Tumor Biol. DOI 10.1007/s13277-015-3725-3
RESEARCH ARTICLE
Dual PI3K/mTOR inhibitor, XL765 (SAR245409), shows superior effects to sole PI3K [XL147 (SAR245408)] or mTOR [rapamycin] inhibition in prostate cancer cell models Giovanni Luca Gravina 1,2 & Andrea Mancini 1 & Luca Scarsella 1 & Alessandro Colapietro 1 & Ana Jitariuc 1 & Flora Vitale 1 & Francesco Marampon 1 & Enrico Ricevuto 3 & Claudio Festuccia 1
Received: 13 May 2015 / Accepted: 28 June 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Deregulation of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway contributes to prostate cancer development and progression. Here, we compared the in vitro effects of the dual PI3K/mTOR inhibitor (XL765) with those observed with the sole PI3K (XL147) or mTOR (rapamycin) inhibition in 2 non-tumor prostate epithelial cell lines, 8 prostate cancer cell lines, and 11 prostate cancer cell derivatives. We demonstrated that the XL765 treatment showed superior and proliferative effects of XL147 or rapamycin. The XL765 effects were associated to increasing the chromosome region maintenance 1 (CRM1)-mediated nuclear localization of glycogen synthase kinase 3 beta (GSK3β) and Foxo-1a with higher induction of apoptosis when compared to those observed in XL147 and rapamycin treatments. IC50 values were calculated in phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-positive and PTEN-negative cell lines as well as after PTEN transfection or PTEN downmodulation by siRNA strategy revealing that the presence of this protein was associated with reduced sensitivity to PI3K and mTOR inhibitors. The comparison of
* Claudio Festuccia
[email protected] 1
Department of Biotechnological and Applied Clinical Sciences, Laboratory of Radiobiology, University of L’Aquila, L’Aquila, Italy
2
Department of Biotechnological and Applied Clinical Sciences, Division of Radiation Oncology, University of L’Aquila, L’Aquila, Italy
3
Department of Biotechnological and Applied Clinical Sciences, Division of Medical Oncology, University of L’Aquila, L’Aquila, Italy
IC50 values was also calculated for androgen-dependent and independent cell lines as well as after androgen receptor (AR) transfection or the AR downmodulation by siRNA strategy revealing that androgen independence was associated with enhanced responsiveness. Our results provide a rationale to use the dual PI3K/Akt/mTOR inhibitors in hormone-insensitive prostate cancer models due to the overactivity of PI3K/Akt/ mTOR in this disease condition. Keywords Prostate cancer . Drug resistance . mTOR . PI3K inhibitors
Introduction Recent epidemiologic studies consider prostate cancer (PCa) a relatively increasing health care problem being the second death cause for solid tumor in men [1] in Europe and the USA. The inhibition of androgen receptor (AR) function is indicated as an effective therapeutic approach in the management of locally advanced or advanced castration-resistant disease (CRPC) [2, 3]. Chemotherapy has limited utility in the treatment of patients with CRPC also if docetaxel- and platinum-based chemotherapy may improve survival in these patient cohorts [4–7] as first- and second-line chemotherapy, respectively. Resistance is showed during chemotherapy. Therefore, it is necessary to improve the efficacy of chemotherapy. Activation of phosphatidylinositol-3-kinase (PI3K)/Akt (protein kinase B) pathway [8, 9] is associated to resistance phenomena vs chemotherapy in several tumors. Commonly, Akt activation triggers survival signals downmodulating the chemotherapy-associated apoptosis. PTEN (phosphatase and tensin homologue deleted on
Tumor Biol.
chromosome 10) is a tumor suppressor gene in solid tumors. This gene could be deleted or mutated in PCa [10–13]. In addition, progression and poor clinical outcome of prostate cancer are associated to increase Akt activity [12, 14–18] due to (i) PTEN loss [10–13] shown in about 30 % cancers, (ii) activation of PIK3 Ca [19], and (iii) activation of PI3K though epidermal growth factor receptor (EGFR) and HER2/neu [13, 20–22] activation. Akt phosphorylates the negative regulator of mammalian target of rapamycin (mTOR), TSC1/TSC2 activating two functionally distinct enzymatic complexes: the mTOR complex 1 (mTORC1) and complex 2 (mTORC2). In addition, mTOR is a main connector of signals from the extracellular microenvironment to intracellular machinery [23]. mTOR activation is associated with solid and hematologic cancers. Therefore, agents that inhibit PI3K, mTOR, or both are considered for cancer treatment of tumors and increasing clinical trials are currently under development. Everolimus (RAD001) is the first mTOR inhibitor [24] used in cancer treatment. This represents an allosteric inhibitor of mTOR and inhibits significantly the activity of TORC1. RAD001 has, however, little or no activity vs TORC2 [24]. It has been demonstrated that RAD001 can determine an increased phosphorylation of AKT in the Ser473 via negative feedback loop involving TORC1 [23]. Although it has been demonstrated that rapamycin and rapanalogues show modest anticancer effects which are not sufficient to be indicative for the rapamycin-based monotherapy in cancer treatment [25, 26], combination strategies involving the use of rapanalogues have been developed. Different PI3K and mTOR inhibitors have been characterized including XL765 (SAR245409), a novel and orally pan-PI3K and mTOR inhibitor [27, 28] and XL147 (SAR245408), a novel pan-PI3K inhibitor [28, 29]. XL147 and XL765 are ATP-competitive reversible enzyme inhibitors currently entered in phase I/II clinical development [28, 29]. In this report, the XL765 effects are compared with those observed with XL147 and rapamycin in hormone-sensitive or CRPC cell models.
