This article has Open Peer Review reports available.
KPT-330, a potent and selective exportin-1 (XPO-1) inhibitor, shows antitumor effects modulating the expression of cyclin D1 and survivin in prostate cancer models
© Gravina et al. 2016
Received: 26 December 2014
Accepted: 16 November 2015
Published: 1 December 2015
The Erratum to this article has been published in BMC Cancer 2016 16:8
Background and aims
Increased expression of Chromosome Region Maintenance (CRM-1)/exportin-1 (XPO-1) has been correlated with poor prognosis in several aggressive tumors, making it an interesting therapeutic target. Selective Inhibitor of Nuclear Export (SINE) compounds bind to XPO-1 and block its ability to export cargo proteins. Here, we investigated the effects of a new class of SINE compounds in models of prostate cancer.
Material and methods
We evaluated the expression of XPO-1 in human prostate cancer tissues and cell lines. Next, six SINE (KPT-127, KPT-185, KPT-205, KPT-225, KPT-251 and KPT-330) compounds having different potency with broad-spectrum, tumor-selective cytotoxicity, tolerability and pharmacokinetic profiles were tested in a panel of prostate cancer cells representing distinct differentiation/progression states of disease and genotypes. Two SINE candidates for clinical trials (KPT-251 and KPT-330) were also tested in vivo in three cell models of aggressive prostate cancer engrafted in male nude mice.
Results and conclusions
XPO-1 is overexpressed in prostate cancer compared to normal or hyperplastic tissues. Increased XPO-1 expression, mainly in the nuclear compartment, was associated with increased Gleason score and bone metastatic potential supporting the use of SINEs in advanced prostate cancer. SINE compounds inhibited proliferation and promoted apoptosis of tumor cells, but did not affect immortalized non-transformed prostate epithelial cells. Nuclei from SINE treated cells showed increased protein localization of XPO-1, survivin and cyclin D1 followed by degradation of these proteins leading to cell cycle arrest and apoptosis. Oral administration of KPT-251 and KPT-330 in PC3, DU145 and 22rv1 tumor-bearing nude mice reduced tumor cell proliferation, angiogenesis and induced apoptosis. Our results provide supportive evidence for the therapeutic use of SINE compounds in advanced/castration resistant prostate cancers and warrants further clinical investigation.
Prostate cancer (PCa) is the second leading cause of cancer mortality in males >40 years of age in the USA and the third most common cause of cancer-related mortality in males . PCa is generally a slow developing cancer, and 5- and 10-year relative survival rates of early stage PCa are 99 and 95 %, respectively . Although hormone therapy is initially very effective, almost all tumors relapse to a hormone refractory stage. In the past, it was presumed that the expression of the androgen receptor (AR) is lost in the cells of advanced, hormone-refractory tumors but AR is rarely lost in human PCa specimens in vivo, even in those of CRPC . Not only that AR is not lost, but it is transcriptionally active in the majority of recurrent CRPC . There is experimental evidence that the Akt, mTOR and glycogen synthase kinase-3 (GSK-3β) pathways are involved in AR signaling [5, 6]. GSK-3β binds to the AR, forming a complex in the cytoplasm that are then imported into the nucleus upon androgenic stimulation. Inhibition of GSK-3β by activation of Akt/mTOR pathways results in increased nuclear export of AR and this export can be abrogated by the inhibition of XPO-1. GSK-3β/XPO-1 activity also regulates the levels of several nuclear and cytoplasmic proteins including survivin [7, 8] and cyclin D1 , which modulate cell division and apoptosis. Advanced castration resistant prostate cancer (CRPC) tumors are characterized by the activation of PI3K/AKT [9, 10]. One of the major effects of the activation of this pathway is XPO-1 dependent nuclear export of the tumor suppressor protein (TSP) FOXO into the nucleus, thus abolishing its activity . Normally, low levels of FOXO protein are found in the cytoplasm. Shortly after SINE treatment, FOXO begins to accumulate in the nucleus where it binds to DNA and induces gene transcription that results in cancer cell death [12, 13].
Cancer cells utilize nuclear-cytoplasmic transport through the nuclear pore complex to effectively evade apoptosis and promote growth [14, 15]. XPO-1-mediated export is increased in various cancers [16–19]. Examples of nuclear proteins that are exported into the cytoplasm in cancer include the drug targets topoisomerase (topo) IIα  and tumor-suppressor proteins such as p53 , p21 , and p27 . Use of XPO-1 inhibition in cancer therapy has been met with limited success. The first studied XPO-1 inhibitor was the anti-fungal natural antibiotic leptomycin B. It was found to efficiently inhibit nuclear export , but induced acute toxicities both in vitro  and in a human phase I trial . Other XPO-1 inhibitors [for review see 14, 15] examined in various studies include compounds such as ratjadone , KOS-2464 , FOXO export inhibitors , valtrate , acetoxychavicol acetate , CBS9106  and SINE (Selective Inhibitors of Nuclear Export) [33–43]. Recent publications have indicated that SINE compounds may be effective against various malignancies, including leukemia , breast cancer [35, 36] kidney cancer , mantle cell lymphoma , melanoma , multiple myeloma (MM) , pancreatic cancer , mesotelioma  and metastatic PCa . For these reasons, we focused our attention of XPO-1 inhibition as therapeutic tool for the treatment of cancer, including PCa. In this study, we show that XPO-1 inhibition using SINE compounds: KPT-185, KPT-205, KPT-225, and KPT-127 reduced, in a panel of PCa cells, XPO-1-dependent nuclear export of different proteins including AR, Foxo3a, and survivin, modulating cell cycle progression through both a G1 and a G2/M-arrest followed by apoptosis. Clinical candidate KPT-251 and KPT-330 were also tested in vivo in three models of aggressive PCa.
