Background
Esophageal cancer (EC) is the leading cause of cancer mortality in China [
1]. The most common variant of esophageal cancer prevalent in China is esophageal squamous cell carcinoma (ESCC). Current therapies are concentrated on surgery, chemotherapy and radiotherapy, which offer poor prognosis with 5-year survival rate less than 20% [
1,
2]. Thus, targeted-therapy based on genetic alterations may give promise.
Cell cycle dysregulation indicated by abnormal expressions and variations (mutations, amplifications, and deletions) were noted to occur frequently in human malignancies [
3,
4]. Given its importance in cell cycle control, Cyclin D1-CDK4/6-Rb pathway is a highly validated anticancer drug target [
5]. Early in the G1 phase of cell cycle, Cyclin D1 activates CDK4/6, and phosphorylates Rb subsequently. Phosphorylation of Rb reduces the inhibitory control of the transcription factor E2F, which enables the cell to pass through the G1 restriction point into S-phase [
6]. Deregulation of the Cyclin D1-CDK4/6-Rb pathway triggered loss of cell-cycle control, one of the hallmark of cancer inducing carcinogenesis [
7]. Targeting CDK4/6 mediated Rb phosphorylation by small molecule inhibitors has the possibility to block cell cycle progression and suppress tumor growth [
8]. CDK4/6 has proven to be an effective target in diseases spanning breast cancer to colon cancer and neuroblastoma [
9‐
13]. CDK4/6 inhibitor has been granted FDA approval as breakthrough therapy of breast cancer. Genomic characterization has demonstrated that ESCC harbour amplification of CDK6 and Cyclin D1, deletion of p16, and mutations of Rb, which are important regulators of cell cycle [
14]. This suggests the potential utility of CDK4/6 inhibitors in ESCC.
Here, we aimed to evaluate the anti-tumor activity of SHR6390, which is an orally bioavailable, small molecule CDK4/6 inhibitor, in ESCC in vitro cell lines and in vivo PDXs models. Moreover, we investigated the possible mechanisms of SHR6390 and the effects of SHR6390 combined with paclitaxel (PTX) or cisplatin (CDDP). Finally, we sought to identify response markers known to be implicated in Cyclin D1-Rb-CDK4/6 signaling. This study will provide direct evidences for the future clinical trials.
Methods
Cell lines and reagents
ESCC cell lines Eca 109, Eca 9706 and KYSE-510 were obtained from the Cell Bank of the Peking Union Medical College (Beijing, China). The cells were cultured in RPMI-1640 media (Gibco-BRL, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and 1% penicillin and streptomycin (Gibco-BRL) in a humidified incubator (37 °C) with 5% CO2. The CDK4/6 inhibitor SHR6390 (purity ≥99%) which is a selective small-molecular CDK4/6 inhibitor was kindly provided by Jiangsu Hengrui Medicine Co., Ltd (Jiangsu, China). Paclitaxel (PTX) (purity ≥ 99.9%) was purchased from Beijing Union Pharmaceutical Factory (Beijing, China), and cisplatin (CDDP) (purity ≥ 99.9%) was purchased from Hospira Australia Pty Ltd (Australia). For in vitro studies, SHR6390 was dissolved in dimethyl sulfoxide at a stock concentration of 10 mmol/L and stored at −20 °C until further use.
Cell viability assay
Eca 109, Eca 9706 or KYSE-510 cells were seeded into 96-well plates at a density of 3–5 × 103 cells/well overnight. Cells were treated the next day with SHR6390 for 72 h, and then assessed for viability using the MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA) according to the manufacturer’s instructions. The absorbance was measured at 490 nm using a spectrophotometer. All experiments were repeated and read three times for each concentration.
RNA interference
The siRNAs targeting CDK6 and Cyclin D1 were purchased from RiBoBio Co., Ltd (Guangzhou, China), which transfected into Eca 109 or Eca 9706 cells using the Lipofectamine® 3000 reagent (Effectene, Qiagen, USA) according to the manufacturer’s protocol. At 24 h after transfections, cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco-BRL) for another 24 h before using for other experiments.
Western blots
Eca 109, Eca 9706, and KYSE-510 cells were starved in serum-free medium overnight, and SHR6390 were added for 24 h. The Cell pellets and tumor tissues of xenografts were lysed using RIPA Lysis Buffer (Beyotime Biotechnology, Jiangsu, China) on ice, containing complete protease inhibitor and phosphatase inhibitor cocktail (Roche, Switzerland). Protein concentrations were measured using the BCA Protein Assay Kit (Beyotime Biotechnology, Jiangsu, China). Protein samples were diluted to equal concentrations (40 μg), and separated by electrophoresis in 10–12% SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ). Antibodies used were against: CDK6 (sc-177) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Rb(#9313S), pRb(#9307S), CDK4(#12790), Cyclin D1 (#2978) (Cell Signaling Technology, Boston, MA, USA); β-actin (#014M4759) (Sigma-Aldrich, USA). All antibody except CDK6 (1:100) dilutions were 1:1000. Proteins were visualized using ECL plus Western Blotting Detection Reagents (GE Healthcare). Densitometry analysis of the Western blot protein was performed using the ImageJ software.
