Tumor Biol. DOI 10.1007/s13277-015-3536-6
RESEARCH ARTICLE
Dual targeting of mTORC1 and mTORC2 by INK-128 potently inhibits human prostate cancer cell growth in vitro and in vivo Shang-jun Jiang 1 & Shuo Wang 2
Received: 29 March 2015 / Accepted: 5 May 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Both mammalian target of rapamycin (mTOR) complexes 1 and 2 (mTORC1/2) are often over-activated in prostate cancer cells and are associated with cancer progression. In the current study, we evaluated the potential antiprostate cancer activity of INK-128, an ATP-competitive mTORC1/2 dual inhibitor, both in vitro and in vivo. Our results showed that INK-128 exerted potent anti-proliferative activity in established (PC-3 and LNCaP lines) and primary (patient-derived) human prostate cancer cells by inducing cell apoptosis. The latter was evidenced by increase of annexin V percentage, formation of cytoplasmic histone-associated DNA fragments, and cleavage of caspase-3. INK-128-induced prostate cancer cell apoptosis and cytotoxicity were alleviated upon pretreatment of cells with the pan-caspase inhibitor z-VAD-FMK or the specific caspase-3 inhibitor z-DVED-FMK. At the molecular level, INK-18 blocked mTORC1/2 activation in PC-3 cells and LNCaP cells and downregulated mTORregulated genes including cyclin D1, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α. ERK-MAPK activation and androgen receptor expression were, however, not affected by INK-128 treatment. In vivo, oral administration of INK-128 significantly inhibited growth of PC-3 xenografts in nude mice. The preclinical results of this study suggest that INK-
* Shang-jun Jiang [email protected] 1
The Department of Urinary Surgery, The People’s Hospital of Fuyang, 4 Gui’hua Road, Fuyang City, Zhejiang Province 311400, China
2
The Department of Urinary Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
128 could be further investigated as a promising anti-prostate cancer agent. Keywords Prostate cancer . mTORC1/2 . INK-128 . Apoptosis . Signaling
Introduction Prostate cancer causes significant cancer-related mortalities in men around the world [1–3]. In the USA, statistical data reveal that one of nine men over the age of 65 is likely to be diagnosed of this disease [1]. Surgery and current chemotherapeutic treatments appear not enough in curing or controlling it, especially for the resistant and metastatic prostate cancer. There is an urgent need for the development of alternative chemotherapeutic strategies [4, 5]. Over-activation of mammalian target of rapamycin (mTOR) has been recognized as an important contributor of prostate cancer initiation and progression [6–8]. Two functionally distinct multi-protein mTOR complexes have been recognized thus far, including mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [9, 10]. mTORC1 is assembled by mTOR, Raptor, mLST8, as well as recently identified PRAS40 and DEPTOR, which phosphorylates p70S6 kinase (S6K1) and 4E-binding protein 1 (4E-BP1) to regulate protein translation and energy metabolism [10, 11]. The activity of mTORC1 could be blocked by rapamycin and its analogs (i.e., RAD001 and others) [10, 11]. mTOR, Rictor, Sin1, as well as others are in the complex of mTORC2, which serves as the upstream kinase for AKT (at Ser-473) and Foxo1/3a, among others [10, 11]. Both mTOR complexes play an important role in regulating cancerous behaviors of prostate cancer [6–8].
Tumor Biol.
