Blockage of bone marrow kinase in chromosome X enhances ABC 294640-induced growth inhibition and apoptosis of colorectal cancer cells

Purpose: To investigate the role of bone marrow kinase in chromosome X (BMX) in colorectal cancer (CRC) cell resistance to ABC294640 treatment. Methods: HCT-116R, LS174T and WiDr cells were transfected with either BMX-specific siRNA or scrambled siRNA, and then BMX mRNA and protein expressions were detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. The cells were treated with ABC294640 and cell viability evaluated using cell counting and colony formation assays. Apoptosis was determined by detecting caspase 3/7 activity. To evaluate tumor growth of HCT-116R cells, a xenograft model was utilized to measure tumor size. Results: Pharmacological inhibition of sphingosine kinase type 2 (SK2) with ABC294640 significantly decreased cell viability (p < 0.001) when compared with control group. SK2 inhibition also remarkably induced apoptosis in HCT-116 CRC cells in a dose-dependent manner (p < 0.01 and p < 0.001). However, no significant effects were observed in HCT-116R, LS174T, or WiDr cells following ABC294640 treatment. BMX mRNA and protein expression increased in ABC294640-resistant cell lines. In addition, silencing BMX expression with siRNA potentiated ABC294640-induced inhibition of tumor growth in CRC cells in vitro and in vivo. Conclusion: ABC294640-induced BMX upregulation impedes the antitumor effect of ABC294640 in CRC cells. Therefore, these results may provide a novel therapeutic strategy for CRC using a combination of ABC294640 treatment and BMX blockade.


INTRODUCTION
Colorectal cancer (CRC) is one of the most common malignancies, ranking second in incidence and mortality among all types of malignant worldwide [1].Without reliable biomarkers, diagnosis of CRC usually occurs in the advanced stage, leading to high mortality in these patients.
Currently, surgery and conventional chemotherapy remain the primary treatments for CRC [2].However, tumor relapse and/or metastasis severely limit their clinical effectiveness, which demonstrates that finding effective cancer therapies remains an important medical challenge [3].Therefore, more investigation of mechanisms of CRC pathogenesis is needed to develop effective treatments for CRC.Sphingosine 1-phosphate (S1P) is a pleiotropic molecule and involved in various intracellular functions, including cell survival, cell motility, and angiogenesis [4].S1P is formed via sphingosine phosphorylation by sphingosine kinase type 1 (SK1) and sphingosine kinase type 2 (SK2) [5].The two kinases are highly conserved but have different subcellular localizations functions, and pharmacology [6].Initially, overexpression of SK2 was reported to induce apoptosis, mediated by its BH3 domain [7].Subsequently, it was found that SK2 downregulation had dramatic antitumor function in several tumor types, including colon cancer [8].Loss of SK2 had stronger anticancer effects than suppression of SK1, providing a rationale for targeting SK2 in cancer therapy [9].ABC294640 is an inhibitor of SK2 which showed very good bioavailability when administered orally as well as a favorable safety profile in various pre-clinical models [10,11].However, the drug resistance of cancer cell to ABC294640 is not clear.Bone marrow kinase in chromosome X (BMX) is a member of Tec family [12].It has two critical domains, a PH-like domain that mediates membrane binding and an SH2 domain important for the binding of tyrosine-phosphorylated proteins [13].BMX is regulated by several oncogenes such as Src and phosphoinositide 3kinase (PI3K), and is activated by inflammatory pathways [14].It been implicated in tumor progression in prostate cancer, bladder cancer, and glioblastoma [15].Additionally, BMX is reported to be involved in drug resistance.For example, overexpression of BMX abolished the miR-495-induced inhibition of drug resistance [16].In addition, it suppressed the ephrin receptor A3 (EPHA3)-induced drug sensitivity of small cell lung cancer [17].In the present study, the effect of BMX on the CRC response to ABC294640 treatment was explored.

EXPERIMENTAL siRNA and transfection
HCT-116R, LS174T and WiDr cells (3×10 5 cells/well) were cultured in 6-well plates overnight.Then the cells were transfected with either BMX-specific siRNA or scrambled siRNA (30 nM; Ribo-Bio, China) using lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.The protein and RNA were extracted after transfection.BMX siRNA sequences were as follows: GUACCAGUCUAGCGCAAUAUU (sense) and UAUUGCGCUAGACUGGUACUU (anti-sense).

Caspase 3/7 activity assay
For detection of apoptosis, caspase-Glo® 3/7 Assay kit from Promega was used.Cells (5×10 3 cells/well) were cultured in 96-well plates and treated with either ABC294640 (0.3 or 3 μM) or left untreated for 48 hr.The Caspase-Glo® 3/7 Reagent was added directly into the wells followed by the luminometer readings.

Tumor xenografts
Six-week old nude mice (Charles River Laboratories) received a single subcutaneous injection of 1×10 6 HCT-116R cells in the right flank.Tumor volumes were assessed with digital calipers and calculated as previously described [19].Tumors were measured 3 times weekly, including total mouse weights.At the end of the study, the tumors were excised, and weighed.The study was approved by Animal Ethic Committee of the First Affiliated Hospital of Wenzhou Medical University (approval ref no.2017-0003), and were in accordance with "Principles of Laboratory Animal Care" (NIH publication no.85 -23, revised 1985) [19].

