Background
Prostate cancer, known as carcinoma of the prostate, is the most commonly diagnosed solid tumor among men. Despite new technology has enabled medical researchers to develop more reliable, less invasive screening and treatment methods, prostate cancer still remains the second leading cause of cancer-related death in male [
1]. The failure of clinical treatment in patients with prostate cancer is often due to the heterogeneous nature of the disease. Comparative understanding of genetic pathways involved in the development and progression of prostate cancer may help define novel therapeutic targets or/and identify treatment regimens that are very likely to provide therapeutic benefit to patients.
Disrupting normal function of microtubules can engage the spindle checkpoint and arrest cell cycle progression at mitosis, leading to cell death [
2]. Therefore, targeting microtubules is one of the efficient strategies for cancer treatment. A bunch of clinical chemo drugs, such as vinblastine, docetaxel, and paclitaxel, have potential to suppress microtubule dynamics without affecting microtubule polymer mass [
2–
4]. Although these microtubule-targeting agents (MTAs) are widely used to treat different types of human cancers [
5–
7], substantial drawbacks such as narrow therapeutic windows and lack of oral bioavailability still remain [
8]. Moreover, the potential side effects (e.g., neural toxicity) and cardio-vascular events (e.g., thromboembolism) largely limit therapeutic efficacy of MTA as single agents against cancer. To overcome these clinical problems, many research efforts have concentrated on developing novel MTAs. CYT997 is a new microtubule-disrupting agent screened from Cytopia’s small molecule library, which has been proven to possess varying degrees of activity against cancers through inhibiting tubulin polymerization and disrupting cellular microtubules [
9–
11]. In phase I clinical trials, the safety, efficacy, and pharmacokinetics of CYT997 in cancer patients have been investigated [
9].
Src, a non-receptor tyrosine kinase, has been implicated in a variety of different cancer types as well as in progression to malignancy [
12–
14]. Activation of Src is associated with its translocation to the plasma membrane in which Src interacts with a number of important effectors in response to extracellular signals. Regardless of which mechanism for Src activation, Src promotes numerous properties associated with metastatic potential once it is phosphorylated on tyrosine residues [
12,
13]. It has been reported that Src trafficking requires both microtubules and actin polymerization [
15], which may link Src functions to MTAs. In hormone-dependent cancers (e.g., breast and prostate cancer), steroid hormones trigger association of the androgen receptor (AR)-estradiol receptor (ER) complex with Src. A synthetic 10 amino-acid peptide that mimics the sequence of the SH3 domain-mediated binding of Src can prevent the AR/ER complex from associating with Src and inhibit the growth of LNCaP xenografts established in nude mice [
16]. A study from Dr. Migliaccio’s group shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells [
17]. Preclinical studies also established a role of Src in progressive stages of prostate cancer and cross talk between Src and AR signaling [
18]. AR can also activate MAP kinase (MAPK) through an early and a late response pathway in a cell type-dependent manner [
19]. The combination of the Src inhibitor PP2 and antiandrogen Casodex does not further decrease the invasion of the LNCaP derivative C4-2 cells [
20], suggesting that Src-assisted invasion may involve nongenomic AR actions whereby MAPK axis stimulation by AR signaling. Recent studies further reported that the Src inhibitors (e.g., dasatinib, saracatinib, and bosutinib) can inhibit motility, migration, and invasion of androgen-dependent and androgen-independent prostate cancer cells in a dose-dependent manner [
21,
22].
In this study, we show that CYT997 effectively inhibits proliferation, viability, and invasion of prostate cancer cells. Genetic or pharmacological blockage of Src sensitizes prostate cancer cells towards CYT997 regardless of AR expression. Synergistic treatment with CYT997 and the Src inhibitor dasatinib exhibits a superior anticancer effect in mouse models of prostate cancer. These findings provide a molecular and cellular basis for CYT997 treatment, suggesting a clinical evaluation of CYT997 in combination with dasatinib for the treatment of patients with prostate cancer.