However, the activity of mTORC1 inhibitors (rapamycin and rapalogs) as anti-cancer agents is generally weak [12, 13]. First, these inhibitors only partially inhibit mTORC1 [12, 13]. Meanwhile, rapalogs are shown to activate AKT and ERKmitogen-activated protein kinases (MAPK) signaling that counteracts their activities in cancer [14, 15]. As a result, these mTORC1 inhibitors may ultimately upregulate cancer cell proliferation [12, 13]. Thus, an agent that dually blocks both mTORC1 and mTORC2 would theoretically result in improved anti-cancer activity [16, 17]. As a matter of fact, ATP-competitive mTOR inhibitors, targeting both mTORC1 and mTORC2, have been developed [18]. These compounds (i.e., AZD-8055, AZD-2014, INK-128, and OSI-027) are more effective mTORC1 inhibitors, which block mTORC1 and mTORC2 simultaneously [18]. Studies have shown that these mTORC1/2 dual inhibitors more completely inhibit 4EBP1 phosphorylation than rapamycin [19, 20]. This explains rapamycin ineffectiveness in cap-dependent protein translation in many cancer cells and also at least partly explains the weak anti-tumor activity by the rapalogs [19, 20]. Several of these compounds are being tested in various tumor models and showed higher efficiencies than rapalogs in suppressing cancer cells [21, 22]. In the current study, we evaluated the potential anti-prostate cancer activity of INK-128, a mTORC1/2 dual inhibitor [23, 24], both in vitro and in vivo.
Material and methods Chemicals and reagents INK-128 was obtained from Selleck China (Shanghai, China). The pan-caspase inhibitor z-VAD-FMK and the caspase-3specific inhibitor z-DVED-FMK were from Calbiochem (Darmstadt, Germany). Anti-tubulin, hypoxia-inducible factor (HIF)-1α, HIF-2α, and cyclin D1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies of cleaved caspase-3, p-AKT (Ser-473), p-AKT (Thr-308), AKT1, p-S6 (Ser-235/236), S6, p-forkhead box O 1a (FoxO1a) (Thr-24), p-ERK1/2 (Thr-202/Tyr-204), ERK1/2, androgen receptor, p-S6K1 (Thr-389), and S6K1 were obtained from Cell Signaling Technologies (Beverly, MA). Antibodies of p-FoxO3a (Thr-32) and FoxO3a were purchased from Abcam (Cambridge, MA). Cell lines Human prostate cancer cell lines PC-3 and LNCaP cells were cultured as monolayer in RPMI 1640 supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Hyclone, Shanghai, China) and antibiotics. RWPE1, a non-transformed prostate epithelial cell line, was obtained from ATCC (CRL-11609)
(Manassas, VA). RWPE1 cells were maintained in Defined Keratinocyte-SFM medium supplemented with growth factors (insulin, epidermal growth factor, and fibroblast growth factor; Invitrogen, Carlsbad, CA), and medium was replaced every other day. The number of viable cells (trypan blue negative) was counted through an automatic cell counter. Primary culture of patient-derived prostate cancer cells Fresh prostate cancer tissues were obtained from two patients (male, 65 and 71 years old) at the prostate resection operation. Neither patient had received prior chemical, hormonal, or radiation therapy. Tissues were finely minced, digested 12 to 18 h in 100 units/ml collagenase I in DMEM, and pipetted to disperse clumps. Cells were washed in PBS and cultured on collagen-coated tissue culture plates (BD Biosciences) in cell culture medium (DMEM, 20 % FBS, 2 mM glutamine, 1 mM pyruvate, 10 mM HEPES, 100 units/ml penicillin/streptomycin, 0.1 mg/ml gentamicin, and 2 g/l fungizone). Cell lines were named T1 to T11 and used within 3 weeks of culture. All tissues were collected under an approved institutional ethics protocol from patients granting informed consent. MTT assay of cell proliferation The methyl thiazolyl tetrazolium (MTT) assay was performed to assess cell proliferation according to instructions from the manufacturer (Sigma-Aldrich Co., St. Louis, MO). Briefly, cells were planted into 96-well plates at a density of 5000 cells per well. At the end of each treatment, 20 μl of MTT (5 mg/ml, Sigma) was added for 2 h. The medium was then discarded carefully, and 150 μl of DMSO per well was added. Absorbance was recorded at 570 nm with the Universal Microplate Reader (Bio-Tek Instruments, Milan, Italy). The value of the treatment group was normalized to that of the control group. Trypan blue staining of Bdead^ cells The number of dead prostate cancer cells (trypan blue positive) after treatment was counted under microscope, and the percentage (%) of Bdead^ cells was calculated by the number of the trypan blue positive cells divided by the total number of the cells. Cell apoptosis assay Apoptosis of prostate cancer cells was quantitatively determined by flow cytometry using the Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA). After treatment, cells were harvested, washed, and incubated with annexin V and propidium iodide (PI) at room temperature for 10 min in the dark. The stained cells were analyzed by FACS using a FACSCalibur instrument (BD Biosciences)
Tumor Biol.