Effect of ABC294640 on CRC cells
Cytotoxicity assays demonstrated that ABC294640 significantly decreased cell viability in a dose-dependent fashion in HCT-116 cells.In contrast, ABC294640 had marginal effect on cell viability in LS174T and WiDr cells, with less than 10-fold change in viability (p < 0.001; Figure 1A).In addition to cytotoxicity, apoptosis was evaluated in the three CRC cell lines following ABC294640 treatment.ABC294640 treated HCT-116 cells displayed higher caspase 3/7 activity relative to the untreated group (p < 0.001 and p < 0.0001; Figure 1B).However, ABC294640 treatment had no effect on apoptosis in LS174T and WiDr cells (Figure 1B).These results demonstrate that HCT-116 cells are sensitive to ABC294640 treatment whereas LS174T and WiDr cells are more resistant.

Upregulation of BMX in CRC cells associated with ABC294640 sensitivity
To reveal the mechanism underlying drug resistance, the ABC294640-resistant cell line, HCT-116R, was established from HCT-116 cells by continuous passage in the presence of sublethal doses of ABC294640.The drug sensitivities of HCT-116R and the parental HCT-116 cells to ABC294640 were determined using the CCK8 cytotoxicity and caspase 3/7 assays.As shown in Figure 2 A, the half maximal inhibitory concentration of ABC294640 treatment in HCT-116 cells was 260 nM, whereas ABC294640 did not affect HCT-116R cell viability (p < 0.001).Consistently, ABC294640 increased the level of caspase 3/7 activity in HCT-116 cells p < 0.001 and p < 0.0001 while showing no effect in HCT-116R cells (Figure 2 B).
To investigate the effect of BMX on CRC resistance to ABC294640, the expression of BMX was evaluated in drug-resistant cells and drug sensitive cells by qPCR and Western blotting.Both protein and mRNA expression levels of BMX were significantly higher in HCT-116R, LS174T, and WiDr compared to HCT-116 cells (p < 0.0001; Figure 2 C and D).

Inhibition of BMX augment ABC294640 potency in CRC
To clarify the role of BMX in CRC resistance to ABC294640, HCT-116R, LS174T and WiDr cells were treated with ABC294640 in the presence or absence of siRNA against BMX (siBMX).Cotreatment with siBMX and ABC294640 significantly inhibited the cell survival of CRC compared to each single treatment group (p < 0.001 and p < 0.0001; Figure 3A).In addition ABC294640 treatment (3 μM) and siBMX induced apoptosis of CRC cells 48 h posttreatment.
Combinational treatment of ABC294640 and siBMX dramatically enhanced apoptosis in CRC cells (p < 0.001 and p < 0.0001; Figure 3B).The results indicate that BMX is resistance molecule during ABC294640 treatment, and specific inhibition of BMX enhanced ABC294640 sensitivity in CRC cells.

BMX inhibition increase ABC294640 potency in CRC in vivo
As ABC294640 treatment and knockdown of BMX showed an additive effect in vitro in CRC,it was investigated that whether this also occurred in vivo.As indicated in Figure 4, neither ABC294640 treatment nor siBMX influenced the HCT-116R CRC xenografts in nude mice when given alone.However, administration of ABC294640 at 5 mpk in combination with siBMX suppressed the growth of HCT-116R tumors (p < 0.01).

DISCUSSION
There S1P rheostat determines the fate of tumor cells.Thus far, two isoforms of SK, SK1 and SK2, have been identified in humans [19].It has been shown that cell proliferation and migration was suppressed by downregulation of SK2, which had a greater affect than SK1 [8].
Recently, phase I clinical data for ABC294640 treatment of solid tumors, a highly specific SK2 inhibitor, have demonstrated variable clinical activity, which may reflect either the lack of biomarkers for patient selection and/or deficiency of knowledge about drug-resistant mechanisms [20].In the current study, the molecular target that contributes to the limited therapeutic efficacy of ABC294640 in CRC was investigated.
ABC294640 has been extensively studied for the treatment of many human tumor types and animal tumor models including CRC.ABC294640 suppressed cell proliferation and induced cell apoptosis in CRC cells in vitro.ABC294640 treatment remarkably inhibited HT-29 tumor growth in vivo, suggesting that ABC294640 might be an effective anti-tumor drug in CRC [21].In the current study, ABC294640 sensitivity in several CRC cell lines was tested, with variable effects among these cell lines, consistent with its clinical activity.This indicates the presence of intrinsic CRC resistance to ABC294640.Bone marrow kinase in chromosome X is reported to be overexpressed in various tumor types [22].It also participates in drug resistance as it suppresses the apoptotic regulator BAK activation via phosphorylation, which facilitates the survival of cells subjected to cytotoxic agents [23].Bone marrow kinase in chromosome X also is suggested to protect prostate cancer cells from photodynamic induced apoptosis [24].In addition, suppressing BMX expression reduced the chemo-resistance of H69/AR cells [25].In this study, BMX mRNA and protein expression were increased in ABC294640-resistant CRC cells and silencing BMX down-regulated cell sensitivity to ABC294640 in ABC294640resistant cells.Further, knockdown of BMX enhanced the effect of ABC294640 treatment on tumor growth both in vitro and in vivo.These findings suggest that BMX upregulation contributes to the resistance of CRC to ABC294640 therapy.

CONCLUSION
These findings demonstrate that BMX upregulation impedes the antitumor effect of ABC294640 in CRC cells, and knockdown of BM augments ABC294640 ability to suppress the tumor growth of CRC in vitro and in vivo.Therefore, these data may present a novel therapeutic strategy for the CRC therapy using a combination of ABC294640 treatment and BMX blockade.