Delanzomib, a novel proteasome inhibitor, sensitizes breast cancer cells to doxorubicin-induced apoptosis.

BACKGROUND: Delanzomib, a novel proteasome inhibitor, has demonstrated promising efficacy and antitumor ability in human multiple myeloma cell lines and patient-derived cells. However, the potential therapeutic effects of delanzomib on breast cancer remain unknown. In this study, we show that delanzomib has antitumor effects and synergizes with doxorubicin (Dox) in human breast cancer cell lines.
METHODS: Cell proliferation assay and flow cytometry were used to evaluate cell viability and apoptosis in eight human breast cancer cell lines after treatment with delanzomib or Dox. Essential molecules of the p53, MAPK, and apoptosis signaling pathways were analyzed by Western blotting.
RESULTS: Delanzomib induced cell death and demonstrated synergism with Dox in all tested breast cancer cell lines. In addition, delanzomib enhanced the Dox-induced phosphorylation of p38/JNK and the expression of transcriptional target proteins of p53, such as p21, p27, NOXA, and PUMA.
CONCLUSION: The combined regimen of the proteasome inhibitor delanzomib with Dox chemotherapy may become an effective strategy for breast cancer therapy.

Introduction

Breast cancer is the most common cancer among women, accounting for 30% of estimated new cancer cases and 14% of cancer related‐deaths among women in the United States (US).1 Currently, standard therapy for breast cancer, according to the 2018 National Comprehensive Cancer Network guidelines, involves the administration of a doxorubicin (Dox)‐containing regimen. Dox is a cytotoxic drug that can induce DNA damage and trigger apoptosis in tumor cells. However, Dox is toxic to many major organs, including the heart, causing treatment to be dose‐limited.2 Thus, targeted therapeutic strategies that enhance the effects of chemotherapy are highly desirable and are actively being tested.

The ubiquitin proteasome pathway (UPP) is a comprehensive and complex protein degradation pathway. In cancer, the UPP plays an essential role in intracellular protein degradation and contributes to the regulation of fundamental cellular events, such as apoptosis, cell‐cycle progression, and DNA repair.3, 4, 5 Deregulation of the UPP results in the downregulation of tumor suppressors, including p53 and p27, and the upregulation of oncogenic proteins, such as NF‐kB. Additionally, these processes contribute to oncogenesis.6, 7, 8, 9 Cancer cells are also more likely to undergo apoptosis after proteasome inhibition when compared to normal cells.8, 9, 10 Preliminary studies have shown that proteasome inhibitors (PIs) can reverse abnormal proliferation and apoptosis in tumor cells. PIs can also stabilize p53 and inhibit the transcriptional activity of the NF‐kB pathway.11, 12, 13

Since the late 1990s, clinical trials of PIs have been carried out on a variety of malignant tumors. The Food and Drug Administration has approved the use of PIs for treating multiple myeloma (MM) and mantle cell lymphoma.14 Although first and second‐generation PIs have significantly improved the prognosis of MM and mantle cell lymphoma,9, 15, 16, 17 disease relapse and acquired treatment resistance remain issues.18 In addition, despite promising results from preclinical studies, sufficient clinical evidence has not yet been obtained for the use of PIs in solid tumors.14, 19 Therefore, optimization of therapeutic regimens and novel PIs may expand the application of PIs in solid tumors. Delanzomib, a novel and reversible P2 threonine boronic acid PI can trigger apoptosis in human MM cell lines and patient‐derived cells. Meanwhile, delanzomib is well tolerated and shows antitumor effects and survival benefits in xenograft and systemic models of human MM.20 Recently, a preclinical study showed that delanzomib could sensitize cervical cancer cells to Dox‐induced apoptosis.21

The potential therapeutic effects of delanzomib on breast cancer remain unknown. We tested a panel of breast cancer cell lines representing the ER/PR+/−, HER2+, and triple negative subtypes to examine the antitumor effects of delanzomib alone and its synergistic effect on Dox‐induced apoptosis in breast cancer (Fig 1).22