Cell cycle assay
After treated with SHR6390 for 24 h, cell pellets were harvested and fixed in 70% cold ethanol overnight at 4 °C. Fixed cells were stained with 50 μg/mL propidium iodide (BD Biosciences), and incubated for 30 min at room temperature in the dark. Cell cycle analysis was performed using a FACS Calibur system (BD Biosciences). Data were analyzed by ModFit 3.0 software (BD Biosciences).
Annexin V apoptosis assay
Cells were exposed to SHR6390 for 24 h and next were conducted by staining with Annexin V-Allophycocyanin (APC) and 7-amino-actinomycin (7-AAD) (BD Biosciences, Erembodegem, Belgium) for 15 min at room temperature in the dark, followed by flow cytometric analysis within 1 h (BD Biosciences). Cell apoptosis was analyzed by using the WinMDI 2.9 software (BD Biosciences).
Xenograft models in immunodeficiency (NOD/SCID) mice
Two kinds of xenograft models were used in our study. Eca 109 cells and Eca 9706 (1–2 × 10
6) cells were suspended in 100 μL of phosphate-buffered saline (PBS), and injected subcutaneously into the flanks of 6-week-old female NOD/SCID mice (Beijing HFK Bio-Technology Co., LTD, Beijing, China). Six patient-derived xenografts (PDX) were obtained according to a previously published report [
15]. Tumors were measured with fine calipers every 2 days. When tumors was about 150–200 mm
3, mice were randomized into six groups (5–6 per group) and treated with saline; SHR6390 (150 mg/kg weekly, oral gavage); PTX (3 mg/kg twice weekly, ip); or CDDP (3 mg/kg twice weekly, ip); SHR6390 and CDDP or PTX (see previous doses). Animals were treated for 3 weeks. Calculations in our study were used as follows:
$$ {\text{Tumor volume }} = {{\left( {{\text{Length }} \times {\text{ Width}}^{2} } \right)}/ 2} $$
$$ {\text{Tumor growth inhibition }}\left( {\text{TGI}} \right) = \Delta {\text{T}}/\Delta {\text{C }} \times 100\% $$
$$\begin{aligned} {\text{Tumor regression rate }}\left( {\text{TRR}} \right) & = {{\left( {{\text{V}}_{\text{pre}} {-}{\text{ V}}_{\text{post}} } \right)} / {{\text{V}}_{\text{pre}} }} \\ & \quad \times 100\% \end{aligned}$$
ΔT = tumor volume change of the drug-treated group on the final day of the study, ΔC = tumor volume change of the control group on the final day of the study, Vpre = pre-drug tumor volume, Vpost = post-drug tumor volume.
The collection of tissue samples was approved and supervised by the Research Ethics Committee of Peking University Cancer Hospital & Institute. All patients signed written informed consent for their samples to be used in the study. All animal experiments were performed in accordance with the animal experimental guidelines of Peking University Cancer Hospital and followed internationally recognized ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.
Statistical analysis
Statistical analysis was performed with SPSS 20.0 software. For in vitro studies, differences between groups were conducted by one-way ANOVA, unpaired two-tailed t test or factorial analysis. For in vivo studies, tumor growth in different groups was compared using repeated measures ANOVA. P < 0.05 was considered statistically significant.
Discussion
The antitumor activity of CDK4/6 inhibitor were observed in many preclinical studies and the encouraging results from these studies have prompted clinicians to evaluate its efficacy and safety in clinical trials. In a single-arm study, five of 17 patients with relapsed mantle cell lymphoma remained progression-free for more than 1 year on CDK4/6 inhibitor therapy, with one complete response (CR) and two partial responses (PRs) [
16]. The phase 2 study PALOMA-1 involved 165 postmenopausal women with advanced ER-positive/HER2-negative breast cancer who had not received any systemic treatment for their advanced disease. All patients were randomly assigned 1:1 to receive letrozole or letrozole plus CDK4/6 inhibitor. After a median follow-up of 29 months, median PFS was 20.2 months for the CDK4/6 inhibitor combined with letrozole group and 10.2 months for the letrozole group [
9]. However, the role of CDK4/6 inhibitor in treating ESCC is still unclear.
