Chemoresistance is associated with overexpression of HAX-1,inhibition of which resensitizes drug-resistant breast cancer cells to chemotherapy

Abstract

Acquired resistance to standard chemotherapy is the common and critical limitation for cancer therapy. Hematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) has been reported to be upregulated in numerous cancers. However, the role of HAX-1 in oncotherapy remains unclear. In this study, we established MDA-MB-231 cell lines which were resistant to cisplatin (MDA-MB-231/CR) or doxorubicin (MDA-MB-231/DR) to study the chemoresistance in breast cancer. As a result, the HAX-1 which is an apoptosis-associated protein was observed to be overexpressed in both MDA-MB-231/CR and MDA-MB-231/DR compared with the routine MDA-MB-231 cells. Moreover, knockdown of HAX-1 via RNA interference decreased IC50 level of cisplatin by 70.91% in MDA-MB-231/CR cells, and the IC50 level of doxorubicin was decreased by 76.46% in MDA-MB-231/DR cells when the HAX-1 was downregulated. Additionally, we found that the knockdown of HAX-1 induced the release of cytochrome C from mitochondria, resulting in the activation of caspases. Taken together, our study indicates that the overexpression of HAX-1 is essential in the development of chemoresistance in breast cancer. Furthermore, we identify that HAX-1 may become the target for cancer therapy.

Keywords

HAX-1 chemoresistance chemotherapeutics apoptosis breast cancer

Introduction

Breast cancer (BC) is the most common malignant cancer in female population worldwide, resulting in the leading cause of cancer deaths because of the metastatic spread of the cancer to vital organs.^1,2 Therapy for primary BC usually involves tumor resection, radiation therapy, and chemotherapy. Surgery usually extends patients’ survival; however, for patients with advanced disease, the chemotherapy is considered as the alternative option.^3,4 The substantial problem of chemotherapy is the drug resistance, reflected in hyposensitivity of cancer cells and subsequent presentations with recurrences.^5,6

Cisplatin (CDDP) and doxorubicin (DOX) are commonly used chemotherapeutic agents for the treatment of multiple cancers including BC.^7,8 Studies revealed that the CDDP as well as DOX belong to the cytotoxic agents, which killed tumor cells directly via lethal DNA damage, initiating the cell death through apoptosis pathway.^9,10 However, one key mechanism of therapy escape in cancer cells is represented by the resistance to undergo apoptosis, through altering the expression profile of related proteins.¹¹

HAX-1 (hematopoietic cell-specific protein 1-associated protein X-1) is a mitochondria-located protein, which has been reported to be an important regulator of apoptosis through mitochondrial pathway.^12,13 It is recognized that HAX-1 protects the cells from the mitochondria collapse, which is characterized by the induction of mitochondrial outer membrane permeabilization (MOMP) and loss of mitochondrial membrane potential (MMP), resulting in the release of cytochrome C (cyto-C) and the subsequent formation of Apaf-1/caspase-9 complex.^14–16 Previous studies have demonstrated that the HAX-1 is overexpressed in a variety of human malignancies and plays important roles. For example, patients with esophageal squamous cell carcinoma overexpressing HAX-1 are more likely to suffer from lymph node metastasis and exhibit a poor prognosis.¹⁷ In BC, previous studies have indicated that overexpression of HAX-1 promotes the development of cancers via improving their survival and is associated with the tumor size, stage, and grade of the disease.^18,19 The HAX-1 in cancers therefore has been reported as the new target for cancer therapy.²⁰ However, since the role of HAX-1 in BC treatment remains unclear, the aim of this study was thus to investigate the relationship between the chemoresistance and HAX-1 in BC.