Figure 1

Delanzomib shows cytotoxic effect in breast cancer cells. (a) Eight human breast cancer cell lines (T‐47D, MCF‐7, MDA‐MB‐361, SK‐BR‐3, HCC‐1954, MDA‐MB‐468, MDA‐MB‐231, and BT‐549) were incubated with medium alone or were treated with increasing concentrations of delanzomib for 72 hours and then subjected to Cell Counting Kit‐8 assay. The absorbance of each well was measured at 450 nm, and the cell viability curve was plotted. The median inhibitory concentration values of delanzomib in breast cancer cell lines are listed. *P < 0.05, **P < 0.01, or ***P < 0.001 (Student's t‐test, two tailed) as indicated. (b) Data from samples treated with doses of 0.01 and 0.1 μM are shown. Photographs of treated cells (magnification, ×200). () MDA‐MB‐231, () MDA‐MB‐468, () MDA‐MB‐361, () BT‐549, () MCF‐7, () HCC‐1954, () SK‐BR‐3 and () T‐47D.

Methods

Antibodies and reagents

Anti‐poly (ADP‐ribose) polymerase (PARP, 9532), anti‐caspase 3 (9662), anti‐caspase 7 (12827), anti‐phospho‐p38 (9211), anti‐p38 (8690), anti‐phospho‐JNK (9251), anti‐JNK (9252), anti‐p27 (3686), anti‐Noxa (14766), anti‐PUMA (12450), anti‐mouse (7076), and anti‐rabbit (7074) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti‐p21 (sc‐53870), anti‐p53 (sc‐126), and anti‐α tubulin (10D8) (sc‐53646) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Delanzomib (CEP‐18770) was purchased from LC Laboratories (Woburn, MA, USA). Doxorubicin (D1515) was purchased from Sigma‐Aldrich Corp. (St. Louis, MO, USA).

Cell lines and cell culture

Human breast cell lines MCF‐7, T‐47D, MDA‐MB‐361, MDA‐MB‐231, MDA‐MB‐468, BT‐549, HCC1954, and SK‐BR‐3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF‐7, MDA‐MB‐361, MDA‐MB‐231, and SK‐BR‐3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Lonza, Walkersville, MD, USA). T‐47D, HCC1954, MDA‐MB‐468, and BT‐549 cells were cultured in RPMI 1640 medium (Lonza), supplemented with 10% (v/v) heat‐inactivated fetal bovine serum (SAFC Biosciences, Lenexa, KS, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin. All cells were maintained in a humidified incubator at a constant temperature of 37°C and 5% CO2.

Cell viability assay

Cell viability was calculated using a Cell Counting Kit‐8 (CCK‐8, WST‐8[2‐(2‐methoxy‐4‐nitrophenyl)‐3‐ (4‐nitrophenyl)‐5‐(2,4‐disulfophenyl)‐2H‐tetrazolium, monosodium salt]; Dojindo Laboratories, Rockville, MA, USA). Cells were seeded in 96‐well clear‐bottom plates at 5 × 103 cells/well. After 24 hours of incubation, increasing concentrations of delanzomib, Dox, or a combination of the two drugs were added to the wells, and the cells were then incubated for 72 hours. A mixture of 10 μL of CCK‐8 and 190 μL of medium with 10% fetal bovine serum was then added into each well. After one hour of incubation, the absorbance was measured at 450 nm, and the data were plotted to generate a cell viability curve. Each experiment was performed with six replicates, and the background reading of the medium was subtracted from each well to standardize the results.

Drug combination analysis

We used the method developed by Chou and Talaly for drug combination analysis.23 Dose‐response curves and half‐maximal inhibitory concentration (IC50) values for delanzomib and Dox in breast cancer cell lines were determined based on proliferation assay. Equipotent ratios of delanzomib and Dox were used to treat 96‐well plates for 72 hours. Individual treatments of the two drugs were used as controls. The combination index (CI) was assessed by CompuSyn software: CI < 1 indicates a synergistic interaction, CI < 1 indicates an antagonistic interaction, and CI = 1 is considered an additive effect.

Cell imaging

A total of eight breast cancer cell lines, MCF‐7, T‐47D, MDA‐MB‐361, MDA‐MB‐231, MDA‐MB‐468, BT‐549, HCC1954, and SK‐BR‐3, were seeded in 96‐well plates at appropriate concentrations. After 72 hours of treatment with the indicated concentrations (0, 0.01, 0.1 μM) of delanzomib, cell morphologies were observed and captured using an optical microscope (Nikon Inc., Melville, NY, USA).