Reagents and drug preparation
All materials for tissue culture were purchased from Hyclone (Cramlington, NE, USA). Plasticware was obtained from Nunc (Roskilde, Denmark). Antibodies including p-GSK3β Ser9 [sc11757], GSK3β [H76, sc-9166], XPO-1 [sc-5595], P53 [sc126], p-Akt Ser473 [sc-135651], p-Akt Thr308 [sc135650] and Rad-51 [sc8349], were purchased from Santa Cruz (SantaCruz, CA, USA). Antibodies including CD3 [ab28364], CD68 [ab955], FoxO3a [ab37409], Iκb [ab32518] were purchased from Abcam (Cambridge UK). An antibody against Cyclin D1 [#2878] was purchased from Cell Signaling (Danvers, MA, USA). A FAS antibody [VP-F702] was purchased from Vector labs (Burlingame, CA, USA). Ki67 antibody (clone MIB-1) was purchased from Dako (Dako Italia, Cernusco sul Naviglio [MI], Italy). Tunel assay kit [S71003] was purchased from Merck KGaA (Darmstadt, Germany). Survivin antibody was purchased from Biorbyt.
SINE compounds (KPT-127, −185, −205, −225, −251, −330) were provided by Karyopharm Therapeutics Inc., Natick, MA. KPT-251 and −330 are suitable for in vivo use. SINE compounds were dissolved in DMSO and stored at −20 °C until use.
A cohort of 50 adult patients with clinically localized PCa, collected as previously described . In addition, we analyzed 4 lymph nodal metastases from PCa patients and 57 samples of two tissue arrays from primary tumors (49 cases) and bone metastases (8 cases) purchased from US Biomax (Rockville, MD, USA). A total of 121 cases including 99 primary tumors, 4 lymph node metastases and 8 bone metastases were included. This research was carried out in accordance with the Helsinki Declaration and the study was approved by the San Salvatore Hospital Ethics Committee, L'Aquila, Italy with Deliberation n. 89/2006. We obtained also a written informed consent of patients.
XPO-1 expression was evaluated on 4 μm tissue sections cut from blocks selected for the presence of representative tumor tissue. The pathologic evaluation and IHC results were interpreted by a uro-pathologist. First, nuclear and cytoplasmic staining of XPO-1 in tumor tissue was scored blindly (LV) using a semi-quantitative immunoreactivity scoring (IRS) system. Category A scored the intensity of immunostaining as 0 (no immunostaining), 1 (weak immunostaining), 2 (moderate immunostaining), and 3 (strong immunostaining). Category B scored the percentage of immunoreactive cells as 0 (none), 1 (<10 %), 2 (10–50 %) and 3 (>50 %). Multiplication of A and B resulted in an IRS from 0 to 9. An IRS of 4 or greater was considered high for expression of XPO-1. This analysis was performed for both nuclear and cytoplasmic staining. Global staining (GIRS) was the sum of nuclear and cytoplasmic staining and ranged between 0 and 18. A GIRS of 8 or greater was considered high for expression of XPO-1.
Four commercial (LnCaP, 22rv1, DU145 and PC3) and twelve non-commercial (LAPC-4 , CWR22, PCb2 , PC3Lymphnode , PC3M, PC3M-pro4  and PC3M-Ln4 , LnCaP-104S , LnCaP104R1 , LnCaP-C81 , C4-2B , VCAP  and DuCaP [49) cell lines were selected for the in vitro experimental studies for their biological characteristics: LAPC-4 (Androgen receptor [AR] positive, androgen dependent with low Akt/mTOR activities, p53 wt); LnCaP (AR positive, androgen dependent with high Akt/mTOR activities, p53 wt); LnCaP-C81 and LnCaP-C4-2B (AR positive, androgen independent with high Akt/mTOR activities, p53 wt); 22rv1 (AR positive, androgen independent with low Akt/mTOR activities, p53 wt); PC3 (AR negative, with high Akt/mTOR activities and no p53 function (p53 del) and DU145 (AR negative, with low Akt/mTOR activities and mutant p53). PC3 and DU145 have been also transfected with AR to obtain PC3AR  and DU-AR . Benign prostatic hyperplasia line (BPH1), and Prostatic epithelial lines (EPN and RWPE-1) were used as non-neoplastic controls. Cells were routinely cultured in appropriate medium supplemented with 10 % fetal bovine serum (FBS), 1 % pen-strep and 1 % L-glutamine (Invitrogen Corporation, Carlsbad, CA). To minimize the risk to work with misidentified and/or contaminated cell lines, DNA profiling was periodically carried out in-house to authenticate cell cultures. DNA was isolated from cell lines using a standard DNA isolation kit. STR profiling was performed by using GenePrint® 10 System (Promega Corporation, Madison, WI). An eight-capillary 3500 Genetic Analyzer (Applied Biosystems Life Technologies Europe BV, Monza, Italy) was used to separate and identify alleles using standard procedures. GenePrint® 10 System allows co-amplification and detection of eight human loci required by the guidelines ASN-0002. For non-commercial cell lines, the authentication process was carried out by comparing STR-fingerprints with those published by Adri van Bokhoven and co-workers . In addition, cell lines were stocked at very low passages and used at <15-20 subcultures.
Cells were seeded at a density of 2 x 104 cells/mL in 24-well plates. Cells were left to attach and grow in 5 % FCS DMEM for 24 h. After this time, cells were maintained in the appropriate culture conditions. Morphological controls were performed every day with an inverted phase-contrast photomicroscope (Nikon Diaphot, Tokyo, Japan) before cell trypsinization and counting. Cells were trypsinized and resuspended in 1.0 ml of saline, then counted using a NucleoCounter™ NC-100 (automated cell counter systems, Chemotec, Gydevang, Denmark). The effect on cell proliferation was measured by taking the mean cell number with respect to controls over time for the different treatment groups.
Cell viability and apoptosis assay
Viable cells were counted by using the NucleoCounter™ NC-100 (automated cell counter systems, Chemotec, Cydevang, DK). Apoptosis was evaluated by using Tali® Apoptosis Kit - Annexin V Alexa Fluor® 488 & Propidium Iodide-based, (Life Technologies Italia, Monza, Italy). Stained cells were then measured on a Tali® Image-Based Cytometer. Apoptosis was further confirmed by FACS analysis following the instructions of the manufacturer.