Materials and methods Reagents All the materials for tissue culture were purchased from Hyclone (Cramlington, NE, USA). Plasticware was obtained from Nunc (Roskilde, Denmark). Anti-PDK1, pPDK1(Ser241), and 70S6K and p-70S6k (Thr388) antibodies were purchased from Antibodies online (Aachen, Germany). Anti-S6, p-S6 (ser235/236) antibodies were purchased from Sigma-Aldrich (St Louis, MA, USA). Anti-PTEN (sc133242), Akt (A112, Sc-377457), p-Akt (Thr308, sc16646R/sc-135650, and Ser473, sc-135651), 4EBP1 (NB19, sc-514073), p-4EBP1 (Thr37/66, sc-18080R), glycogen synthase kinase 3 beta (GSK3β) (H76 sc-9166), p-GSK3β (Ser9, sc-11757), Foxo1a (C9, sc-374427), p-Foxo1a (Ser256, sc-
16307), MDM2 (C18, sc-412), p-MDM2 (Ser166, sc293105), cyclin D1 (H295, sc-753), cyclin B1 (GNS1, sc245), cyclin E (M20, sc-481), cdk1/cdk2 (sc-53219), cdk4 (sc-23896), cdk6 (B10, sc-7961), p21 (sc-56335), p27 (C19, sc-528), p14ARF (sc-53392), cytochrome C (C20, sc-8385), Smac/Diablo (sc-1363302), Bax (B9, sc-7480), Bcl2 (C21, sc783), BclXl (A20, sc-7122), Bad (K17, sc-942) and p-Bad (Ser112, sc-7998), and ATG5 (N18, sc-8666) antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies against LC3-II and beclin were purchased from Biorbyt Ltd (Cambridge, UK). Akt/protein kinase B (PKB) kinase activity was performed using a non-radioactive assay kit which was purchased from Stressgene Bioreagents (Victoria, BC, Canada). The antibody recognizing the 46 kDa (pro-form) and 35 kDa (cleaved form) of caspase-9 was purchased from Epitomics (Burlingame, CA, USA). SiRNA for PTEN was purchased from Santa Cruz. Caspase3 activity was assessed using a commercially available kit (Trevigen, Gaithersburg, MD). Cell lines Analogously to those presented in a previous our report [29], 2 non-tumor prostate epithelial cell lines [BPH-1 and EPN], 8 PCa cells lines (CWR22, 22rv1, LAPC-4, LNCaP, DU145, PC3, VCaP and DuCaP), as well as 11 cell derivatives (CWR22R-2152, CWR22R-2272, CWR22R2274, and CWR22R-2524, LNCaP-C81, LNCaP-104S and LNCaP-104R1, C4-2B, PTEN-transfected PC3 and ARtransfected PC3 and AR-transfected DU145) were used. A complete characterization of these cell models is reviewed in Pienta et al., 2008 [30]. The RAD001-resistant PC3 sub-line was kindly provided from Dr. Roman Blaheta (Department of Urology, Goethe-University, Frankfurt am Main, Germany) [31]. To minimize the risk of misidentified and/or contaminated cell lines, a DNA profiling was periodically carried out in house to authenticate cell cultures by STR-fingerprints comparing these with those published by ATCC and DSMZ or with those published by Adri van Bokhoven and co-workers [32]. Growth assays Cells were seeded at a density of 2×104 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. Cells were trypsinized and resuspended in 1.0 ml of saline; thus, viable cells were counted using the NucleoCounter™ NC-100 (Chemotec, Cydevang, DK). All experiments were conducted in triplicate. IC50 values were calculated by the GraFit method (Erithacus Software Limited, Staines, UK). Drugs and preparation XL147 and XL765 (kindly provided by Exelixis Inc. So San Francisco, CA) and rapamycin (Selleck Chemicals LLC, Houston, TX, USA) were dissolved in DMSO and stored at 4 °C on the day of use when were suspended in complete medium at considered concentrations.