Materials and methods

Cell culture and establishment of chemoresistant MDA-MB-231

Human normal breast epithelial cells MCF-10A²¹ and BC cell lines MDA-MB-231, T-47D, and MCF-7 were purchased from American Type Culture Collection (ATCC, Manassas, USA). All these cell lines were cultured according to the instruction provided by ATCC. CDDP-resistant MDA-MB-231 (MDA-MB-231/CR) and DOX-resistant MDA-MB-231 (MDA-MB-231/DR) cells were established by stepwise exposure to increasing concentrations of CDDP and DOX. Briefly, MDA-MB-231 cells were initially treated with CDDP at 1 µM (DOX at 0.1 µg/mL) for 3 months. Then, the CDDP concentration was increased every 3 weeks by 0.1 µM up to a final concentration of 2 µM, and the DOX concentration was increased every 3 weeks by 0.01 µg/mL up to a final concentration of 0.2 µg/mL. The chemoresistant MDA-MB-231 cells were exposed to CDDP or DOX over a period of 12 months. Before the experiments were performed, chemoresistant MDA-MB-231 cells were moved to the drug-free medium for 2 weeks.

Real-time polymerase chain reaction for detecting HAX-1 messenger RNA expression

Total RNA from MDA-MB-231 cells was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized with 500 ng total RNA, using M-MLV Reverse Transcriptase (Invitrogen). Real-time polymerase chain reaction (PCR) was performed using SYBR green (Promega, USA) on an Applied Biosystems 7500 thermal cycler. Quantification was performed using the 2^−ΔΔCT method,²² taking the gene of β-actin as the internal control.

Gene knockdown

Human HAX-1 siRNA was purchased from Guangzhou RiboBio Co., Ltd (China) to knock down the expression of HAX-1. For transfection of HAX-1 (or control siRNA, RiboBio), cells were plated at approximately 80% confluence, and then the siRNA (50 pmol/mL) were introduced into the tumor cells using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions.

Drug sensitivity assay

About 5 × 10³ cells per well were seeded in 96-well plates containing Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). After 24 h, tumor cells were transfected with siRNA. After 24 h of transfection, cells were treated with different kinds of antitumor drugs including CDDP, DOX, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand, R&D Systems, USA), and 5-fluorouracil (5-FU; Sigma-Aldrich, USA). After incubating for 48 h, 5 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich] was added to each well and incubated for another 4 h. The absorbance was measured at 540 nm using reference wavelength of 690 nm using an automated enzyme-linked immunosorbent assay (ELISA) plate reader. The 50% inhibitory concentrations (IC50) for CDDP and DOX were calculated according to the survival curves.

Cell proliferation assay

To determine the cell proliferation, cancer cells were seeded onto 6-well plate. During the last 6 h of treatment, the ³H thymidine was added into the culture medium, and then the ³H thymidine incorporation assay was performed to determine the cell proliferation.

Preparation of mitochondrial and cytosolic fractions

Cells were washed with phosphate-buffered saline (PBS) and then resuspended in digitonin lysis buffer (75 µg/mL digitonin dissolved in 250 mM sucrose solutions, cocktail of protease inhibitors were added). After 15 min incubation on ice, unbroken cells were removed by centrifugation at 2500g for 10 min. The supernatant was then centrifuged at 15,000g for 30 min at 4°C. The resultant supernatant and the pellet were designated as the cytosolic and microsomal fractions, respectively.

Immunoprecipitation

Cells were lysed in RIPA (radioimmunoprecipitation assay) buffer with protease inhibitor cocktail (Cell Signaling Technology, USA), and then the lysates were incubated with primary antibody of Apaf-1 (Cell Signaling Technology) overnight at 4°C followed by 2 h of incubation of protein A agarose beads. After washing the beads with cold RIPA buffer, proteins were removed from the beads by boiling in sodium dodecyl sulfate (SDS) sample buffer and analyzed by western blotting analysis.

SDS-polyacrylamide gel electrophoresis and western blotting

Cells were lysed in RIPA buffer with protease inhibitor cocktail (Cell Signaling Technology). About 25 µg proteins were then separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, samples were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, USA), blocked with 5% milk/Tris-Buffered Saline Tween-20 (TBS-T) and incubated with primary antibodies including anti-HAX-1, BCL-2, BCL-xL, BCL-w, BIM, BAX, BAK, cytochrome C, caspase-9, caspase-7, caspase-3, and anti-β-actin. Bound antibodies were detected with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology), and the protein levels were analyzed by enhanced chemiluminescence detection kit (Pierce, USA).