Cytoplasmic and nuclear protein extracts were obtained by using the Nuclear/Cytosol Fractionation Kit from Biovision Inc. (Milpitas, CA, USA). Cell extracts and conditioned media from treated and untreated cells were electrophoresed under reducing conditions and transferred to nitrocellulose filter (Schleicher and Schuell GmbH, Dassel, Germany). Non specific binding sites were blocked for 1 h in 5 % non-fat dried milk in a Tris buffer containing 20 mM Tris and 137 mM NaCl (pH 7.6). Blots were incubated with 1 μg/ml of primary antibody diluted in blocking solution for 1 h at room temperature, washed and then incubated for 1 h in secondary antibody diluted 1:3000 in blocking solution. Following an additional wash, reactive bands were visualized by a chemiluminescent detection kit (Supersignal, Perbio Science, Tattenhall, UK) using Bio-Rad gel Doc™ (Bio-Rad Laboratories S.r.l., Milan, Italy).
Male CD1 nude mice (Charles River, Milan, Italy) were maintained under the guidelines established by the University of L’Aquila, Medical School and Science and Technology School Board Regulations. Experiments on animals have been approved by your local IRB in compliance with the Italian government regulation n.116 January 27, 1992 for the use of laboratory animals which is line with ARRIVE guidelines. All mice received subcutaneous flank injections of 1 x 106 PC3, DU145 or 22v1 cells. Tumor growth was measured bi-weekly with a Vernier caliper (length x width). Tumor weight was calculated according to the formula: TW (mg) = tumor volume (mm3) = d2 x D/2, where d and D are the shortest and longest diameters, respectively. The effects of the treatments were examined as previously described . Animals were sacrificed by carbon dioxide inhalation and tumors were subsequently removed surgically. A piece of tumor was frozen in liquid nitrogen for protein analysis and another piece was fixed in paraformaldehyde overnight for immunohistochemical analyses.
Martius yellow-brilliant crystal scarlet blue technique
Stains were purchased from HD Supplies (Aylesbury, UK) and used to analyze the presence of red cells in tumor tissue and blood vessels, as well as to better evaluate the presence of micro-thrombi and bleeding zones. Martius yellow, a small molecule dye, together with phosphotungstic acid in alcoholic solution stains red cells. Early fibrin deposits may be colored, but the phosphotungstic acid blocks the staining of muscle, collagen and connective tissue fibers. Brilliant crystal scarlet, a medium sized molecule, stains muscle and mature fibrin. Phosphotungstic acid removes any red stain from the collagen. The large molecule dye aniline blue stains the collagen and old fibrin.
Indirect evaluation of angiogenesis was performed by using tumor hemoglobin levels as previously described . Tumors were homogenized in double-distilled water. Eighty microliters of homogenates were mixed with 1 ml of Drabkin’s solution and incubated for 15 min at room temperature. After centrifugation at 400 x g for 5 min, the supernatants were subjected to absorbance measurement at 540 nm. The absorption, which is proportional to hemoglobin concentration, was divided by tumor weight.
Mice were treated by oral gavage with either vehicle control (Pluronic F-68/PVP-K29/32), KPT330 or KPT251. Before tumor injection, animals were randomized into seven treatment groups as follows: Group 1: mice (10 animals) receiving 100 μl vehicle PO; Group 2: mice (10 animals) receiving 100 mg/kg KPT-251 every two days (Monday and Friday)/week PO; Group 3: mice (10 animals) receiving 30 mg/kg KPT-251 every two day/week PO; Group 4: mice (10 animals) receiving 10 mg/kg/day PO; Group 5: mice (10 animals) receiving 30 mg/kg KPT-330 every two days/week PO; Group 6: mice (10 animals) receiving 10 mg/kg KPT-330 every two days/week PO; Group 7: mice (10 animals) receiving 3 mg/kg/day KPT-330 PO. Treatments were started when tumor volumes reached approximately 80 mm3 (Day 0) and were stopped after 28 days. The following parameters were used to quantify the antitumor effects upon different treatments: (1) tumor volume measured during and at the end of experiments, (2) tumor weight measured at the end of experiment, (3) complete response (CR) defined as the disappearance of the target lesion with respect to baseline, (4) partial response (PR) defined as a reduction of greater than 50 % of tumor volume with respect to baseline, (5) stable disease (SD) defined as a reduction of less than 50 % or an increase of less than 100 % of tumor volume with respect to baseline, (6) tumor progression (TP) defined as an increase of greater than 50 % of tumor volume with respect to baseline, (7) time to progression (TTP). In vivo, combinational studies were evaluated by CalcuSyn (Biosoft). For the calculation of CI, the values of cell kill for a fixed tumor volume were considered (determined by the log cell kill (LCK)). LCK was determined as LCKZ (TKC)/(3.3KTd), where Td represents the mean control group doubling time required to reach a fixed tumor volume, expressed in days, whereas T and C are the same values as described above .
Continuous variables were summarized as the mean and SD or 95 % CI for the mean. Statistical comparisons between controls and treated groups were established by carrying out the ANOVA test or by Student’s t-test for unpaired data (for two comparisons). Dichotomous variables were summarized by absolute and/or relative frequencies. For dichotomous variables, statistical comparisons between control and treated groups were established by carrying out the exact Fisher’s test. For multiple comparisons, the level of significance was corrected by multiplying the P value by the number of comparisons performed (n) according to the Bonferroni correction. Overall survival was determined by Kaplan–Meier analysis and a Gehan's generalized Wilcoxon test. When more than two survival curves were compared, the Logrank test for trend was used. This tests the probability that there is a trend in survival scores across the groups. All tests were two-sided and were determined by Monte Carlo significance. P values <0.05 were considered statistically significant. In the figures in which statistical analysis was performed, significance is indicated by an asterisk. SPSS (statistical analysis software package, IBM Corp., Armonk, NY, USA) version 10.0 and StatDirect (version. 2.3.3, StatDirect Ltd, Altrincham, Manchester, UK) were used for statistical analysis and graphical presentation.
