Mcl‐1 inhibition overcomes BET inhibitor resistance induced by low FBW7 expression in breast cancer

Abstract While the promise of bromodomains and extraterminal (BET) protein inhibitors (BETis) is emerging in breast cancer (BC) therapy, resistance in these cells to BETis conspicuously curbs their therapeutic potential. FBW7 is an important tumour suppressor. However, the role of FBW7 in BC is not clear. In the current study, our data indicated that the low expression of FBW7 contributes to the drug resistance of BC cells upon JQ1 treatment. shRNA‐mediated FBW7 silencing in FBW7 WT BC cells suppressed JQ1‐induced apoptosis. Mechanistically, it was revealed that this diminished FBW7 level leads to Mcl‐1 stabilization, while Mcl‐1 upregulation abrogates the killing effect of JQ1. Mcl‐1 knockdown or inhibition resensitized the BC cells to JQ1‐induced apoptosis. Moreover, FBW7 knockdown in MCF7 xenografted tumours demonstrated resistance to JQ1 treatment. The combination of JQ1 with a Mcl‐1 inhibitor (S63845) resensitized the FBW7 knockdown tumours to JQ1 treatment in vivo. Our study paves the way for a novel therapeutic potential of BETis with Mcl‐1 inhibitors for BC patients with a low FBW7 expression.


| INTRODUC TI ON
Globally, breast cancer (BC) has emerged as a ubiquitous malignancy and predominant cause of death in women. 1 While their survival has been augmented by early detection and adjuvant therapy, advanced BC remains challenging due to the limited efficacy of current therapies. 2 The clinically vital categorization of BC ubiquitously is based on the expression of the estrogen receptor (ER) and the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2) amplification status. 3 This categorization guides the first-line of the therapeutic approach that is adopted. 4,5 However, a challenge emerges for triple-negative breast cancer (TNBC) patients, where all three of these receptors are absent with no FDA-approved targeted therapies yet available, necessitating traditional cytotoxic chemotherapy as the only solution. 6 Epigenetic regulators have emerged as being resourceful for treating hematologic malignancies and solid tumours. 7 The crucial involvement of the bromodomain and extraterminal (BET) family of proteins in gene expression via recruitment of transcriptional proteins is known. 8 Targeting these proteins by employing BET inhibitors (BETis) has recently emerged as a potent approach to quell malignancies 9 with documentation of its efficacy in various tumours, including breast cancer. 10 Apoptosis is one of the major mechanisms for the killing effect exerted by BETis in cancer cells. 11,12 The involvement of the GSK3β/Fbw7/proteasome pathway has also been reported in BETi-mediated cell death in various cancers. 13,14 However, the involvement of this pathway in the therapeutic effect of BETis in BC remains to be corroborated.
The tumour-suppressive functioning of FBW7 in several mouse and human cancers is known. 15,16 It is an F-box protein comprising a key element of the SCF (SKP1-CUL1-F-box) E3 ligase complex that functions in protein ubiquitination and degradation, 17 including cyclin E1, c-Jun, c-Myc, Notch1 and Mcl-1. 18 The inactivation of FBW7 in numerous human malignancies entails gene mutation 19,20 and downregulation. 21 While BC rarely demonstrates FBW7 mutations, 22 the FBW7 genetic locus is frequently deleted in TNBC, 23 and the FBW7 promoter is highly hypermethylated in 51% of primary BC tumours. 24 Loss of FBW7 expression has been documented in several breast carcinomas and is associated with poor a prognosis. 25 This is suggestive of the vital role of FBW7 in BC. Furthermore, the involvement of FBW7 in several distinct signaling pathways facilitates its use as a plausible and attractive therapeutic target for BC.