Methods
Cell lines, reagents, antibodies, and standard assays
Prostate cancer cell lines PC3, DU145, LNCaP, and 22Rv1 were obtained from ATCC and passage <5 were used in this study. LNCaP-derivative C4-2 and C4-2B cells were a gift from Dr. Daqing Wu (Georgia Cancer Center). All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Luciferase stable PC3 cells were generated by transduction of pGL4.5 vector (Promoga, Madison, WI) encoding the luciferase reporter gene luc2 and selection of hygromycin. pLKO.1 lentiviral vectors harboring small hairpin RNAs (shRNAs) targeting Src were obtained from Open Biosystems (Huntsville, AL). CYT997 and dasatinib were purchased from Selleckchem (Houston, TX). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) and D-Luciferin bioluminescent substrate were purchased from Sigma-Aldrich (St Louis, MO). Antibodies that recognize p62, p-AKT (Ser473), p-ERK1/2 (Thr202/Tyr204), p-STAT3 (Tyr705), p-Src (Tyr416), AKT, ERK1/2, STAT3, Src, LC3B I/II, and cleaved (c)-PARP were purchased from Cell Signaling Technology (Beverly, MA). β-Actin and Ki67 antibodies were purchased from Sigma-Aldrich (St Louis, MO) and Abcam (Cambridge, MA), respectively. Western blot, cell proliferation, and electrochemical detection were carried out as described previously [
23–
28]. Matrigel invasion assays were performed by transwells from BD biosciences (San Jose, CA) as described previously [
23–
26]. Briefly, 5 × 10
4 serum-starved cells were seeded into Matrigel-coated Boyden chambers in the presence or absence of the indicated concentrations of CYT997, and DMSO containing 10% FBS was added to the lower chamber. After 24-h treatment, the membranes that contained invading cells were fixed in methanol and stained with 0.2% Crystal violet. The dye was dissolved in 10% acetic acid and read colorimetrically at 590 nm for quantification of invasion. Cell viability was determined by CellTiter-Glo® Luminescent cell viability assay (Promega, Madison, MI) and Zombie Aqua™ fixable viability kit (BioLegend, San Diego, CA). Flow cytometry data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Detection of apoptosis
The Cell Death Detection Elisa kit (Roche, Indianapolis, IN) was used to determine apoptosis by measuring mono- and oligonucleosomes in the lysates of apoptotic cells according to the manufacturer’s protocol. Briefly, the cells treated with different drugs were lysed and placed into a streptavidin-coated microplate and incubated with a mixture of anti-histone-biotin and anti-DNA-peroxidase. The amount of peroxidase retained in the immunocomplex was photometrically determined with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as the substrate. Absorbance was measured at 405 nm (492 nm as reference wavelength).
Animal models
Six-week-old male NSG (NOD.Cg
-Prkdc
scid
Il2rg
tm1Wjl
/SzJ) mice were purchased from the Jackson Laboratory (Bar Harbor, ME), and all animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Augusta University. To generate a xenotransplantation model, exponentially growing PC3 cells (1 × 10
6 cells) were suspended in 100 μl PBS/matrigel (1:1) and injected subcutaneously into the right flanks of NSG mice. To generate an intracardiac model, NSG mice were injected via intracardiac with 1 × 10
6 luciferase-containing PC3 cells.
Drug administration and immunohistochemistry
Four days after PC3 cell implantation, mice were randomized to receive control vehicle and drug(s) (
n = 5). The treatment groups received followed equal volume treatment of CYT997 (20 mg/kg), dasatinib (10 mg/kg), or in combination of CYT997 (20 mg/kg) and dasatinib (10 mg/kg), respectively. CYT997 was administered by garage twice per day for a total of 3 weeks, and dasatinib was intraperitoneally (i.p.) administered 5 days per week for a total of 4 weeks. The control mice were injected i.p. with 100 μl sterile saline. Tumor growth was measured externally every 4 to 7 days using vernier calipers as length × width
2 × 0.52. The mice were sacrificed on treatment day 42, and the lungs were removed and processed for immunohistochemistry (IHC) with Ki67 antibody and pathological analysis by HE staining as described previously [
29,
30]. For intracardiac models, mice were randomized for vehicle (sterile saline) or combined (20 mg/kg CYT997 with 10 mg/kg dasatinib) treatment. Mice were imaged for luciferase signal by an intraperitoneal injection of luciferin (15 μg in 100 μl PBS) every other week for 4 weeks using a Xenogen IVIS-200 In Vivo Imaging System (PerkinElmer, Waltham, MA).
Statistical analysis
Treatment effects were evaluated using a two-tailed Student
t test at each measurement time point. To assess the longitudinal effect of treatment, a mixed model was employed to test the overall difference across all groups as well as between each pair of groups during the whole study period. The data were presented as means ± SD from three or more independent experiments, and a
p value less than 0.05 was considered significant.