Expression of XPO-1 in human prostate samples
Expression of XPO-1 in human prostate cancer lines
Next, we analyzed and quantified the expression of XPO-1 in prostate cancer, normal or neoplastic prostate epithelial cells by immunoblots and by adjusted densitometry units. XPO-1 protein levels were high in prostate cancer when compared to non neoplastic prostate epithelial cells (Fig. 1d, e). Statistical analyses reveal that the XPO-1 expression levels were statistically lower in AR positive (LAPC-4, CWR22, LnCaP, LnCaP-104S, LnCaP-104R1, LnCaP-C81, C4-2B, 22rv1, DuCaP, VCaP, PC3AR and DU145AR) when compared to those observed in AR negative (PC3 and PC3 variants [PC3PTEN, PC3M-pro4, PC3M-Ln4, PC3Me, PCb2] and DU145) PCa cells lines (1.39 ± 0.38 vs 2.57 ± 0.39, P = 0.0015, Fig. 1f). The XPO-1 levels were statistically lower in androgen dependent (LAPC-4, CWR22, LnCaP, LnCaP-104S, DuCaP, VCaP, PC3AR and DU145AR) when compared to those observed in androgen independent/CRPC (LnCaP-104R1, LnCaP-C81, C4-2B, 22rv1, PC3 and PC3 variants [PC3PTEN, PC3M-pro4, PC3M-Ln4, PC3Me, PCb2] and DU145) PCa cell lines (1.41 ± 0.47 vs 2.44 ± 0.52, P = 0.0150, Fig. 1g) The comparison in PC3 cell derivatives showed higher XPO-1 levels in more aggressive/metastatic cells.
Inhibition of XPO1 blocks growth of PCa cells
Molecular changes induced by XPO-1 inhibition
Western blots and ELISAs revealed a reduced nuclear export of survivin in PCa cell lines after KPT-330 administration. In Fig. 4b and c, we show the early reduction of cytoplasmic survivin and increase of nuclear accumulation in PC3 cells treated for different times with 100 nM KPT-330. A decrease in cytosolic an increase in nuclear survivin protein expression was observed as early as 4 hours after treatment. Survivin continued to accumulate within the nucleus until 12 hours after treatment, after which time it decreased dramatically in all cellular compartments.
Immunoblot analysis also showed changes in the Bcl-2 family proteins: Bax, Bcl-2, Bcl-Xl and Bcl-Xs, suggesting a role in apoptosis (Fig. 4d). This was associated with an increased caspases-3 dependent PARP cleavage (Fig. 4e-g). FACS and Histone/DNA ELISA analyses indicated an increased apoptosis. In Fig. 4h we show the dose and time-dependent apoptosis measured in aggressive PC3, 22rv1, DU145 and C4-2B cell lines.
XPO-1 inhibition slows prostate tumor growth in vivo
Multiple tumor suppressor proteins are mislocalized in cancer cells by overexpressed XPO-1 [13–23]. Here, we demonstrate that nuclear and cytoplasmic expression of XPO-1 is elevated in prostate tumors compared to normal and hyperplastic tissue. In addition, XPO-1 IR is higher in Gleason score > 7 and metastases, which could be associated to its function. In vitro aggressive/metastatic PCa cells show higher levels of XPO-1 when compared to less aggressive cells, and castration resistant cells show higher XPO-1 levels when compared to androgen dependent cells.
Our results show that SINE compounds, which inhibit XPO1 activity, have anticancer effects in in vitro and in vivo models of human cancer. In this report, we also demonstrate that inhibition of XPO-1 is a potential target for the treatment of aggressive/castration resistant PCa. We tested a battery of SINE compounds in PCa cell lines and KPT-330 and KPT-251 were tested in vivo using two models of aggressive PCa. The results of our study demonstrate that SINE compounds are potent anticancer agents in these models. The clinical candidate, KPT-330, significantly reduced tumor cell growth to approximately one-third the volume of tumors observed in docetaxel–treated animals (internal control, data not shown). Reduction in tumor growth was dose-dependent and associated with inhibition of cellular proliferation and activation of apoptosis, which correlated with our in vitro findings showing PARP and caspase-3 cleavage. SINE compounds are potent therapeutic tools to treat aggressive/castration resistant PCa cells. This appears to be due to the modulation of a multiple signaling pathways.
It has been demonstrated that cyclin D1 overexpression increases the progression of PCa  and cyclin D1 knockdown reduce cell proliferation and increases sensitivity to radiotherapy and chemotherapy . Cyclin D1 nuclear overexpression induces differentiated phenotype in B-cell lymphoma in transgenic mice  and drives the oncogenic transformation of murine fibroblasts . Cyclin D1 is sequestered in the cytoplasm of mammalian cancer cells , where the enforced nuclear localization of cyclin D1 induces apoptosis. Thus, the subcellular localization of cyclin D1 may play a role in cell survival. The competing processes of nuclear import and export induce cyclin D1 localization . Here, we observed that SINE compounds inhibit cell cycle progression, increase early nuclear expression and reduce late cyclin D1 expression PCa cells. Cyclin D1 overexpression and abnormalities in cell-cycle inhibitory genes p21WAF1, p16INK4a, and p27KIP1 have been reported in PCa . P21WAF1 mainly localizes to the cytoplasm in many tumor cells, and cytoplasmic P21WAF1 is anti-apoptotic. P21WAF1 prevents cell cycle progression at the G1 phase. KPT-330 and KPT-251 reduce the export of P21WAF1 from the nucleus and increase nuclear expression of this protein. This was associated with reduced Ki67 and increased tunnel staining. Our data is in agreement with those observed by Van der Watt et al.,  which found that XPO-1 inhibition significantly reduces cell proliferation and increases apoptosis and P21 nuclear localization.
Exogenous and endogenous stress can activate ATM, a DNA damage sensor that activates the tumor suppressor p53, which, in turn, inhibits cell cycle progression and activates DNA repair mechanisms. p53 is often inactivated in PCa due to its deletion or mutation . However, p53 activity can be also regulated by its subcellular localization. p53 mis-localization arising from an aberrant import mechanism, hyperactive export, or sequestration with a cytoplasmic factor has been observed in several cancers. Normally, the nuclear-cytoplasmic transportation of p53 is tightly regulated. Here, we demonstrated that KPT-330 or KPT-251 are able to block transport of p53 from the nucleus, leading to its activation, cell cycle arrest, and apoptosis in p53 wt 22rv1 cells as previously demonstrated with leptomycin B . Furthermore, down regulation of both p21/Cip1 and p27/Kip1 produces a more aggressive PCa phenotype . The nuclear localization of P27KIP1 enables this regulatory function. However, the nuclear export of P27KIP1 is mediated by the XPO-1 export receptor. Hence, XPO-1 inhibition may restore the negative regulatory function of P27KIP1 in prostate cell cycle progression.