Tumor Biol. Table 1 Enzymatic inhibition by XL147 of different activities belonging to PI3K/Akt/mTOR pathways: IC50 values (μM) were calculated through Western blot analyses performed on cell extracts from cultures treated with different doses of XL147 Cell line
PDK1 (Ser241)
Akt (Thr308)
Akt (Ser 473)
GSK3β (Ser 19)
Foxo1a (Ser256)
S6K (Ser 235/236)
4EBP1 (Thr 27/66)
MDM2 (Ser166)
PC3
0.33±0.03
0.38±0.01
1.48±0.45
2.33±0.38
3.43±0.94
3.23±0.30
2.20±0.65
2.56±0.60
LNCaP
0.31±0.02
0.52±0.07
1.50±0.21
1.75±0.12
3.66±0.84
3.75±0.57
2.88±0.92
2.19±0.83
C-81 C4-2B
0.35±0.04 0.41±0.05
0.62±0.11 0.51±0.07
1.60±0.12 1.64±0.03
2.80±0.66 2.55±0.76
3.60±0.37 2.15±0.64
1.80±0.58 2.14±0.74
1.55±0.43 0.88±0.12
2.66±0.68 2.33±0.58
22rv1 DU145
0.72±0.14 1.08±0.09
1.67±0.23 1.44±0.32
3.40±0.67 3.00±0.51
4.20±0.88 4.55±1.09
4.72±0.14 4.08±0.09
4.67±0.23 4.44±0.32
6.40±0.67 6.00±0.51
7.20±0.88 7.55±1.09
RAD001-res PC3
0.30±0.03
0.41±0.03
1.88±0.37
2.75±0.42
4.11±0.52
3.70±0.35
2.78±0.53
3.00±0.45
Preparation of cell lysates, Western blot, and enzymatic analysis Following treatments, cells grown in 90-mm diameter Petri dishes were washed with cold PBS and immediately lysed with 1 ml lysis buffer containing a proteinase and phosphatase inhibitor cocktail. Cell lysates were separated by electrophoresis in 7 % SDS-PAGE, and proteins transferred onto nitrocellulose and probed with the appropriate antibodies using the conditions recommended by the suppliers. ECLtreated filters were analyzed using the Gel Doc™ XRplus system (BioRad laboratories, Milan, Italy). Parallely, lysates were analyzed for Akt activity by AlphaScreen® SureFire® phospho-PI3K/Akt/mTOR signaling protein (codes TGR4ES500, TGR70S500, TGRA3S500, TGRGBS500, TGRA4S500) Kits (Perkin Elemer, Waltham, MA). Gsk3β activity was measured by cell-based Elisa assay (R&D Systems Inc., Minneapolis, MN, USA). For cell-based ELISA assays, cells were plated in Corning® 96 or 384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates. Cell seeding densities of 40,000 cells/well (96‐ well format) or 10,000 cells/well (384‐well format) are generally sufficient for most cell lines. Sub-confluent cell cultures were starved for 24 h and successively activated with 50 ng/ml EGF (controls) for 20 min. Experimental points were made pre-treating cells with different doses of XL765, XL147, and rapamycin.
Cell cycle and apoptosis analysis Apoptosis was analyzed by using Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Life Technologies Europe BV, Monza, Italy). All cells were then measured on Tali® Image-Based Cytometer measuring the fluorescence emission at 530 nm (e.g., FL1) and >575 nm. The results were expressed as the percentage of cell death by apoptosis in controls and in treated cultures. Statistics Continuous variables were summarized as mean and standard deviation (SD) or as median and 95 % CI for the median. For continuous variables not normally distributed, statistical comparisons between control and treated groups were established by carrying out the Kruskal-Wallis tests. For continuous variables normally distributed, statistical comparisons between control and treated groups were established by carrying out the ANOVA test or by Student P test for unpaired data (for two comparisons).