Apoptosis assay

After treatment, cells were harvested, washed with PBS, and stained with Annexin V and propidium iodide (PI) according to the manufacturer’s instructions (Sigma-Aldrich). Cells were analyzed using flow cytometry (Becton Dickinson, USA), and the ones positive for Annexin V were classified as apoptotic.

MMP (ΔΨ_m) analysis

After treatment, cells were harvested, washed with PBS, and stained with 5 µM 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl imidacarbo cyanine iodide (JC-1, Molecular Probes, USA) as an indicator. Following a 20-min incubation period at 37°C in the dark, ΔΨ_m was determined by flow cytometry analysis.

Statistical analysis

Data are given as means ± standard error of the mean (SEM). For statistical comparison, Student’s t test was used using SPSS 11.0 statistical software; p < 0.05 was considered to be statistically significant. All of the data were derived from at least three independent experiments.

Results

Expression of HAX-1 in the chemoresistant MDA-MB-231 cells

Consistent with the findings that HAX-1 is overexpressed in tumor cells,¹⁸ we observed that the expression of HAX-1 in BC cells was higher than that in the nontumorigenic MCF-10A cells (Figure 1(a)). The results suggested an important role of HAX-1 in BC. In order to assess the relevance of chemoresistance and expression of apoptosis-associated proteins in BC, the CDDP-resistant MDA-MB-231 (MDA-MB-231/CR) and DOX-resistant MDA-MB-231 (MDA-MB-231/DR) cell lines were established. As shown in Figure 1(b), the MDA-MB-231/CR as well as MDA-MB-231/DR cells were resistant to the CDDP or DOX therapy. In contrast, their parental MDA-MB-231 cells were sensitive to the treatment of both CDDP and DOX. To investigate the alteration of apoptosis-related proteins when the MDA-MB-231 became chemoresistant, western blot analysis was performed. We observed that the prosurvival protein of HAX-1, but not the Bcl-2 family proteins, was significantly upregulated in MDA-MB-231/CR and MDA-MB-231/DR cells (Figure 1(c)). Meanwhile, the expression of HAX-1 at messenger RNA (mRNA) level was also upregulated in MDA-MB-231/CR and MDA-MB-231/DR cells (Figure 1(d)). These results suggested that the overexpression of HAX-1 is associated with the chemoresistance in BC.

Figure 1.

HAX-1 is upregulated in MDA-MB-231 cells which are cisplatin-resistant or doxorubicin-resistant. (a) Expression of HAX-1 in MCF-10A, MDA-MB-231, T-47D, and MCF-7 cells was analyzed by western blot. (b) MTT assay was used to evaluate the sensitivity of MDA-MB-231, MDA-MB-231/CR, and MDA-MB-231/DR cells to cisplatin or doxorubicin. (c) Expression of HAX-1 and the Bcl-2 family proteins in MDA-MB-231, MDA-MB-231/CR, and MDA-MB-231/DR cells was analyzed by western blot. (d) Expression of HAX-1 was analyzed by real-time PCR.

HAX-1 is required for the resistance of CDDP and DOX in CDDP/DOX-resistant MDA-MB-231 cells

To determine whether HAX-1 plays a role in drug resistance, we knocked down the HAX-1 expression in MDA-MB-231/CR and MDA-MB-231/DR cells using its specific siRNA and assessed the resulting change of IC50 (IC50 was determined by the survival curves performed by MTT assay). As shown in Figure 2(a) and (b), knockdown of HAX-1 decreased IC50 level of CDDP by 70.91% in MDA-MB-231/CR cells, and the IC50 level of DOX was decreased by 76.46% in MDA-MB-231/DR cells when the HAX-1 was downregulated. Furthermore, our results revealed that knockdown of HAX-1 evoked the ability of CDDP/DOX to induce the apoptosis in CDDP/DOX-resistant MDA-MB-231 cells (Figure 2(c)). In addition, knockdown of HAX-1 also enhanced the proliferation inhibition induced by the CDDP or DOX in the CDDP/DOX-resistant MDA-MB-231 cells (Figure 2(d)). Taken together, we indicated that HAX-1 knockdown resensitizes drug-resistant BC cells to chemotherapeutics-induced cytotoxicity.