The constitutive activation of the PI3K pathway is key to PCa cell survival [9, 10] due to growth factor activation or phosphatase and tensin homolog (PTEN) loss. In normal cells, PTEN, the cellular PI3K antagonist, can inhibit PI3K activation, resulting in the nuclear localization of Forkhead Box O (FOXO) transcription factors, involved in multiple signaling pathways and having tumor-suppressor functions. In PCa deregulation of oncogenic kinases, including Akt, extra-signal-regulated kinase, or IκB kinase, is frequently observed, which may potentially inactivate FOXO activity. FOXO3a is, indeed, in a constant inactive state due to its cytoplasmic localization. In the nucleus, FOXO can activate the transcription of genes that promote cell cycle arrest and apoptosis . Thus, localizing FOXO to the nucleus is beneficial to controlling cell survival. The constitutive activation of PI3K constitutively activates protein kinase B (AKT) which phosphorylates FOXO transcription factors at multiple sites, thereby preventing FOXO-DNA binding and transcriptional activities, as well as promoting the XPO-1-dependent export of FOXO from the nucleus. We observed that KPT-251 and KPT-330 significantly induce nuclear localization of FOXO3a. FOXO re-localization to the nucleus, where it becomes active, is a promising method of controlling cell proliferation.
The transcriptional activator nuclear factor-κB (NF-κB) has been implicated in tumorigenesis and resistance therapy. Advanced/aggressive PCa cells express constitutively activated NF-κB . Here we observed that IkBα expression is increased in the nuclei of cells when 22rv1- or PC3-bearing mice were treated with KPT-330. Therefore, the perturbation of the XPO-1-dependent nuclear export of IκBα may attenuate constitutively activated NF-κB and cause immediate apoptosis in PCa cells.
The Fas/FasL system is significant in tumorigenesis and a previous investigation has indicated that the impairment of the Fas/FasL system in cancer cells may lead to apoptosis resistance and contribute to tumor progression . We observed that KPT-330 and KPT-251 induced the expression of FAS in vitro and in vivo. This increase could be responsible for the elevation of caspase 8 activity observed in vitro. Therefore FAS/FASL system could be activated and FASL produced in the tumor microenvironment by pro-inflammatory and resident stromal cells could participate to the XPO-1 mediated cell death. We also observed increased fibrosis and necrosis associated to increased percentage of CD68 monocytes/macrophages. Taken together these observations could further increase local FAS/FASL activity. In addition, it is previously shown that FOXO3A when active (nuclear localization) induces the expression of the FAS ligand protein .
High levels of survivin expression are independent risk factors for poor prognosis in cancer . Cytoplasmic survivin has been shown to be particularly high in prostate tumors and to be an independent predictor of poor prognosis, whereas nuclear survivin has been a favorable factor [68, 69] in some studies. These clinical results support the notion that nuclear survivin is suppressive for tumor growth, and further that targeting the cytoplasmic, antiapoptotic fraction of survivin would be an ideal therapeutic avenue. As survivin requires XPO-1/XPO1-RanGTP for its nuclear exit, inhibiting the activity of this complex could directly address this therapeutic need by increasing the tumor-suppressive nuclear survivin and reducing the antiapoptotic cytoplasmic survivin. Our results suggest that survivin is an essential component of the downstream signaling pathway of XPO-1 inhibition in prostate cancer cells, intriguingly an increase in the total concentration of survivin can interfere with these drugs' antitumor effects. Treatments with KPT-330 or KPT-251 led to an initial decrease in cytoplasmic survivin protein levels with a corresponding increase in its nuclear expression. This result was transient however, as the major effect was to deplete total survivin levels. Interestingly, other reports have shown a decrease in total survivin following treatment with LMB , and more recently it was reported that the NF-κB–dependent survival factor, Mcl1, is depleted in response to KPT-185 . Taken together, these results provided unequivocal proof of the potential SINEs as new class of anticancer drugs. Our results suggest that SINEs initially promote survivin nuclear localization, but at later time points leads to a reduction in its protein levels correlating with the timing of cellular antitumor effects of these compounds and supporting a hypothesis that XPO-1 inhibition leads to a loss of survivin levels which tend to lead to inhibition of tumor cell growth and enhanced tumor cell apoptosis.
We demonstrated that the SINE KPT-330, recently demonstrated to inhibit bone metastases formation in PCa  and currently in phase I clinical studies in humans (NCT01607905), is an interesting therapeutic target for advanced/castration resistant prostate tumors.