Colony formation assay

The soft agar assay for detecting colony formation abilities was performed as previously described.24 A 5% (w/v) base agar layer was made by mixing agar (214220, Difco Laboratories, Detroit, MI, USA) with distilled water and then autoclaving the mixture for 50 minutes before cooling in a 56°C water bath. The solution was then mixed with medium to a final concentration of 0.5%. To make the bottom agar layer, 2 mL of the 0.5% agar/medium solution was added to each well and cooled until semisolid. The top agar layer was made of 1.5 mL of 0.3% agar, and each breast cancer cell line was counted and added to the mixture at 1 × 104 cells/well along with the indicated concentrations of delanzomib. Cells were grown at 37°C for two weeks and then stained with 500 μL of 0.005% crystal violet (C3886, Sigma) for four hours. Images were captured by microscopy, and the colonies were counted using Quantity One software. Each assay was performed in triplicate.

Immunoblotting assay

After each treatment, breast cancer cells were washed twice with ice‐cold phosphate buffered saline. Cell pellets were collected after centrifuging for five minutes at 6000 rpm and then lysed on a rotator at 4°C for 30 minutes in cooled radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 50 mM Tris‐HCl at pH 7.4, 50 mM sodium fluoride, 1 mM ethylene‐diamine‐tetraacetic acid, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.1 mM sodium orthovanadate, 10 μg/mL leupeptin, 1% NP‐40, 0.25% sodium deoxycholate, and phosphatase inhibitor cocktail 2 and 3; p5726 and p0044, Sigma). Cell lysates were collected by centrifuging for 15 minutes at 13 000 rpm. Protein concentration in cell lysates was measured using Bradford reagent (Bio‐Rad Laboratories, Hercules, CA, USA), and samples were mixed with 4× loading buffer before being heated at 100°C for six minutes. Lysates were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Bio‐Rad), blocked with 5% milk for one hour at room temperature (25°C), and probed with appropriate dilutions of the indicated primary antibodies overnight at 4°C. The membranes were then incubated with anti‐mouse or rabbit immunoglobulin G conjugated with horseradish peroxidase at room temperature for one hour. An ECL‐Plus Western detection system (GE Health Care, Buckinghamshire, UK) was then used for chemiluminescent visualization. The anti‐α tubulin antibodies were used as a loading control for whole cell extracts in all samples.

Flow cytometry and proteasome inhibitor staining

The experiments measuring apoptosis were performed following procedures described previously.25 Briefly, breast cancer cell lines were treated with delanzomib at 0 and 0.5 μM for 24 hours. Cells were then washed with ice‐cold phosphate buffered saline three times and incubated with PI staining solution (51‐66211E; BD Biosciences) at 50 μL/mL for 15 minutes at real time (25°C). The samples were analyzed using flow cytometry. Unstained cells were used as a negative control, and untreated cells were used as a control for the treated cells.

To test cell cycle arrest, breast cancer cells were treated with the indicated concentrations of delanzomib. Cells were then harvested in PI solution and analyzed by flow cytometry. The G1, S, and G2/M cell cycle phases are shown.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5 software. All experiments were repeated three times. Values are presented as the mean ± standard deviation (SD). Statistical significance (P < 0.05, *) was determined by Student's t‐test.

Results

Delanzomib inhibits the proliferation of breast cancer cells

To assess the antitumor effect of delanzomib on breast cancer cells, eight breast cancer cell lines (T‐47D, MCF7, MDA‐MB‐361, SK‐BR‐3, HCC1954, MDA‐MB‐468, MDA‐MB‐231, and BT‐549) were selected, which represent the major molecular subtypes of breast cancer.22 Cells were either incubated with medium alone (control) or with delanzomib at the indicated concentrations (0.001–1 μM) for 72 hours before being subjected to CCK‐8 assay. Delanzomib reduced the viability of all types of breast cancer cells in a dose‐dependent manner, but with varying efficacy (Fig 1a).

The median IC50 values of delanzomib on breast cancer cell lines were calculated (Table 1). The IC50 values of T‐47D and MDA‐MB‐361 were < 0.02 μM, but the IC50 value of MCF‐7 was > 0.5 μM. Triple‐negative cells (MDA‐MB‐468, MDA‐MB‐231, and BT‐549) had higher IC50 values (0.013, 0.027, and 0.1 μM). The IC50 values of HER2+ cells (SK‐BR‐3 and HCC1954) were intermediate (range: 0. 008–0.038 μM). The cytotoxic effect of delanzomib was further confirmed by morphological imaging of the cells after treatment for 72 hours (Fig 1b). As the IC50 values were between 0.01 and 0.1 μM for all cell lines, we only show the data at 0.01 and 0.1 μM.