equipped with CellQuest 3.3 software. The percentage of annexin V-positive cells was recorded as a quantitative indicator of cell apoptosis. Histone DNA apoptosis ELISA assay The Cell Death Detection ELISA method quantifies apoptotic cell death in cellular systems by measuring cytoplasmic histone-associated DNA fragments. These DNA fragments in vehicle control and INK-128-treated cells were quantified using a commercially available ELISA kit from Roche Diagnostics (Mannheim, Germany), according to recommended procedures. Caspase-3 activity assay Cytosolic proteins from 1×106 cells were extracted in hypotonic cell lysis buffer [25]. Twenty micrograms of cytosolic extracts was added to caspase assay buffer (312.5 mM HEPE S, pH 7.5; 31.25 % sucrose; 0.3125 % CHAPS) with AcDEVD-AFC (15 μg/ml) (Calbiochem, Darmstadt, Germany) as the substrate. After incubation at 37 °C for 1 h, the amount of AFC liberated was measured using a spectrofluorometer (Thermo-Labsystems, Helsinki, Finland) with excitation of 380 nm and emission wavelength of 460 nm. The value of the treatment group was normalized to that of the control group. Western blots After treatment, cells were collected and lysed. Protein lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and were transferred onto a PVDF membrane. The membrane was incubated with a solution containing Tris-buffered saline, 0.05 % Tween 20, and 10 % (w/v) non-fat dry milk, and then exposed for 3 h at room temperature to desired primary antibody. Following incubation with appropriate secondary antibody, the immunoreactive bands were visualized using enhanced chemiluminescence (ECL) method. The blots were stripped and re-probed with the loading control. Band intensity was quantified through ImageJ software and normalized to the loading. Xenograft assay Male nude mice (6–8 weeks old) were purchased from the author institutional animal facility and maintained in accordance with Institutional Animal Care Use Committee guidelines. PC-3 cells were mixed in a 1:1 ratio with Matrigel (Becton Dickinson, Bedford, MA), and a 0.1-ml suspension containing 3×106 cells was injected subcutaneously on the right flank of each mouse. Mice were randomized into two groups of ten mice/group. Treatment began 4 weeks post
tumor implant when the tumor reached around 100 mm3 in volume. Experimental animals were treated orally with INK128 (1 mg/kg in 0.1 ml PBS) daily for 21 days [26]. Control mice received an equal volume of the vehicle. Tumor volumes, mice body weights, as well wet tumor weights were determined as described [27]. At the termination of the experiment, the tumor tissues were harvested and divided into two pieces. A portion of the tumor tissue was processed for immunohistochemistry for analysis of apoptotic bodies, whereas the second piece was used for Western blots [27]. For immunohistochemistry, tumor tissues were embedded in paraffin, sectioned (4 μm), de-paraffinized, and processed for determination of apoptotic bodies using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Intergen, NY) according to the manufacturer’s instructions. Brown-colored apoptotic bodies in tumor sections of vehicle control and INK-128treated mice were counted under a Nikon microscope at ×20 magnification. Ten randomly selected fields on each tumor section were counted for apoptotic bodies. For Western blots, tumor tissues were minced, suspended in PBS, and homogenized. The homogenate was centrifuged, and the supernatant fraction was collected and tested for Western blots. Statistical analysis All experiments were repeated at least two to three times, and similar results were obtained. Data were expressed as mean± standard deviation (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) using SPSS 18.0 software. Multiple comparisons were performed using Tukey’s honestly significant difference procedure. A p value