The authors thank the Dr Giovanna Di Emidio for the linguistic check of the manuscript.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Trama A, Foschi R, Larrañaga N, Sant M, Fuentes-Raspall R, Serraino D, et al. Survival of male genital cancers (prostate, testis and penis) in Europe 1999-2007: Results from the EUROCARE-5 study. Eur J Cancer. 2015. S0959-8049(15)00707-8.Google Scholar
- Feuer EJ, Rabin BA, Zou Z, Wang Z, Xiong X, Ellis JL, et al. The Surveillance, Epidemiology, and End Results Cancer Survival Calculator SEER*CSC: Validation in a Managed Care Setting. J Natl Cancer Inst Monogr. 2014;2014:265–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhou Y, Bolton E, Jones JO. Androgens and androgen receptor signaling in prostate tumorigenesis. J Mol Endocrinol. 2014. pii: JME-14-0203.Google Scholar
- Shafi AA, Putluri V, Arnold JM, Tsouko E, Maity S, Roberts JM, et al. Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively active splice variant, AR-V7, in prostate cancer cells. Oncotarget. 2015;6:31997–2012.PubMedPubMed CentralGoogle Scholar
- Darrington RS, Campa VM, Walker MM, Bengoa-Vergniory N, Gorrono-Etxebarria I, Uysal-Onganer P, et al. Distinct expression and activity of GSK-3α and GSK-3β in prostate cancer. Int J Cancer. 2012;131:E872–83.View ArticlePubMedGoogle Scholar
- Schütz SV, Cronauer MV, Rinnab L. Inhibition of glycogen synthase kinase-3beta promotes nuclear export of the androgen receptor through a CRM1-dependent mechanism in prostate cancer cell lines. J Cell Biochem. 2010;109:1192–200.PubMedGoogle Scholar
- Li J, Xing M, Zhu M, Wang X, Wang M, Zhou S, et al. Glycogen synthase kinase 3beta induces apoptosis in cancer cells through increase of survivin nuclear localization. Cancer Lett. 2008;272:91–101.View ArticlePubMedPubMed CentralGoogle Scholar
- Shimura T. Acquired radioresistance of cancer and the AKT/GSK3β/cyclin D1 overexpression cycle. J Radiat Res. 2011;52:539–44. Review.View ArticlePubMedGoogle Scholar
- Dal Col J, Dolcetti R. GSK-3beta inhibition: at the crossroad between Akt and mTOR constitutive activation to enhance cyclin D1 protein stability in mantle cell lymphoma. Cell Cycle. 2008;7:2813–6.View ArticlePubMedGoogle Scholar
- Festuccia C, Gravina GL, Muzi P, Pomante R, Ventura L, Vessella RL, et al. Bicalutamide increases phospho-Akt levels through Her2 in patients with prostate cancer. Endocr Relat Cancer. 2007;14:601–11.View ArticlePubMedGoogle Scholar
- Kau TR, Schroeder F, Ramaswamy S, Wojciechowski CL, Zhao JJ, Roberts TM, et al. A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells. Cancer Cell. 2003;4:463–76.View ArticlePubMedGoogle Scholar
- Fluteau A, Ince PG, Minett T, Matthews FE, Brayne C, Garwood CJ, et al. Simpson JE; MRC Cognitive Function Ageing Neuropathology Study Group. The nuclear retention of transcription factor FOXO3a correlates with a DNA damage response and increased glutamine synthetase expression by astrocytes suggesting a neuroprotective role in the ageing brain. Neurosci Lett. 2015;609:11–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang JY, Hung MC. Deciphering the role of forkhead transcription factors in cancer therapy. Curr Drug Targets. 2011;12:1284–90. Review.View ArticlePubMedPubMed CentralGoogle Scholar
- Senapedis WT, Baloglu E, Landesman Y. Clinical translation of nuclear export inhibitors in cancer. Semin Cancer Biol. 2014;27:74–86. Review.View ArticlePubMedGoogle Scholar
- Gravina GL, Senapedis W, McCauley D, Baloglu E, Shacham S, Festuccia C. Nucleo-cytoplasmic transport as a therapeutic target of cancer. J Hematol Oncol. 2014;7:85. Review.View ArticlePubMedPubMed CentralGoogle Scholar
- Turner JG, Dawson J, Sullivan DM. Nuclear export of proteins and drug resistance in cancer. Biochem Pharmacol. 2012;83(8):1021–32.View ArticlePubMedGoogle Scholar
- Takeda A, Yaseen NR. Nucleoporins and nucleocytoplasmic transport in hematologic malignancies. Semin Cancer Biol. 2014;27:3–10.View ArticlePubMedGoogle Scholar
- Noske A, Weichert W, Niesporek S, Roske A, Buckendahl AC, Koch I, et al. Expression of the nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 is a prognostic factor in human ovarian cancer. Cancer. 2008;112:1733–43.View ArticlePubMedGoogle Scholar
- Mendonca J, Sharma A, Kim HS, Hammers H, Meeker A, De Marzo A, et al. Selective inhibitors of nuclear export (SINE) as novel therapeutics for prostate cancer. Oncotarget. 2014;5:6102–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Turner JG, Dawson J, Emmons MF, Cubitt CL, Kauffman M, Shacham S, et al. CRM1 Inhibition Sensitizes Drug Resistant Human Myeloma Cells to Topoisomerase II and Proteasome Inhibitors both In Vitro and Ex Vivo. J Cancer. 2013;4:614–25.View ArticlePubMedPubMed CentralGoogle Scholar
- Santiago A, Li D, Zhao LY, Godsey A, Liao D. p53 SUMOylation promotes its nuclear export by facilitating its release from the nuclear export receptor CRM1. Mol Biol Cell. 2013;24:2739–52.View ArticlePubMedPubMed CentralGoogle Scholar
- Alt JR, Gladden AB, Diehl JA. p21(Cip1) Promotes cyclin D1 nuclear accumulation via direct inhibition of nuclear export. J Biol Chem. 2002;277:8517–23.View ArticlePubMedGoogle Scholar
- Wang Y, Wang Y, Xiang J, Ji F, Deng Y, Tang C, et al. Knockdown of CRM1 inhibits the nuclear export of p27(Kip1) phosphorylated at serine 10 and plays a role in the pathogenesis of epithelial ovarian cancer. Cancer Lett. 2014;343:6–13.View ArticlePubMedGoogle Scholar