Figure 2.

Knockdown of HAX-1 resensitizes the drug-resistant MDA-MB-231 cells to CDDP and DOX. (a) MDA-MB-231 and MDA-MB-231/CR cells were treated with HAX-1 siRNA and/or different concentrations of CDDP, and the IC50 of CDDP was determined by the survival curves performed by MTT assay, *p < 0.05. (b) MDA-MB-231 and MDA-MB-231/DR cells were treated with HAX-1 siRNA and/or different concentrations of DOX, and the IC50 of DOX was determined by the survival curves performed by MTT assay, *p < 0.05. (c) After the MDA-MB-231/CR and MDA-MB-231/DR cells were treated with 50 pmol/mL HAX-1 siRNA, 10 µM CDDP, and 1 µg/mL DOX, the cell apoptosis was measured using Annexin V/PI staining on flow cytometry. (d) After the MDA-MB-231/CR and MDA-MB-231/DR cells were treated with 50 pmol/mL HAX-1 siRNA, 10 µM CDDP and 1 µg/mL DOX, the cell proliferation was detected by ³H thymidine incorporation assay. *p < 0.05 versus CDDP/DOX + control siRNA treatment group.

Cross-resistance of TRAIL and 5-FU in the CDDP/DOX-resistant MDA-MB-231 cells

Since the CDDP/DOX-resistant MDA-MB-231 cells were resistant to CDDP or DOX, respectively, we next investigated whether the cells were also resistant to some other anticancer chemotherapeutics such as TRAIL and 5-FU, antitumor mechanism of which was inducing the apoptosis in tumor cells.^23,24 As we observed, the sensitivity of MDA-MB-231/CR cells to DOX, TRAIL, and 5-FU was also reduced significantly compared with their parental MDA-MB-231 cells. And similarly, we found the MDA-MB-231/DR cells were resistant to CDDP, TRAIL, and 5-FU (Figure 3(a)). Interestingly, we found the cross-chemoresistance of MDA-MB-231/CR as well as MDA-MB-231/DR cells was inhibited by transfection of HAX-1 siRNA (Figure 3(b)), suggesting the HAX-1 plays a key role in the drug resistance of apoptosis-inducing chemotherapeutics.

Figure 3.

The cross-resistance to antitumor drugs in CDDP/DOX-resistant MDA-MB-231 cells. (a) MDA-MB-231/CR and MDA-MB-231/DR cells were treated with 10 µM CDDP, 1 µ/mL DOX, 5 ng/mL TRAIL, and 2 µM 5-FU, respectively. After 48 h incubation, the cell viability was determined by MTT assay, *p < 0.05 versus DOX/CDDP treatment group, ^#p < 0.05 versus TRAIL treatment group, ^Δp < 0.05 versus 5-FU treatment group. (b) MDA-MB-231/CR and MDA-MB-231/DR cells were treated with HAX-1 siRNA and various kinds of antitumor drugs, and the cell viability was determined by MTT assay. *p < 0.05.

Role of mitochondrial pathway in the HAX-1 siRNA-promoted apoptosis

Previous researches have proved HAX-1 functions as antiapoptotic protein by inhibiting the MOMP.²⁵ Therefore, we posited that HAX-1 might function as a resistance factor against chemotherapy through mitochondrial pathway. To test this possibility, the MMP (ΔΨ_m) was detected after the CDDP/DOX-resistant MDA-MB-231 cells were treated with CDDP/DOX combined with HAX-1 siRNA. As expected, although the HAX-1 siRNA alone almost did not influence the ΔΨ_m, it significantly facilitated the CDDP/DOX to damage the mitochondrial of CDDP/DOX-resistant MDA-MB-231 cells, respectively (Figure 4(a)). As a result, the cytochrome C, which is the mitochondria-derived apoptogenic protein, was released into the cytoplasm from the mitochondria in CDDP/DOX-resistant MDA-MB-231 cells co-treated with CDDP/DOX combined with HAX-1 siRNA (Figure 4(b)).