Table 1

IC50 values of delanzomib on breast cancer cell lines

Cell lines	IC50 values (μM)	Subtypes	TP53 status
MDA‐MB‐231	0.027	TNB	MUT
MDA‐MB‐468	0.013	TNB	MUT
MDA‐MB‐361	0.005	Luminal B	MUT
BT‐549	0.1	TNB	MUT
MCF‐7	0.59	Luminal A	WT
HCC‐1954	0.038	HER2 overexpression	MUT
SK‐BR‐3	0.008	HER2 overexpression	MUT
T47D	0.007	Luminal A	MUT

IC50, half‐maximal inhibitory concentration; MUT, mutation type; TNB, triple negative breast cancer; WT, wild type.

Delanzomib induces apoptosis in breast cancer cells

Delanzomib induces apoptosis in a variety of cancers, including MM and cervical cancer. To examine the effect of delanzomib on apoptosis in human breast cancer, cell lines were treated with delanzomib at concentrations of 2 μM for 2, 4, 8, and 24 hours. Cells were subjected to immunoblotting assays. Because MCF‐7 cells are caspase 3‐deficient, caspase 7 was measured. We found that delanzomib induced PARP and caspase 3 (or caspase 7) cleavage in tested cell lines (Fig 2a). To further verify whether delanzomib could induce apoptosis in the eight breast cancer cell lines, all cell lines were treated with the drug at a concentration of 2 μM for 24 hours before undergoing flow cytometry (Fig 2b). Consistent with the findings of immunoblotting assays, delanzomib induced apoptosis in all tested cell lines. Overall, these results suggest that delanzomib alone triggered apoptosis in breast cancer cells.

Figure 2

Delanzomib induces apoptosis in breast cancer cells. (a) Breast cancer cells (MDA‐MB‐231, MDA‐MB‐468，MDA‐MB‐361, BT‐549, MCF‐7, HCC‐1954, SK‐BR‐3, and T‐47D) were treated with 2 μM for 2, 4, 8, and 24 hours. Untreated cells were used as controls. Whole‐cell lysates were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblotted with antibodies against poly (ADP‐ribose) polymerase (PARP) and Caspase 3 (or Caspase 7) to test apoptosis. α‐tublin was used as a loading control. (b) Breast cancer cell lines were treated with 2 μM delanzomib for 24 hours. Cells were then washed with ice‐cold phosphate buffered saline and incubated with proteasome inhibitor (PI) staining solution. The samples were analyzed by flow cytometry. Untreated cells were used as a control for treated cells. OD, optical density.

Delanzomib induces G2/M phase arrest in breast cancer cells

To investigate the effect of delanzomib on the cell cycle, four cell lines (MCF‐7, MDA‐MB‐231, MBA‐MB‐361, and HCC‐1954 cells) were treated with delanzomib (0.5 μM) for 12 hours. Cells were evaluated by flow cytometry. The cell cycle distributions of the four cell lines are presented as percentages. After 12 hours of treatment, significant accumulation of G2/M cell populations in these cancer cell lines was observed (Fig 3).

Figure 3

Delanzomib induces G2/M phase arrest in breast cancer cells. Breast cell lines (MCF‐7, MDA‐MB‐231, MBA‐MB‐361, and HCC‐1954 cells) were treated with delanzomib (0.5 μM) for 12 hours. Cells were tested by flow cytometry. The cell cycle distributions of four cell lines are presented as percentages. MCF‐7: Vehicle – () G1: 60.2%, () S: 19.9%, () G2: 16.6%. Delantinib ‐ () G1: 7.12%, () S: 35.1%, () G2: 60.4%. MDA‐MB‐231: Vehicle – () G1: 62.9%, () S: 10.9%, () G2: 25.3%. Delantinib ‐ () G1: 27.9%, () S: 26.3%, () G2: 42.5%. MBA‐MB‐361: Vehicle – () G1: 54.3%, () S: 25.7%, () G2: 20.1%. Delantinib ‐ () G1: 30.2%, () S: 38.3%, () G2: 31.1. HCC‐1954: Vehicle – () G1: 51.7%, () S: 28.1%, () G2: 19.6%. Delantinib ‐ () G1: 30.7%, () S: 25.2%, () G2: 46.2%. () G2, () S and () G1.