- Hamamoto T, Seto H, Beppu T. Leptomycins A and B, new antifungal antibiotics. II. Structure elucidation. J Antibiot (Tokyo). 1983;36:646–50.View ArticleGoogle Scholar
- Roberts BJ, Hamelehle KL, Sebolt JS, Leopold WR. In vivo and in vitro anticancer activity of the structurally novel and highly potent antibiotic CI-940 and its hydroxy analog (PD 114,721). Cancer Chemother Pharmacol. 1986;16:95–101.View ArticlePubMedGoogle Scholar
- Newlands ES, Rustin GJ, Brampton MH. Phase I trial of elactocin. Br J Cancer. 1996;74:648–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Kalesse M, Christmann M, Bhatt U, Quitschalle M, Claus E, Saeed A, et al. The chemistry and biology of ratjadone. Chembiochem. 2001;2:709–14.View ArticlePubMedGoogle Scholar
- Mutka SC, Yang WQ, Dong SD, Ward SL, Craig DA, Timmermans PB, et al. Identification of nuclear export inhibitors with potent anticancer activity in vivo. Cancer Res. 2009;69:510–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Zanella F, Rosado A, Blanco F, Henderson BR, Carnero A, Link W. An HTS approach to screen for antagonists of the nuclear export machinery using high content cell-based assays. Assay Drug Dev Technol. 2007;5:333–41.View ArticlePubMedGoogle Scholar
- Li X, Chen T, Lin S, Zhao J, Chen P, Ba Q, et al. Valeriana jatamansi constituent IVHD-valtrate as a novel therapeutic agent to human ovarian cancer: in vitro and in vivo activities and mechanisms. Curr Cancer Drug Targets. 2013;13:472–83.View ArticlePubMedGoogle Scholar
- Watanabe K, Takatsuki H, Sonoda M, Tamura S, Murakami N, Kobayashi N. Anti-influenza viral effects of novel nuclear export inhibitors from Valerianae Radix and Alpinia galanga. Drug Discov Ther. 2011;5:26–31.View ArticlePubMedGoogle Scholar
- Sakakibara K, Saito N, Sato T, Suzuki A, Hasegawa Y, Friedman JM, et al. CBS9106 is a novel reversible oral CRM1 inhibitor with CRM1 degrading activity. Blood. 2011;118:3922–31.View ArticlePubMedGoogle Scholar
- Kalid O, Toledo Warshaviak D, Shechter S, Sherman W, Shacham S. Consensus Induced Fit Docking (cIFD): methodology, validation, and application to the discovery of novel Crm1 inhibitors. J Comput Aided Mol Des. 2012;26:1217–28.View ArticlePubMedGoogle Scholar
- Savona M, Garzon R, Brown PN, Yee K, Lancet JE, Gutierrez M, et al. Phase I Trial of Selinexor (KPT-330), A First-In-Class Oral Selective Inhibitor Of Nuclear Export (SINE) In Patients (pts) With Advanced Acute Myelogenous Leukemia (AML). Blood. 2013;122:1440.Google Scholar
- Cheng Y, Holloway MP, Nguyen K, McCauley D, Landesman Y, Kauffman MG, et al. XPO1 (CRM1) inhibition represses STAT3 activation to drive a survivin-dependent oncogenic switch in triple-negative breast cancer. Mol Cancer Ther. 2014;13:675–86.View ArticlePubMedPubMed CentralGoogle Scholar
- Ginestier C, Liu S, Diebel ME, Korkaya H, Luo M, Brown M, et al. CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts. J Clin Invest. 2010;120:485–97.View ArticlePubMedPubMed CentralGoogle Scholar
- Inoue H, Kauffman M, Shacham S, Landesman Y, Yang J, Evans CP, et al. CRM1 blockade by selective inhibitors of nuclear export attenuates kidney cancer growth. J Urol. 2013;189:2317–26.View ArticlePubMedGoogle Scholar
- Zhang K, Wang M, Tamayo AT, Shacham S, Kauffman M, Lee J, et al. Novel selective inhibitors of nuclear export CRM1 antagonists for therapy in mantle cell lymphoma. Exp Hematol. 2013;41:67–78. e4.View ArticlePubMedGoogle Scholar
- Yang J, Bill MA, Young GS, La Perle K, Landesman Y, Shacham S, et al. Novel small molecule XPO1/CRM1 inhibitors induce nuclear accumulation of TP53, phosphorylated MAPK and apoptosis in human melanoma cells. PLoS One. 2014;9, e102983.View ArticlePubMedPubMed CentralGoogle Scholar
- Tai YT, Landesman Y, Acharya C, Calle Y, Zhong MY, Cea M, et al. CRM1 inhibition induces tumor cell cytotoxicity and impairs osteoclastogenesis in multiple myeloma: molecular mechanisms and therapeutic implications. Leukemia. 2014;28:155–65.View ArticlePubMedGoogle Scholar
- Azmi AS, Aboukameel A, Bao B, Sarkar FH, Philip PA, Kauffman M, et al. Selective inhibitors of nuclear export block pancreatic cancer cell proliferation and reduce tumor growth in mice. Gastroenterology. 2013;144:447–56.View ArticlePubMedGoogle Scholar
- De Cesare M, Cominetti D, Doldi V, Lopergolo A, Deraco M, Gandellini P, et al. Anti-tumor activity of selective inhibitors of XPO1/CRM1-mediated nuclear export in diffuse malignant peritoneal mesothelioma: the role of survivin. Oncotarget. 2015;6:13119–32.View ArticlePubMedPubMed CentralGoogle Scholar
- Gravina GL, Tortoreto M, Mancini A, Addis A, Di Cesare E, Lenzi A, et al. XPO1/CRM1-selective inhibitors of nuclear export (SINE) reduce tumor spreading and improve overall survival in preclinical models of prostate cancer (PCa). J Hematol Oncol. 2014;7:46.View ArticlePubMedPubMed CentralGoogle Scholar
- Craft N, Shostak Y, Carey M, Sawyers CL. A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med. 1999;5:280–5.View ArticlePubMedGoogle Scholar
- Angelucci A, Gravina GL, Rucci N, Festuccia C, Muzi P, Vicentini C, et al. Evaluation of metastatic potential in prostate carcinoma: an in vivo model. Int J Oncol. 2004;25:1713–20.PubMedGoogle Scholar
- Chu LW, Pettaway CA, Liang JC. Genetic abnormalities specifically associated with varying metastatic potential of prostate cancer cell lines as detected by comparative genomic hybridization. Cancer Genet Cytogenet. 2001;127:161–7.View ArticlePubMedGoogle Scholar