Given the presence of highly mutation loads in Cyclin D1-CDK4/6-Rb signalling pathway-related molecules of ESCC [
9‐
11], we investigated the effect of CDK4/6 inhibitor SHR6390 in our study. Treatment of SHR6390 suppressed cell proliferation and tumor growth in ESCC cell lines and xenografts by inhibiting levels of pRb and effectively arrested the cell cycle at the G1 phase with the expression levels of p53 and p21 were upregulated. Hypophosphorylation of Rb due to CDK4/6 inhibition lead to the inhibition of E2F activity which is crucial for DNA synthesis and progression of cell cycle [
17]. Li et al. reported that CDK4/6 inhibitor treatment induces cell cycle arrest at the G1 phase and thus suppresses the proliferation of colorectal carcinoma cells [
18], which was similar to our results. p21 is one of the CDK inhibitor, which considered as the most potent regulator of cell cycle. It involved in blocking G1 cell cycle progression, which is controlled in a tumor suppressor p53-dependent manner [
19]. On the other hand, SHR6390 treatment failed to induce significant apoptosis in Eca 109 cells, dedicating that SHR6390 inhibited tumor growth mainly through arresting cell cycle at G1 phase.
Our data demonstrated that combination therapy yield greater anti-tumor activity than individual treatment in Eca 9706 xenografts. It has been reported that CDDP could induce DNA damage-mediated cytotoxicity in cells that are arrested in the G1 phase [
20]. Thus, SHR6390 mediated G1 accumulation of cells may sensitize them to CDDP. Also, Zhang et al. reported that combined CDK4/6 inhibition and paclitaxel produced synergistic antitumor activity and increased apoptosis through reduced Cyclin D1 and Bcl-2 in cancer cells [
21]. Modeling studies indicate that combinations of effective cytostatic and cytotoxic drugs should increase cure rates by delaying drug resistance and preventing tumor growth between treatments with cytotoxic agents [
5]. However, CDK4/6 inhibitors combined with standard cytotoxic chemotherapy showed conflicting results in preclinical studies. It has been reported that dual inhibition of CDK4/6 inhibitor and gemcitabine enhanced the antitumor effect in a xenograft model of lung cancer [
22] and CDK4/6 inhibition also sensitized neuroblastoma cells to doxorubicin-induced apoptosis [
23]. In contrast, CDK4/6 inhibitor reduced the cytotoxicity of antimitotic and platinum agents in preclinical models [
24‐
26]. In future, we will optimize the combination regimen and search for other possible targets in pathways of PI3K-mTOR [
27,
28] or MEK-ERK [
10,
29], which may enhance the efficacy of CDK4/6 blockade.
The combination of CDK4/6 inhibitors and other therapy is being used in the clinic in unselected patient populations, but not all patients will benefit from such therapy [
9]. Thus, there is an urgent need to identify biomarkers that predict response to CDK4/6 inhibitor. Preclinical studies have defined a series of biomarkers that respond to CDK4/6 inhibitors, of which the Cyclin D1-CDK4/6-Rb pathway has been the best one till now [
30,
31]. Alterations in the expression of genes that related to the cell cycle are important in determining drug sensitivity to anticancer agents. We therefore sought to validate biomarkers that predict in vitro response to SHR6390 in cell cycle signaling. Our results show that low expression of CDK6 may correlated with high sensitivity of SHR6390. Similarly, Yang et al. reported that knockdown of CDK6 restored CDK4/6 inhibitor sensitivity, while enforced overexpression of CDK6 was sufficient to mediate drug resistance in breast cell lines. High CDK6 expression may affect response to CDK4/6 inhibition by preferentially binding to Cyclin D3, creating a resistant complex than the Cyclin D1-CDK4 complex [
30]. Meanwhile, our results showed that higher expression of Cyclin D1 was correlated with SHR6390 higher sensitivity. However, in the PALOMA-1 study, patient selection based on Cyclin D1 amplification was not correlated with better outcome [
9]. In addition to Cyclin D1 and CDK6 status, CDK4 has been proposed as a predictive biomarker of response to CDK4/6 inhibition [
31]. However, our results indicated that level of CDK4 expression has no relationship with the sensitivity to SHR6390 (data not shown). Also, Rb status has been proposed as a selective biomarker of CDK4/6 inhibitor utility [
17]. It has been reported that the efficacy of CDK4/6 inhibitors requires functional Rb expressing in tumor cells. However, variable growth inhibition among Rb-proficient cell lines and PDXs models suggests the existence of other factors that influence tumor cell sensitivity to SHR6390.
Authors’ contributions
LS and JG conceived and designed the study. JW and QL performed the experiments. JY, JW and QL analyzed the data. ZC, ZL, JW, ZL and YL contributed reagents, materials, and analysis tools. JW and JG wrote the manuscript. All authors read and approved the final manuscript.