Delanzomib suppresses anchorage‐independent growth of breast cancer cells

Anchorage‐independent growth in soft agar is a key characteristic of cancer cells. To examine the relationship between delanzomib and anchorage‐independent breast cancer cell growth, soft agar assays were performed. We used a range of concentrations for treatment to account for the different IC50 values of delanzomib: MDA‐MB‐361, SK‐BR‐3, HCC‐1954, and T‐47D were cultured with delanzomib at concentrations of 0.01 or 0.1 μM; MDA‐MB‐468, MDA‐MB‐231, and BT‐549 were cultured with the drug at concentrations of 0.05 or 0.5 μM; and MCF‐7 was cultured with the drug at concentrations of 0.1 or 1 μM. After three weeks, visible colonies were fixed and stained. Untreated cells were used as controls (Fig 4a).

Figure 4

Delanzomib suppresses anchorage‐independent growth of breast cells. (a) The anchorage‐independent growth ability of breast cancer cells was assessed by soft agar assay. According to the different half‐maximal inhibitory concentration values, diverse concentrations for treatment were used: MDA‐MB‐361, SK‐BR‐3, HCC‐1954, and T‐47D were cultured with delanzomib at concentrations of 0.01 or 0.1 μM; MDA‐MB‐468, MDA‐MB‐231, and BT‐549 were cultured with the drug at concentrations of 0.05 or 0.5 μM; and MCF‐7 was cultured with the drug at concentrations of 0.1 or 1 μM. After three weeks, visible colonies were fixed and stained. Cells were then stained with crystal violet and photographed. (b) The colonies were counted, and the data were plotted. Data are represented as means ± standard deviations (SD). *P < 0.05, **P < 0.01, and ***P < 0.001 (analysis of variance and Dunnett multiple comparison post‐test).

In all eight cell lines, a significant decrease in the ability to form colonies was observed after delanzomib treatment compared to the vehicle‐treated control. Quantitative analysis also showed decreased colony numbers in delanzomib‐treated cell lines compared to those of controls (Fig 4b). Our results clearly indicate that delanzomib greatly restricts anchorage‐independent growth in breast cancer cells.

Delanzomib synergizes with doxorubicin to induce apoptosis of breast cancer cells

To explore the ability of delanzomib to synergize with Dox in breast cancer cells, we analyzed the effects of cotreatment on apoptosis in cancer cells. Using Chou and Talaly's method for synergistic analysis,23 delanzomib was combined with Dox in seven equipotent ratios based on the IC50 values derived from the single treatment of the eight breast cancer cell lines. CIs at median effective doses of 50 (ED50) and ED90 were derived from the tested cell lines using CompuSyn software. We observed synergistic antitumor effects at almost all EDs in tested cells (Table 2). For deeper molecular mechanistic insights, an immunoblotting assay was performed to detect cell apoptosis induced by the delanzomib‐Dox regimen. Breast cancer cells (MDA‐MB‐231, MDA‐MB‐468, MDA‐MB‐361, BT‐549, MCF‐7, HCC‐1954, SK‐BR‐3, and T‐47D) were treated with Dox (0.05 μM) alone, delanzomib (0.1 μM) alone, or a Dox‐delanzomib regimen for 24 hours. Untreated cells were used as controls. Delanzomib enhanced Dox‐induced apoptosis, as there were significant increases in caspase 3 (or caspase 7) cleavage and elevation of PARP levels compared to those of controls (Fig 5).

Table 2

Combination indexes of delanzomib and Dox

Cell line		Combination index
		ED50	ED75	ED90
MDA‐MB‐231	Delanzomib + Dox	0.79725	0.74373	0.69477
MDA‐MB‐468	Delanzomib + Dox	0.73738	0.39167	0.21027
MDA‐MB‐361	Delanzomib + Dox	0.53280	0.40423	0.26290
BT‐549	Delanzomib + Dox	0.70242	0.55134	0.43765
MCF‐7	Delanzomib + Dox	0.62501	0.43649	0.37025
HCC‐1954	Delanzomib + Dox	0.81880	0.58339	0.44687
SK‐BR‐3	Delanzomib + Dox	0.57779	0.63977	0.70868
T47D	Delanzomib + Dox	0.35585	0.23094	0.17036

Chou and Talaly's method for synergistic analysis was used. Combination indexes at the median effective doses 50 (ED50), ED75, and ED90 were derived from tested cell lines using CompuSyn software. Dox, doxorubicin.