RESEARCH ARTICLE
Dual targeting of mTORC1 and mTORC2 by INK-128 potently inhibits human prostate cancer cell growth in vitro and in vivo Shang-jun Jiang 1 & Shuo Wang 2
Received: 29 March 2015 / Accepted: 5 May 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Both mammalian target of rapamycin (mTOR) complexes 1 and 2 (mTORC1/2) are often over-activated in prostate cancer cells and are associated with cancer progression. In the current study, we evaluated the potential antiprostate cancer activity of INK-128, an ATP-competitive mTORC1/2 dual inhibitor, both in vitro and in vivo. Our results showed that INK-128 exerted potent anti-proliferative activity in established (PC-3 and LNCaP lines) and primary (patient-derived) human prostate cancer cells by inducing cell apoptosis. The latter was evidenced by increase of annexin V percentage, formation of cytoplasmic histone-associated DNA fragments, and cleavage of caspase-3. INK-128-induced prostate cancer cell apoptosis and cytotoxicity were alleviated upon pretreatment of cells with the pan-caspase inhibitor z-VAD-FMK or the specific caspase-3 inhibitor z-DVED-FMK. At the molecular level, INK-18 blocked mTORC1/2 activation in PC-3 cells and LNCaP cells and downregulated mTORregulated genes including cyclin D1, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α. ERK-MAPK activation and androgen receptor expression were, however, not affected by INK-128 treatment. In vivo, oral administration of INK-128 significantly inhibited growth of PC-3 xenografts in nude mice. The preclinical results of this study suggest that INK-
* Shang-jun Jiang [email protected] 1
The Department of Urinary Surgery, The People’s Hospital of Fuyang, 4 Gui’hua Road, Fuyang City, Zhejiang Province 311400, China
2
The Department of Urinary Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
128 could be further investigated as a promising anti-prostate cancer agent. Keywords Prostate cancer . mTORC1/2 . INK-128 . Apoptosis . Signaling
Introduction Prostate cancer causes significant cancer-related mortalities in men around the world [1–3]. In the USA, statistical data reveal that one of nine men over the age of 65 is likely to be diagnosed of this disease [1]. Surgery and current chemotherapeutic treatments appear not enough in curing or controlling it, especially for the resistant and metastatic prostate cancer. There is an urgent need for the development of alternative chemotherapeutic strategies [4, 5]. Over-activation of mammalian target of rapamycin (mTOR) has been recognized as an important contributor of prostate cancer initiation and progression [6–8]. Two functionally distinct multi-protein mTOR complexes have been recognized thus far, including mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [9, 10]. mTORC1 is assembled by mTOR, Raptor, mLST8, as well as recently identified PRAS40 and DEPTOR, which phosphorylates p70S6 kinase (S6K1) and 4E-binding protein 1 (4E-BP1) to regulate protein translation and energy metabolism [10, 11]. The activity of mTORC1 could be blocked by rapamycin and its analogs (i.e., RAD001 and others) [10, 11]. mTOR, Rictor, Sin1, as well as others are in the complex of mTORC2, which serves as the upstream kinase for AKT (at Ser-473) and Foxo1/3a, among others [10, 11]. Both mTOR complexes play an important role in regulating cancerous behaviors of prostate cancer [6–8].
Tumor Biol.