Discussion
Microtubules are essential for cell growth, division, motility, intracellular trafficking, and the ability to adapt to a variety of shapes to interact with the environment [
2]. Suppression of microtubule dynamics is a common mechanism of chemotherapeutic agents to block mitosis and kill tumor cells. CYT997, a novel anticancer MTA with a favorable combination of pharmacologic and pharmacokinetic properties and oral bioavailability, is currently undergoing clinical trials in a variety of cancer indications [
9–
11]. This work demonstrates the therapeutic efficacy of CYT997 in either cultured prostate cancer cells or mouse models of prostate cancer and reveals the possible mechanisms involved in drug action.
The therapeutic goal of cancer treatment has been to trigger tumor-selective cell death by apoptosis, and drug-mediated autophagy is increasingly recognized as an important factor in tumor apoptosis or survival [
31]. Autophagic changes were not seen in the CYT997-treated prostate cancer cells, excluding the possibility that CYT997 promotes apoptosis in prostate cancer cells by accumulation of autophagy. Oxidative stress is one of the other contributing factors to apoptosis. CYT997 induces an increase of ROS levels and O
2
·− release in DU145 cells, but not in PC3 cells. We also determined oxidative stress in AR-positive LNCaP cells with or without CYT997 treatment and observed the similar results seen in PC3 cells (data not shown). This discrepancy may be due to genetic background of the cell lines used in our study. One of our follow-up investigations is to discover possible molecular mechanisms mediating the apoptotic effect of CYT997.
SRC kinase activity has been found to be upregulated in a number of preclinical models of prostate cancer, and a potentially effective strategy to inhibit prostate cancer growth and metastasis is to target SRC kinases [
21,
22]. Dasatinib inhibits multiple tyrosine kinases including SRC kinases, which are implemented to promote androgen independence in metastatic castration-resistant prostate cancer (mCRPC) that makes malignant cells unresponsive to therapies [
32,
33]. Some of the common chemo drugs used to treat prostate cancer are MTAs. For example, docetaxel (Taxotere) stabilizes microtubules through binding to polymerized microtubules at the inner surface of the β subunit [
34,
35]. As suggested by the phase III docetaxel plus dasatinib study, combination treatment is most effective in delaying and/or preventing skeletal metastases from prostate cancer [
36]. However, no survival benefit was observed for the addition of dasatinib to docetaxel versus docetaxel/placebo [
36,
37]. Despite the disappointing results seen from studies evaluating dasatinib in combination with other agents, clinical trials are needed to address whether dasatinib may be used as a single agent or in combination with chemotherapy mediated by novel MTAs.
There is a clear potential for CYT997 to be used in all prostate tumors carrying AR or not and perhaps all clinical stages of prostate cancer since it can greatly inhibit nearly all prostate cancer cell lines. Our data indicate that CYT997 possess highly potent cytotoxic activity through inhibiting the PI3K/AKT and MAPK oncogenic signaling cascades. However, CYT997 cannot block the Src pathway at the examined concentrations. Considering that monotherapy frequently fails to produce an adequate response and combining cancer drugs with disparate mechanisms of action is a feasible strategy to achieve high anticancer activities, we treated prostate cancer cells with CYT997 and dasatinib. This drug combination shows synergistically decreased cell proliferation and increased apoptosis, leading to distinctly improved anticancer activities in vitro and in vivo. In the present study, we generated two types of prostate cancer mouse models using PC3 cells. The combination of CYT997 and dasatinib inhibits both prostate tumor growth and metastasis in these mouse models, providing a rational basis for the clinical treatment for patients with prostate cancer. Since the majority of mCRPC contains AR or even overexpresses it, use of different cells such as C4-2B (a cell line can produce osteoblastic metastases in the lumbar spine) would provide another model to explore the mechanisms of drug action and add value to the obtained findings. Future trials should identify patient subpopulations with elevated activation of Src in prostate cancer and better define the biologic processes of prostate cancer progression that are specifically “Src-driven.” Combination treatment of CYT997 and dasatinib may be developed as a novel therapy to improve clinical outcomes for prostate cancer carrying high levels of activated Src.
Acknowledgements
We would like to thank Kongxiang Yang and Tian Jiang for the assistance with the data collection.