported synergistic anticancer effects of treatment with a combination of a drug and a signaling pathway inhibitor in vitro and in vivo cancer models. 34 agents such as CDDP, oxaliplatin, docetaxel, and 5-fluorouracil. 16,19,29 However, to the best of our knowledge, this is the first study to show that  Bax and inhibition Bcl-2, and suppressed AKT phosphorylation, leading to improve chemotherapeutic agents responsiveness. In the present study, the expression of cleaved caspase-3, 8 and 9, cleaved PARP and cytochrome c were increased and p-AKT were decreased by CDDP and DAPT treatment, especially in combination of these two drugs.
ERK is a major effect target of the mitogen-activated protein kinase (MAPK) pathway, and the MEK/ERK signaling pathway plays a critical role in proliferation, cell cycle, apoptosis and differentiation, and is an important downstream pathway of angiogenesis. 46 Previous data obtained in in vitro cultured cells showed that the Notch pathway upregulates the levels of ERK phosphorylation and GSI treatment could decrease p-ERK which presented therapeutic potential of targeting γ-secretase in non-small cell lung carcinoma. 28 And p-ERK also can be suppresses by CDDP alone and then inhibit cell proliferation. 45 In the present study, it was also found that p-ERK protein expression was downregulated by treatment with DAPT and CDDP alone or in combination.
So, it could be that CDDP plus DAPT inhibited the Notch, PI3K/ AKT, and MEK/ERK pathway, which induced the cell apoptosis and cell cycle arrest and inhibited cellular motility, and then leaded to tumor arrest ( Figure 7A). are involved in the maintenance of stem cells. 22,48 . Notch-activated mutation acted as a second hit in a p53-loss-driven OS model. 49 Mu et al 50 noted that Notch signaling may influence OSCs via suppression of ALDH and inhibited the Notch signaling pathway could reduce the OSCs properties. In this study, we also found that DAPT, Notch signaling pathway specific inhibitor, could reduce the number and volume of primary and secondary sphere assay. In addition, we used recombinant lentiviral specific blocking Notch1 signaling pathway, compared with the control group, it was found that: (1) the stem cell related gene expression was significantly decreased; and (2) the ability

FIGURE 7
Schematic view depicting mechanisms of action of combination CDDP and DAPT. A, Signaling pathways for anti-tumor effects by CDDP and/or DAPT. The combination of CDDP and DAPT induced the cellular apoptosis and G1 phase arrest through pAKT-Bax/Bcl-2, pAKT-p21-Cyclin E1/CDK2, and pERK-c-Myc pathways. Meanwhile, the combination decreased VEGF and MMPs to inhibit cellular migration. B, Diagram illustrating that CDDP does not target Notch+ chemotherapy resistance or OSC cells, but DAPT could partially eliminated Notch+ chemotherapy resistance or OSC cells, so as to play a synergistic anticancer effect with CDDP and emphase the need to target residual drugresistant or OSC cells to eliminate all cancer cells to form sarcospheres decreased signifivantly. Furthermore, we found that activation of the Notch signaling pathway is involved in osteosarcoma resistance, whereas Notch signaling pathway activity is positively related to stem cell-like phenotype (Figure 1). Taken together, all these results indicate that blocking the Notch pathway can be targeted to kill OSCs, therefore reducing the resistance of osteosarcoma. So the combination of CDDP and DAPT show a surprising therapeutic effect, the mechanism may be that CDDP/ DAPT combination is effectively eliminates both OCSs and the bulk of tumor cells, indicating that a dual combination targeting both populations is needed for tumor eradication ( Figure 7B)