- Kokontis JM, Hsu S, Chuu CP, Dang M, Fukuchi J, Hiipakka RA, et al. Role of androgen receptor in the progression of human prostate tumor cells to androgen independence and insensitivity. Prostate. 2005;65:287–98.View ArticlePubMedGoogle Scholar
- Lin DL, Tarnowski CP, Zhang J, Dai J, Rohn E, Patel AH, et al. Bone metastatic LNCaP-derivative C4-2B prostate cancer cell line mineralizes in vitro. Prostate. 2001;47:212–21.View ArticlePubMedGoogle Scholar
- Lee HL, Pienta KJ, Kim WJ, Cooper CR. The effect of bone-associated growth factors and cytokines on the growth of prostate cancer cells derived from soft tissue versus bone metastases in vitro. Int J Oncol. 2003;22:921–6.PubMedGoogle Scholar
- Bonaccorsi L, Carloni V, Muratori M, Salvadori A, Giannini A, Carini M, et al. Androgen receptor expression in prostate carcinoma cells suppresses alpha6beta4 integrin-mediated invasive phenotype. Endocrinology. 2000;141:3172–82.PubMedGoogle Scholar
- Scaccianoce E, Festuccia C, Dondi D, Guerini V, Bologna M, Motta M, et al. Characterization of prostate cancer DU145 cells expressing the recombinant androgen receptor. Oncol Res. 2003;14:101–12.PubMedGoogle Scholar
- van Bokhoven A, Varella-Garcia M, Korch C, Johannes WU, Smith EE, Miller HL, et al. Molecular characterization of human prostate carcinoma cell lines. Prostate. 2003;57:205–25.View ArticlePubMedGoogle Scholar
- Gravina GL, Marampon F, Petini F, Biordi L, Sherris D, Jannini EA, et al. The TORC1/TORC2 inhibitor, Palomid 529, reduces tumor growth and sensitizes to docetaxel and cisplatin in aggressive and hormone-refractory prostate cancer cells. Endocr Relat Cancer. 2011;18:385–400.View ArticlePubMedGoogle Scholar
- Bruzzese F, Di Gennaro E, Avallone A, Pepe S, Arra C, Caraglia M, et al. Synergistic antitumor activity of epidermal growth factor receptor tyrosine kinase inhibitor gefitinib and IFN-alpha in head and neck cancer cells in vitro and in vivo. Clin Cancer Res. 2006;12:617–25.View ArticlePubMedGoogle Scholar
- He Y, Franco OE, Jiang M, Williams K, Love HD, Coleman IM, et al. Tissue-specific consequences of cyclin D1 overexpression in prostate cancer progression. Cancer Res. 2007;67:8188–97.View ArticlePubMedGoogle Scholar
- Marampon F, Gravina GL, Ju X, Vetuschi A, Sferra R, Casimiro MC, et al. Cyclin D1 silencing suppresses tumorigenicity and radiosensitizes androgen-independent prostate cancer cells by impairing DNA double strand break repair pathways. Oncotarget 2015 in pressGoogle Scholar
- Shimura T, Ochiai Y, Noma N, Oikawa T, Sano Y, Fukumoto M. Cyclin D1 overexpression perturbs DNA replication and induces replication-associated DNA double-strand breaks in acquired radioresistant cells. Cell Cycle. 2013;12:773–82.View ArticlePubMedPubMed CentralGoogle Scholar
- Lovec H, Sewing A, Lucibello FC, Müller R, Möröy T. Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras: link between cell cycle control and malignant transformation. Oncogene. 1994;9(1):323–6.PubMedGoogle Scholar
- Alao JP, Gamble SC, Stavropoulou AV, Pomeranz KM, Lam EW, Coombes RC, Vigushin DM. The cyclin D1 proto-oncogene is sequestered in the cytoplasm of mammalian cancer cell lines. Mol Cancer. 2006;5:7Google Scholar
- Alt JR, Cleveland JL, Hannink M, Diehl JA. Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation. Genes Dev. 2000;14:3102–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Roy S, Singh RP, Agarwal C, Siriwardana S, Sclafani R, Agarwal R. Downregulation of both p21/Cip1 and p27/Kip1 produces a more aggressive prostate cancer phenotype. Cell Cycle. 2008;7:1828–35.View ArticlePubMedPubMed CentralGoogle Scholar
- van der Watt PJ, Maske CP, Hendricks DT, Parker MI, Denny L, Govender D, et al. The Karyopherin proteins, Crm1 and Karyopherin beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation. Int J Cancer. 2009;124:1829–40.8.View ArticlePubMedGoogle Scholar
- Gravina GL, Marampon F, Sherris D, Vittorini F, Di Cesare E, Tombolini V, et al. Torc1/Torc2 inhibitor, Palomid 529, enhances radiation response modulating CRM1-mediated survivin function and delaying DNA repair in prostate cancer models. Prostate. 2014;74:852–68.View ArticlePubMedGoogle Scholar
- Ceballos MP, Parody JP, Quiroga AD, Casella ML, Francés DE, Larocca MC, et al. FoxO3a nuclear localization and its association with β-catenin and Smads in IFN-α-treated hepatocellular carcinoma cell lines. J Interferon Cytokine Res. 2014;34:858–69.View ArticlePubMedPubMed CentralGoogle Scholar
- Jin R, Yi Y, Yull FE, Blackwell TS, Clark PE, Koyama T, et al. NF-κB gene signature predicts prostate cancer progression. Cancer Res. 2014;74:2763–72.View ArticlePubMedPubMed CentralGoogle Scholar
- Villa-Morales M, Fernández-Piqueras J. Targeting the Fas/FasL signaling pathway in cancer therapy. Expert Opin Ther Targets. 2012;16:85–101. Review.View ArticlePubMedGoogle Scholar
- Behzad H, Jamil S, Denny TA, Duronio V. Cytokine-mediated FOXO3a phosphorylation suppresses FasL expression in hemopoietic cell lines: investigations of the role of Fas in apoptosis due to cytokine starvation. Cytokine. 2007;38:74–83.View ArticlePubMedGoogle Scholar
- Shariat SF, Lotan Y, Saboorian H, Khoddami SM, Roehrborn CG, Slawin KM, et al. Survivin expression is associated with features of biologically aggressive prostate carcinoma. Cancer. 2004;100:751–7.View ArticlePubMedGoogle Scholar
- Adisetiyo H, Liang M, Liao CP, Aycock-Williams A, Cohen MB, Xu S, et al. Loss of survivin in the prostate epithelium impedes carcinogenesis in a mouse model of prostate adenocarcinoma. PLoS One. 2013;8:e69484. 67.View ArticlePubMedPubMed CentralGoogle Scholar