| Cell viability and apoptosis
For viability assays, cells were plated in 96-well plates at 1 × 10 4 cells/ml in RPMI-1640 medium (Gibco) supplemented with 10% FCS and 10 µg/ml insulin followed by JQ1 treatment. Cell viability was determined by MTT assays (Promega, Madison, WI, USA) based on the manufacturer's protocol. For apoptosis analysis, Hochst-33258based staining was employed to probe apoptosis as described previously. 26,27

| Plasmids and transfections
Short hairpin RNA (shRNA) transfection and subsequent screening were conducted as reported previously. 13

| Real-time PCR
The real-time PCR assay was performed as described in previous studies. 28,29 Briefly, total RNA was extracted using TRIzol RNA extraction reagent (Invitrogen) and treated with DNaseI.
DNA-free RNA was reverse transcribed using an RNA-to-cDNA kit (Invitrogen). Total cDNA was amplified using iTaq Universal SYBR Green Supermix (Bio-Rad). The results were normalized to GAPDH expression. The primers used for this study in-

| Western blotting
The western blotting assay was performed as described in previous studies. 30
The cell suspension was sonicated and centrifuged at 13,000 g for 20 min to prepare cell lysates. For the IP experiment, the IP antibody (1 mg) was incubated with protein G/A-agarose beads (Invitrogen) for 20 min at room temperature. The beads were washed twice with PBS containing 0.02% Tween-20, incubated with cell lysates on a rocker for 4 h at room temperature, and then washed with PBS three times. Beads were then boiled in 2× Laemmli sample buffer and subjected to SDS-PAGE and western blotting.

| Xenograft mouse model
The housing of the animals (5-to 6-week-old female Nu/Nu mice) was in a sterile environment in micro isolator cages with access to water and chow ad libitum. MCF7 cells (4 × 10 6 ) stably transfected with control or FBW7 shRNA were subcutaneously injected on the right. After tumour growth for 7 days, the mice were treated with JQ1 (i.p.; 30 mg/kg every other day for 10 days). For the combination treatment. MCF7 cells (4 × 10 6 ) stably transfected with FBW7 shRNA were subcutaneously injected on the right. After tumour growth for 7 days, mice were treated with JQ1 (i.p.; 30 mg/ kg every other days for 10 days) and/or Mcl-1 inhibitor (S63845, 15 mg/kg intravenously, every 3 day for 10 days). Calipers were employed to measure the tumour size every 2 days, with the formula: 1/2 × length × width 2 employed for computing the tumour volumes.
All animal experiments and research plans were approved by the Animal Research Committee of China Medical University.

| Patient-derived xenograft (PDX) mouse model
The PDX model was established using primary tumours resected from patients with written informed consent and an approved Institutional Review Board agreement. Then, tumours were implanted subcutaneously and passaged. In short, within 4 h after tumour removal, the breast cancer sample from an unidentified patient was transported to the laboratory in Antibiotic/Antimycotic Solution (Invitrogen). Tissues were cut into 25-mg pieces and directly implanted subcutaneously on both flanks of the NOD. Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice. Tumours that were passaged and expanded for two generations (P2) in NSG mice were used for the experiments. Mice were treated with JQ1 (i.p.; 10 mg/kg every other day for 10 days) and/or Mcl-1 inhibitor (S63845, 15 mg/kg intravenously, every 3 days for 10 days). The caliper was used to monitor the tumour volume, and the tumour volume was calculated as: The ethical endpoint was defined as the point in time when the tumour reached 2 cm or more in any dimension.
The tumour was dissected and fixed in 10% formalin and embedded in paraffin. By using an AlexaFluor 488-conjugated secondary antibody (Invitrogen) for detection and 4' 6-Diamidino-2-phenylindole (DAPI) for nuclear counter staining, the 5μm paraffin-embedded tumour sections were subjected to active caspase 3 staining.

| Statistical analysis
All assays illustrated in the figures were conducted in duplicate with two or three independent runs. The final figures are inclusive of the representative results. The computation of p values entailed was performed by the two-tailed distribution Student's t-test on paired or unpaired datasets. p < 0.05 was considered statistically significant.

| Low FBW7 expression in breast cancer patients
To determine the functional role of FBW7 in BC, we first probed the

| FBW7 decreases breast cancer cell growth in vitro and in vivo
We next considered whether FBW7 had any effect on the biological behavior of breast cancer cells. As shown in Figure 1D (Figure 2G and H). The above data indicate that FBW7 overexpression suppresses tumour growth in BC.