Figure 5

Delanzomib synergizes with Dox in breast cancer cells to induce apoptosis. Breast cancer cells (MDA‐MB‐231, MDA‐MB‐468, MDA‐MB‐361, MCF‐7, SK‐BR‐3, HCC‐1954, and BT‐549) were treated with Dox (0.05 μM) or delanzomib (0.1 μM) alone or a combination of the two drugs for 24 hours. Untreated cells were used as controls. Whole‐cell lysates were then subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblotted with antibodies against poly (ADP‐ribose) polymerase (PARP) and Caspase 3 (or Caspase 7) to detect apoptosis. α‐tublin was used as a loading control.

Delanzomib enhances Dox‐induced apoptosis through p53 stabilization, p21, p27, PUMA, and NOXA upregulation, and p38/JNK activation in breast cancer cells

Our results have shown that delanzomib works synergistically with Dox. To elucidate the potential mechanisms driving this effect, we assessed the effects of the delanzomib‐Dox combination on the activity of p53 and MAPK using immunoblotting analysis in four breast cancer cell lines (MCF7, MDA‐MB‐361, MDA‐MB‐231, and HCC‐1954), which represent four major molecular subtypes of breast cancer. Cells were cultured with Dox (0.05 μM) alone or in combination with delanzomib (0.1 μM) for 6, 8, or 12 hours. Untreated cells were used as controls.

In MCF‐7, a p53 wild‐type cell line, the combination treatment increased p53 levels, consequently upregulating the p53 transcriptional targets p21, p27, PUMA, and NOXA. In p53 mutant cell lines (MDA‐MB‐231, MDA‐MB‐361, and HCC‐1954), p53 levels exhibited no significant change before or after drug treatments. However, both single treatment with delanzomib and combination treatment resulted in the upregulation of p53 transcriptional targets p21, p27, PUMA, and NOXA compared to those of controls. In addition, delanzomib enhanced the Dox‐induced activation of p38/JNK in four cell lines compared to that of controls (Fig 6).

Figure 6

Delanzomib enhances Dox‐induced apoptosis via p53 stabilization, p21, p27, PUMA, and NOXA upregulation, and p38/JNK activation in breast cancer cells. Four breast cancer cell lines (MCF‐7, MDA‐MB‐361, MDA‐MB‐231, and HCC‐1954) which represent the four major molecular subtypes of breast cancer, were cultured with Dox (0.05 μM) alone or in combination with delanzomib (0.1 μM) for 6, 8, or 12 hours. Untreated cells were used as controls. Whole‐cell lysates were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblotted with indicated antibodies. α‐tublin was used as a loading control.

Discussion

It has been demonstrated that dysregulation of the UPP could lead to the upregulation of oncogenic proteins, such as NF‐kB, and the downregulation of tumor suppressor proteins, such as p53, in MM and breast cancer cells.6, 7, 14, 26, 27 Blocking proteasome activity interferes with the regulation and degradation of essential proteins in cells, such as cyclins, cell‐cycle dependent kinases, and proapoptotic proteins.28 In addition, misfolded protein aggregation and the abundance of short‐lived regulatory proteins lead to cell destruction.29 Therefore, UPP inhibition has become a therapeutic strategy for some cancers.14

In this study, we tested the cytotoxic effects of delanzomib, a novel PI, in breast cancer cells. Delanzomib demonstrated consistent antitumor activity in breast cancer cell lines. The IC50 values for all of the cell lines treated with delanzomib were at the submicromolar level. These values were consistent with previously reported IC50 values of carfilzomib (an approved PI for MM) but were lower than that of ixazomib (another approved PI for MM) in breast cancer cell lines.25, 30 We hypothesize that breast cancer cell lines of different subtypes may exhibit unique responses to PIs. It is worth noting that triple‐negative cells (MDA‐MB‐468, MDA‐MB‐231, and BT‐549) had more consistent and higher IC50 values than those of the other subtypes, indicating that delanzomib had lower efficacy for triple‐negative breast cancer.