Results The dual PI3K/mTOR inhibitor, XL765, exerts enhanced effects on suppression of the mTOR signaling when compared to XL147 or rapamycin We performed a series of Western blotting and enzymatic determinations for member
Table 2 Enzymatic inhibition by XL765 of different activities belonging to PI3K/Akt/mTOR pathways: IC50 values (mM) were calculated through Western blot analyses performed on cell extracts from cultures treated with different doses of XL765 Cell line
PDK1 (Ser241)
Akt (Thr308)
Akt (Ser 473)
GSK3β (Ser 19)
Foxo1a (Ser256)
S6K (Ser 235/236)
4EBP1 (Thr 27/66)
MDM2 (Ser166)
PC3
0.25±0.02
1.38±0.31
0.33±0.02
0.55±0.01
0.33±0.02
0.63±0.01
2.13±0.44
0.70±0.04
LNCaP C-81 C4-2B 22rv1 DU145 RAD001-res PC3
0.31±0.04 0.30±0.02 0.55±0.08 1.24±0.12 0.85±0.07 0.28±0.04
1.51±0.33 1.06±0.07 0.98±0.13 1.28±0.55 2.25±0.44 1.62±0.55
0.51±0.06 1.20±0.08 0.48±0.05 1.30±0.07 1.04±0.11 0.88±0.08
0.51±0.01 0.44±0.03 0.48±0.06 0.60±0.05 0.52±0.04 0.79±0.04
0.58±0.08 0.60±0.08 0.48±0.05 1.30±0.07 1.04±0.11 0.37±0.02
0.81±0.04 0.66±0.07 0.78±0.09 1.84±0.11 2.28±0.18 0.85±0.13
0.82±0.10 0.77±0.09 0.58±0.06 1.71±0.20 0.98±0.07 2.65±0.55
0.82±0.12 0.55±0.05 0.66±0.07 0.81±0.07 0.78±0.09 1.44±0.05
Tumor Biol. Table 3 Enzymatic inhibition by rapamycin of different activities belonging to PI3K/Akt/mTOR pathways: IC50 values (nM) were calculated through Western blot analyses performed on cell extracts from cultures treated with different doses of rapamycin Cell line
PDK1 (Ser241)
Akt (Thr308)
Akt (Ser 473)
GSK3β (Ser 19)
Foxo1a (Ser256)
PC3
>3000
LNCaP
>3000
C-81 C4-2B
S6K (Ser 235/236)
4EBP1 (Thr 27/66)
>3000
183±26
288±44
>3000
177±15
351±11
>3000 >3000
>3000 >3000
184±12 192±24
22rv1 DU145
>3000 >3000
>3000 >3000
230±38 222±36
RAD001 -res PC3
>3000
>3000
1940±215
318±6
43±6
66±12
246±21
337±19
37±10
44±11
277±27
282±29 284±34
265±42 388±18
60±17 71±12
77±27 77±15
115±18 133±15
377±13 497±22
424±32 414±53
35±7 28±5
122±25 128±44
145±26 183±31
2520±244
>3000
>3000
>3000
of the PI3K/Akt/mTOR cascade. We found that the inhibition of several members of PI3K/Akt cascade was maintained for some hour, and successively, phenomena of degradation reduced the effects. Due to this observation, we chose the time of 3 h for our analyses. Cells were lysed with 100 μl of lysis
MDM2 (Ser166)
2560±285
buffer and stored at 80 °C until to be used or immediately tested for enzymatic activity for PDK1 (Ser241), Akt (Thr308 and Ser473), GSK3β (Ser19), and 4EBP1 (Thr27/ 66). Cells were pre-treated with XL147, XL765, or rapamycin 30 min before EGF administration. Phosphorylation levels of
A
D B
5
C
IC50 (M)
4
3 2 1
XL147 XL765 RAD001
0
Fig. 1 IC50 values calculated in different cell lines cultured with different concentrations of XL765 (a), XL147 (b), and rapamycin (c). In the bottom of this figure are the summarized phenotype of used cell models. Cells were classified for PTEN expression, PIK3CA mutation, Akt activity, androgen receptor (AR) expression, androgen dependence (AD) or independence (AI). Therefore, (i) PC3 and LnCaP cell lines and their cell derivatives were PTEN mutated (M) or deleted (D) possessed basally activated Akt (a); (ii) 22rv1 cell line and its cell derivative presented the H1047R PIKCA mutation (M) determining PI3K basal
activity but weak Akt activity (+/−); (iii) AR expression was present in normal and benign BPH-1 cells as well as in LAPC-4, CWR22, VCaP and DUCaP, LnCaP (and it cell derivatives), PC-3AR, 22rv1 (and its cell derivatives), and DU-AR cells (+); (iv) androgen-dependent cell lines (AR+/AD) were EPN, BPH-1, PC3AR, DU-AR, CWR22, LnCaP, LnCaP-104S, VCaP, DuCaP and LAPC4 (+); LnCaP-R1, LnCaP-C81, C4-2B, 22rv1 (and its derivatives) cells were (v) AR+/ AI cell lines (filled squares); PC3, PC3-PTEN and DU145 were AR-/AI cell lines (+)
Tumor Biol. Table 4 Correlation between XL765, XL147, and rapamycin effects (IC50) vs levels of Akt phosphorylation Correlation
Coefficient
Statistics
XL147 vs Akt ser 473
r=−0.707
P