Figure 4.

Knockdown of HAX-1-promoted CDDP/DOX-induced apoptosis through mitochondrial pathway. (a) CDDP/DOX-resistant MDA-MB-231 cells were transfected with 50 pmol/mL HAX-1 siRNA for 24 h. Then, the cells were treated with 10 µM CDDP or 1 µ/mL DOX for incubation of another 24 h. The mitochondrial membrane potential (ΔΨm) was detected using JC-1 staining on flow cytometry. (b) After the treatment as above, the mitochondria fraction and cytoplasm were separated, and the level of cytochrome C in mitochondria or cytoplasm was evaluated by western blot analysis.

Activation of cytochrome C/caspase-9 axis is required for the HAX-1 siRNA-promoted apoptosis

Since the previous studies demonstrated that the mitochondrial apoptosis is induced by release of cytochrome C and the subsequent formation of Apaf-1/casapase-9 complex,¹⁶ we next investigated the role of HAX-1 siRNA in the cytochrome C/caspase-9 axis. As shown in Figure 5(a), although the HAX-1 siRNA alone almost did not induce the formation of Apaf-1/casapase-9 complex, it significantly facilitated the formation of this apoptosome in the resistant BC cells treated with CDDP/DOX. As a result, the caspase-9 was activated, following the cleavage of caspase-7 and caspase-3 (Figure 5(b)). These data emphasize the essential role of cytochrome C/caspase-9 axis in HAX-1 siRNA-promoted apoptosis in chemoresistant tumor cells.

Figure 5.

Knockdown of HAX-1 promoted the mitochondrial pathway through triggering the cytochrome C/caspase-9 axis. (a) After treatment, the interaction of Apaf-1 and caspase-9 was determined by co-immunoprecipitation of Apaf-1. (b) Western blot analysis was performed to measure the cleaved caspase-9, caspase-7, and caspase-3.

Discussion

The human HAX-1 gene is located on chromosome 1 (1q21.3), producing at least five splice variants with the same open reading frame. Studies indicate that variant II and variant III levels are reproducibly higher in tumors rather than normal tissues.¹⁹ In addition, monoallelic loss of FBXO25 stabilizes HAX-1 in tumor cells.²⁶ Mechanisms were developed in tumor cells to overexpress the HAX-1. Recently, studies have demonstrated that chemoresistance is related to the overexpression of HAX-1 in a variety of cancers. For example, esophageal squamous carcinoma cells with overexpressed HAX-1 developed chemoresistance to CDDP.²⁷ SW480 colorectal cancer cells whose HAX-1 is overexpressed exhibited increased resistance to camptothecin in vitro.²⁸ Consistent with these findings, our data indicated that both CDDP- and DOX-resistant MDA-MB-231 BC cells showed a significantly higher level of HAX-1 compared with the routine MDA-MB-231 cell line, suggesting the chemoresistance is commonly accompanied with the HAX-1 overexpression in BC.

TRAIL is a member of tumor necrosis factor (TNF) family and belongs to the group of chemotherapeutic agents selectively targeting a wide variety of cancer cells without affecting the normal cells. Therefore, the TRAIL is considered as a possible new anticancer drug that induces apoptosis in tumor cells.²⁹ 5-FU is a chemotherapeutic drug, which leads to the DNA damage and the subsequent apoptosis in tumor cells.³⁰ Interestingly, in our research, we found that the CDDP- and DOX-resistant MDA-MB-231 BC cells exhibited the cross-resistance to TRAIL and 5-FU. We speculated that the mechanism of this phenomenon was due to the overexpression of HAX-1 in chemoresistant cells. To prove the hypothesis, we used the RNA interference to knock down the HAX-1 in MDA-MB-231/CR and MDA-MB-231/DR cells. We found the HAX-1 siRNA overcame the cross-resistance in CDDP- and DOX-resistant MDA-MB-231 BC cells by decreasing the expression of HAX-1, suggesting the essential role of HAX-1 in the cross-resistance of BC.