Anchorage‐independent growth correlates strongly with tumorigenicity and invasiveness in several cell types. Although we used three concentration ranges of delanzomib according to IC50 results that were previously measured in different cell lines (0.01 and 0.1 μM; 0.05 and 0.5 μM; 0.1 and 1 μM), we found that the colony formation of all cell lines treated with the drugs decreased significantly, and in a dose‐dependent manner, compared to those of controls (Fig 3b). These results suggest that delanzomib alone can restrict the proliferation and anchorage‐independent growth of breast cancer cells in a dose‐dependent manner. In addition, we provide strong in vitro evidence that delanzomib can suppress breast cancer cell proliferation by inducing apoptosis.

Doxorubicin is a commonly used chemotherapy in breast cancer patients. However, severe side effects and chemoresistance limit its dosage.31, 32 Therefore, novel target drugs that enhance the cytotoxicity of Dox are highly desirable in clinical practice. We demonstrated that delanzomib enhances Dox‐induced cell apoptosis in breast cancer cell lines. We also found a strong synergistic effect of combination treatment in breast cancer cell lines. Our data suggest that the combination treatment of delanzomib and Dox is a potential therapeutic strategy for breast cancer.

JNK and p38 are essential mediators of Dox‐induced cell death. Cellular stress can activate MAPK and phosphorylate both the JNK and p38 proteins, which induces apoptosis via mitochondria‐dependent mechanisms involving cytochrome c release and caspase‐3/‐9 activation. We found that delanzomib enhanced Dox‐induced phosphorylation of JNK and p38 and intensified Dox‐induced apoptosis in breast cancer cells.

TP53 is a tumor suppressor gene that regulates many crucial cellular processes, such as the maintenance of genomic stability, cell cycle arrest, and apoptosis.33, 34 As a transcription factor, the essential function of p53 is to regulate the expression of its target genes. p53 transactivates a series of genes, such as p21, PUMA, Gadd45, and the Bcl‐2 family genes, which are responsible for cell cycle arrest and apoptosis.35, 36, 37

TP53 is the most commonly mutated gene in human tumors; mutated TP53 is present in nearly 50% of malignant tumors.38, 39 The oncogenic function of mutant p53 is a viable target for antitumor therapy.40 We hypothesize that inhibiting proteasomal degradation can stabilize p53 protein levels and upregulate p53 transcriptional targets. Our study found substantial upregulation of the downstream p53 target genes with relatively low concentrations of delanzomib treatment in the MCF‐7 cell line, a p53 wild‐type breast cancer cell line. Our data strongly suggest that delanzomib can stabilize p53 while upregulating the downstream targets of p53 in the wild‐type p53 cell line. Moreover, some studies have reported delanzomib enhanced p53 expression in p53 wild‐type tumor cells;21, 41 our results were consistent with this finding. Therefore, we speculate that the potential mechanism of action for the synergistic effect of combined treatment in wild‐type cells is initiated with increased p53 stability and expression, followed by upregulation of the p53 downstream target genes, causing apoptosis and cell cycle arrest.

The p53 levels did not significantly change in the mutant cell lines after treatment compared to those of the wild‐type cell line. Both delanzomib‐only and combined treatment led to p53 transcriptional target protein stability, including that of p21, p27, PUMA and NOXA. A previous study reported that delanzomib sensitized cervical cancer cell lines to Dox‐induced apoptosis by stabilizing suppressor proteins in the p53 pathway; our findings support this result.21 In addition, we suggest that delanzomib has potentially broad clinical applications because of its ability to cause cell cycle arrest in both wild type and mutant cell lines.

Recent studies have claimed that PIs inhibit Dox‐induced NF‐kB activation and enhance Dox‐induced cytotoxicity in some tumor cell lines.25, 30, 42 A lack of IKK2 activity and the consequent inability to activate NF‐kB facilitated p53 stabilization and promoted cell death has also been observed.11 The mechanism of the synergistic effects of delanzomib and Dox is complex and broad; efforts continue to define the exact mechanism of the synergistic antitumor effect of delanzomib.

In conclusion, this study demonstrates that the novel PI delanzomib has antitumor effects on diverse types of breast cancer cells and induces cell apoptosis in both p53 wild‐type and mutant cell lines. Delanzomib enhances Dox cytotoxicity by intensifying the JNK and p38 phosphorylation pathways and by upregulating p53 transcriptional targets in a panel of breast cancer cell lines. Our findings suggest that the combination of delanzomib and Dox is an improved strategy for breast cancer therapy.

Disclosure

No authors report any conflict of interest.