However, the activity of mTORC1 inhibitors (rapamycin and rapalogs) as anti-cancer agents is generally weak [12, 13]. First, these inhibitors only partially inhibit mTORC1 [12, 13]. Meanwhile, rapalogs are shown to activate AKT and ERKmitogen-activated protein kinases (MAPK) signaling that counteracts their activities in cancer [14, 15]. As a result, these mTORC1 inhibitors may ultimately upregulate cancer cell proliferation [12, 13]. Thus, an agent that dually blocks both mTORC1 and mTORC2 would theoretically result in improved anti-cancer activity [16, 17]. As a matter of fact, ATP-competitive mTOR inhibitors, targeting both mTORC1 and mTORC2, have been developed [18]. These compounds (i.e., AZD-8055, AZD-2014, INK-128, and OSI-027) are more effective mTORC1 inhibitors, which block mTORC1 and mTORC2 simultaneously [18]. Studies have shown that these mTORC1/2 dual inhibitors more completely inhibit 4EBP1 phosphorylation than rapamycin [19, 20]. This explains rapamycin ineffectiveness in cap-dependent protein translation in many cancer cells and also at least partly explains the weak anti-tumor activity by the rapalogs [19, 20]. Several of these compounds are being tested in various tumor models and showed higher efficiencies than rapalogs in suppressing cancer cells [21, 22]. In the current study, we evaluated the potential anti-prostate cancer activity of INK-128, a mTORC1/2 dual inhibitor [23, 24], both in vitro and in vivo.
Material and methods Chemicals and reagents INK-128 was obtained from Selleck China (Shanghai, China). The pan-caspase inhibitor z-VAD-FMK and the caspase-3specific inhibitor z-DVED-FMK were from Calbiochem (Darmstadt, Germany). Anti-tubulin, hypoxia-inducible factor (HIF)-1α, HIF-2α, and cyclin D1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies of cleaved caspase-3, p-AKT (Ser-473), p-AKT (Thr-308), AKT1, p-S6 (Ser-235/236), S6, p-forkhead box O 1a (FoxO1a) (Thr-24), p-ERK1/2 (Thr-202/Tyr-204), ERK1/2, androgen receptor, p-S6K1 (Thr-389), and S6K1 were obtained from Cell Signaling Technologies (Beverly, MA). Antibodies of p-FoxO3a (Thr-32) and FoxO3a were purchased from Abcam (Cambridge, MA). Cell lines Human prostate cancer cell lines PC-3 and LNCaP cells were cultured as monolayer in RPMI 1640 supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Hyclone, Shanghai, China) and antibiotics. RWPE1, a non-transformed prostate epithelial cell line, was obtained from ATCC (CRL-11609)
(Manassas, VA). RWPE1 cells were maintained in Defined Keratinocyte-SFM medium supplemented with growth factors (insulin, epidermal growth factor, and fibroblast growth factor; Invitrogen, Carlsbad, CA), and medium was replaced every other day. The number of viable cells (trypan blue negative) was counted through an automatic cell counter. Primary culture of patient-derived prostate cancer cells Fresh prostate cancer tissues were obtained from two patients (male, 65 and 71 years old) at the prostate resection operation. Neither patient had received prior chemical, hormonal, or radiation therapy. Tissues were finely minced, digested 12 to 18 h in 100 units/ml collagenase I in DMEM, and pipetted to disperse clumps. Cells were washed in PBS and cultured on collagen-coated tissue culture plates (BD Biosciences) in cell culture medium (DMEM, 20 % FBS, 2 mM glutamine, 1 mM pyruvate, 10 mM HEPES, 100 units/ml penicillin/streptomycin, 0.1 mg/ml gentamicin, and 2 g/l fungizone). Cell lines were named T1 to T11 and used within 3 weeks of culture. All tissues were collected under an approved institutional ethics protocol from patients granting informed consent. MTT assay of cell proliferation The methyl thiazolyl tetrazolium (MTT) assay was performed to assess cell proliferation according to instructions from the manufacturer (Sigma-Aldrich Co., St. Louis, MO). Briefly, cells were planted into 96-well plates at a density of 5000 cells per well. At the end of each treatment, 20 μl of MTT (5 mg/ml, Sigma) was added for 2 h. The medium was then discarded carefully, and 150 μl of DMSO per well was added. Absorbance was recorded at 570 nm with the Universal Microplate Reader (Bio-Tek Instruments, Milan, Italy). The value of the treatment group was normalized to that of the control group. Trypan blue staining of Bdead^ cells The number of dead prostate cancer cells (trypan blue positive) after treatment was counted under microscope, and the percentage (%) of Bdead^ cells was calculated by the number of the trypan blue positive cells divided by the total number of the cells. Cell apoptosis assay Apoptosis of prostate cancer cells was quantitatively determined by flow cytometry using the Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA). After treatment, cells were harvested, washed, and incubated with annexin V and propidium iodide (PI) at room temperature for 10 min in the dark. The stained cells were analyzed by FACS using a FACSCalibur instrument (BD Biosciences)
Tumor Biol.