Apoptosis is important for maintaining cellular homeostasis by eliminating the excess and damaged cells. However, cancer cells usually achieve the ability to escape from the apoptosis pathway. Therefore, induction of apoptosis is one important mechanism of action of various chemotherapeutic agents.³¹ Bcl-2 family proteins are a group of intracellular factors regulating the mitochondrial pathway of apoptosis, which divide into two main subclasses of Bcl-2 proteins: antiapoptotic (BCL-2, BCL-xL, BCL-w) and proapoptotic (Bad, Bak, Bax, Bid, Bim, Noxa, and Puma).³² However, in this study, we found that the HAX-1, but not the Bcl-2 family proteins, was significantly upregulated in CDDP/DOX-resistant MDA-MB-231 cells. The function of the overexpressed HAX-1, which locates on the mitochondria, is suppressing the loss of MMP and the formation of mitochondrial membrane permeability transition pore (PTP). Pore formation on mitochondrial membrane results in the release of proapoptotic factors (such as cytochrome C) to cytosol, activating caspase-9 and effecter of caspase-3 by cleavage.^16,25,33

Based on the present data, we found that knockdown of HAX-1 by its inhibitors strongly promoted the CDDP/DOX to damage the mitochondrial in CDDP/DOX-resistant MDA-MB-231 cells. We next found the knockdown of HAX-1 reversed the chemoresistance by inducing the release of cytochrome C which is the key inducer of mitochondrial apoptosis.³⁴ Furthermore, as a result of cytochrome C release, we demonstrated that the removal of chemoresistance by knockdown of HAX-1 is dependent on the intrinsic apoptotic pathway characterized by the formation of Apaf-1/casapase-9 complex and the subsequent activation of caspase-9, caspase-7, and caspase-3.¹⁶

In summary, our findings have provided strong evidence that the chemoresistance in BC is dependent on the overexpression of HAX-1. Moreover, inhibiting HAX-1 levels could successfully chemosensitize BC cells to chemotherapy via mitochondrial cell death pathway. Therefore, manipulation of the HAX-1 may represent a new strategy for treatment of BC and/or other malignancies which were chemoresistant and overexpressing HAX-1.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

This study was approved by the Ethics Committee of First Affiliated Hospital, School of Medicine, Zhejiang University.

Funding

This study was supported by the National Clinical Key Specialty Construction Project of Geriatrics Department and the General Research Project of Zhejiang Provincial Medical Science and Technology (grant nos 2015KYB032 and 2015KYB159).

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Chemoresistance is associated with overexpression of HAX-1,inhibition of which resensitizes drug-resistant breast cancer cells to chemotherapy

Abstract

Keywords

Introduction

Materials and methods

Cell culture and establishment of chemoresistant MDA-MB-231

Real-time polymerase chain reaction for detecting HAX-1 messenger RNA expression

Gene knockdown

Drug sensitivity assay

Cell proliferation assay

Preparation of mitochondrial and cytosolic fractions

Immunoprecipitation

SDS-polyacrylamide gel electrophoresis and western blotting

Apoptosis assay

MMP (ΔΨm) analysis

Statistical analysis

Results

Expression of HAX-1 in the chemoresistant MDA-MB-231 cells

HAX-1 is required for the resistance of CDDP and DOX in CDDP/DOX-resistant MDA-MB-231 cells

Cross-resistance of TRAIL and 5-FU in the CDDP/DOX-resistant MDA-MB-231 cells

Role of mitochondrial pathway in the HAX-1 siRNA-promoted apoptosis

Activation of cytochrome C/caspase-9 axis is required for the HAX-1 siRNA-promoted apoptosis

Discussion

Footnotes

Declaration of conflicting interests

Ethical approval

Funding

References

MMP (ΔΨ_m) analysis