| High expression of FBW7 contributes to BET inhibitor sensitivity in breast cancer cell lines
To further scrutinize the impacts of FBW7 in BC, we probed the sensitivity of cell lines to small molecule drugs with FBW7 knockdown or overexpression ( Figure 3A and B). This entailed an initial treatment of FBW7 knockdown or overexpressing BC cells with several small molecules, followed by analysis of the drug sensitivity by computing the IC50 for each group. The normalized IC50 values of these inhibitors in the FBW7 knockdown/overexpressed group vs. those in the control group are illustrated as a heatmap ( Figure 3A Figure 4F). In FBW7-KD cells, following JQ1 treatment, Mcl-1 binding to PUMA was augmented, causing a lowered interaction of the latter with Bcl-XL, while the Bax-Bcl-XL interaction was increased ( Figure 4G and H). These observations therefore demonstrate Mcl-1 upregulation is due to the loss of FBW7, which leads to PUMA induction via JQ1 to cause resistance to JQ1 in BC.

| Mcl-1 inhibitor enhances the killing effect of JQ1 in vivo
The in vivo assessment of the effect of FBW7 in BETi chemosensitization entailed the use of the athymic nude mouse system harbouring xenograft tumours as outlined in the materials section. Seven days post-tumour inoculation, the mice were administered JQ1 for 10 days. A conspicuous decrease in MCF7 xenograft tumour growth was observed after JQ1 injection ( Figure 6A and B).  shown lead to cell proliferation arrest and apoptosis in BC cells. 35 Addressing of therapy resistance has also entailed targeting FBW7 in multiple cancers, including BC. 36,37 Resistance to the BET inhibitors JQ1 and OTX-015 was documented in T-ALL tumour cells with FBW7 mutations. 13 Our work also corroborates of the involvement of FBW7 in BETis sensitivity in BC cells. The lowered expression of FBW7 leads to Mcl-1 upregulation, which makes the BC cells assessed in our work less sensitive to BETis-induced apoptosis.
The negative regulation of apoptosis in malignant and healthy cells by Mcl-1 (of the Bcl-2 family) is known. 38 Mcl-1 has a very short half-life, and its expression is tightly regulated by its interaction with FBW7, which also mediates its degradation. 32,39,40 FBW7 mutations in tumours have been shown to increase Mcl-1 expression to augment resistance to both standard chemotherapy and targeted therapy. 32,38,41,42 The pathogenesis and poor prognosis arising from elevated Mcl-1 protein levels in refractory cancers are suggestive of manipulating the protein rather than the mRNA to boost apoptosis and thereby target a malignancy. Given that Mcl-1 is predominantly involved in resistance to BETis in HCC cells, the use of drugs that down-regulate Mcl-1 emerged as promising for augmenting HCC therapy. 43 The results of this work are also indicative of the inhibition or depletion of Mcl-1 to overcome BETis resistance in BC cells with a low level of FBW7 expression. In addition, our findings also indicate that JQ1 upregulates PUMA, which promotes apoptosis by releasing Bax from Bcl-XL, which is consistent with a previous study showing that the BET inhibitor I-BET151 induces PUMA and sensitizes gliomas to TMZ. 33 Previous studies have shown that the transcription factor c-Myc is the target of bromodomain proteins. 44,45 Furthermore, FBW7 controls proteasome-mediated degradation of c-Myc. 46,47 Thus, c-Myc may also be involved in FBW7-mediated BETi resistance in breast cancer. Therefore, targeting Mcl-1 emerges as a plausible option for BC patients with low levels of FBW7 or with mutated FBW7.
In conclusion, our results suggest the crucial involvement of the This paves the way for the genetic characterization and expression profile of FBW7 to also be taken into account for personalized BC treatment in the future.

E TH I C A L A PPROVA L
The study was approved from the Institutional Review Board, the First Affiliated Hospital of China Medical University, Shenyang, China.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.