equipped with CellQuest 3.3 software. The percentage of annexin V-positive cells was recorded as a quantitative indicator of cell apoptosis. Histone DNA apoptosis ELISA assay The Cell Death Detection ELISA method quantifies apoptotic cell death in cellular systems by measuring cytoplasmic histone-associated DNA fragments. These DNA fragments in vehicle control and INK-128-treated cells were quantified using a commercially available ELISA kit from Roche Diagnostics (Mannheim, Germany), according to recommended procedures. Caspase-3 activity assay Cytosolic proteins from 1×106 cells were extracted in hypotonic cell lysis buffer [25]. Twenty micrograms of cytosolic extracts was added to caspase assay buffer (312.5 mM HEPE S, pH 7.5; 31.25 % sucrose; 0.3125 % CHAPS) with AcDEVD-AFC (15 μg/ml) (Calbiochem, Darmstadt, Germany) as the substrate. After incubation at 37 °C for 1 h, the amount of AFC liberated was measured using a spectrofluorometer (Thermo-Labsystems, Helsinki, Finland) with excitation of 380 nm and emission wavelength of 460 nm. The value of the treatment group was normalized to that of the control group. Western blots After treatment, cells were collected and lysed. Protein lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and were transferred onto a PVDF membrane. The membrane was incubated with a solution containing Tris-buffered saline, 0.05 % Tween 20, and 10 % (w/v) non-fat dry milk, and then exposed for 3 h at room temperature to desired primary antibody. Following incubation with appropriate secondary antibody, the immunoreactive bands were visualized using enhanced chemiluminescence (ECL) method. The blots were stripped and re-probed with the loading control. Band intensity was quantified through ImageJ software and normalized to the loading. Xenograft assay Male nude mice (6–8 weeks old) were purchased from the author institutional animal facility and maintained in accordance with Institutional Animal Care Use Committee guidelines. PC-3 cells were mixed in a 1:1 ratio with Matrigel (Becton Dickinson, Bedford, MA), and a 0.1-ml suspension containing 3×106 cells was injected subcutaneously on the right flank of each mouse. Mice were randomized into two groups of ten mice/group. Treatment began 4 weeks post
tumor implant when the tumor reached around 100 mm3 in volume. Experimental animals were treated orally with INK128 (1 mg/kg in 0.1 ml PBS) daily for 21 days [26]. Control mice received an equal volume of the vehicle. Tumor volumes, mice body weights, as well wet tumor weights were determined as described [27]. At the termination of the experiment, the tumor tissues were harvested and divided into two pieces. A portion of the tumor tissue was processed for immunohistochemistry for analysis of apoptotic bodies, whereas the second piece was used for Western blots [27]. For immunohistochemistry, tumor tissues were embedded in paraffin, sectioned (4 μm), de-paraffinized, and processed for determination of apoptotic bodies using ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Intergen, NY) according to the manufacturer’s instructions. Brown-colored apoptotic bodies in tumor sections of vehicle control and INK-128treated mice were counted under a Nikon microscope at ×20 magnification. Ten randomly selected fields on each tumor section were counted for apoptotic bodies. For Western blots, tumor tissues were minced, suspended in PBS, and homogenized. The homogenate was centrifuged, and the supernatant fraction was collected and tested for Western blots. Statistical analysis All experiments were repeated at least two to three times, and similar results were obtained. Data were expressed as mean± standard deviation (SD). Statistical analyses were performed by one-way analysis of variance (ANOVA) using SPSS 18.0 software. Multiple comparisons were performed using Tukey’s honestly significant difference procedure. A p value
