miR-99b-targeted mTOR induction contributes to irradiation resistance in pancreatic cancer
- Feng Wei†1,
- Yan Liu†2, 3,
- Yanhai Guo3,
- An Xiang3,
- Guangyi Wang1,
- Xiaochang Xue4Email author and
- Zifan Lu3Email author
© Wei et al.; licensee BioMed Central Ltd. 2013
Received: 8 April 2013
Accepted: 22 July 2013
Published: 25 July 2013
Radiation exerts direct antitumor effects and is widely used in clinics, but the efficacy is severely compromised by tumor resistance. Therefore uncovering the mechanism of radioresistance might promote the development of new strategies to overcome radioresistance by manipulating activity of the key molecules.
Immunohistochemistry were used to find whether mTOR were over-activated in radioresistant patients’ biopsies. Then Western blot, real-time PCR and transfection were used to find whether radiotherapy regulates the expression and activity of mTOR by modulating its targeting microRNA in human pancreatic cancer cell lines PANC-1, Capan-2 and BxPC-3. Finally efficacy of radiation combined with mTOR dual inhibitor AZD8055 was assessed in vitro and in vivo.
Ionizing radiation promoted mTOR expression and activation in pancreatic cancer cells through reducing miR-99b expression, which negatively regulated mTOR. Novel mTOR inhibitor, AZD8055 (10 nM, 100 nM, 500 nM) synergistically promoted radiation (0–10 Gy) induced cell growth inhibition and apoptosis. In human pancreatic cancer xenografts, fractionated radiation combined with AZD8055 treatment further increased the anti-tumor effect, the tumor volume was shrinked to 278 mm3 after combination treatment for 3 weeks compared with single radiation (678 mm3) or AZD8055 (708 mm3) treatment (P < 0.01).
Our data provide a rationale for overcoming radio-resistance by combined with mTOR inhibitor AZD8055 in pancreatic cancer therapy.
KeywordsRadiation resistance mTOR AZD8055 Pancreatic cancer
Mammalian target of rapamycin
Pancreatic cancer is the fourth leading cause of cancer death, and is amongst the deadliest of human cancers. Only 10-15% patients undergo surgery due to late diagnosis, therefore radiotherapy becomes the major way in the treatment of pancreatic cancers in clinics, either alone or in combination with chemotherapy. Local control of tumor growth is partly achieved by radiation-induced cell death as a result of damage to cell membranes and DNA[2, 3]. However, the efficacy of radiotherapy remains limited due to intense tumor resistance. The molecular mechanisms underlying radiation resistance of pancreatic cancer are not fully understood.
The mammalian target of rapamycin (mTOR), a well-known serine/threonine kinase, is identified as a downstream target of PI3K/Akt survival pathway and functions as a central regulator of cell growth, proliferation and survival[5, 6]. Accumulating evidence demonstrated that mTOR was dysregulated in various cancers, its over-expression and over-activation contribute to cancer progression and drug-resistance[7, 8]. As a result, mTOR inhibitors represent a promising therapeutic approach for cancer and solid tumors[9, 10].
The first generation mTOR inhibitors, like rapamycin and its analogs everolimus (RAD001), temsirolimus (CCI-779) and ridaforolimus (AP23573), have been developed as cancer therapeutic agents[10, 11]. However, they are insufficient for achieving a broad and robust anticancer effect due to the feedback of AKT activation via up-regulating insulin-like growth factor-1 (IGF-1). AZD8055, a novel ATP-competitive inhibitor of mTOR kinases, besides preventing feedback to AKT, potently showed excellent selectivity (about 1,000 fold) against all class I PI3K isoforms and other members of the PI3K-like kinase family. AZD8055 is currently tested in phase I clinical trials as an anti-tumor drug[13, 14]. Prior studies reported that combination of mTOR inhibitor RAD001 with radiotherapy can delay solid tumor growth in vitro and in vivo due to synergistic anti-angiogenic and anti-vascular effects, but the detail mechanisms remain poorly defined. Here, we wonder whether mTOR inhibitor AZD8055 can also amplify the radiotherapeutic effects in pancreatic cancers.
MicroRNAs (miRNAs) are a class of small non-coding RNAs which play important roles in gene regulation by targeting mRNA in a sequence-specific manner, and their dysregulations are a common feature in tumorigenesis and drug-resistance[16, 17]. Numerous studies have shown that miR-99b, miR-100, miR-199a-3p, miR-451, miR-144 and miR-101 can directly or indirectly mediate mTOR expression[18–23], and reduction of these miRNAs was connected with the elevated levels of mTOR in prostate cancer and endometrial carcinoma[18, 24]. However, it is still not clear whether these miRNAs can be regulated by radiation and be connected with aberrant mTOR activation in pancreatic cancer.
In this study, we identified that mTOR is positively regulated by radiation in both human pancreatic biopsy specimens and cell lines, and this mTOR upregulation is promoted by radiation induced miR-99b downregulation. We further provided evidence that dual mTOR inhibitor AZD8055 significantly reversed the aberrant mTOR activation, consequently sensitized pancreatic cancer cell lines and xenografts to radiotherapy. Thus, our data provide a rationale for overcoming radio-resistance by combined with mTOR inhibitor AZD8055 in pancreatic cancer therapy.
mTOR was upregulated in pancreatic cancer patients subjected to radiotherapy
Ionizing radiation upregulates mTOR in pancreatic cancer cells at both transcriptional and protein levels
mTOR is a critical factor in pancreatic cancer radioresistance
Downregulation of miR-99b, a key mediator of mTOR kinase, contributes to radiation induced mTOR upregulation
In order to validate whether miR-99b could affect the cell sensitivity towards radiotherapy, PANC-1 cells were treated with radiation before and after miR99b precursor/inhibitor transfection. As shown in Figure 4C and D, cell growth and proliferation were significantly inhibited after downregulation of mTOR expression by miR-99b precursor whereas cells were more resistant to radiation after upregulation of mTOR by miR-99b inhibitor. All these data suggested that downregulation of miR-99b might induce cell resistance to ionizing radiation via enhanced mTOR expression.
Inhibition of mTORC1/2 activity by AZD8055 sensitizes pancreatic cancer cells to ionizing radiation
AZD8055 enhances radiation induced cell cycle disruption and cell apoptosis
Then Annexin V assay was employed to test whether the combination treatment was accompanied with increased programmed cell death. As shown in Figure 6B, Radiation or AZD8055 alone merely induced a small number of cells apoptosis by 18.4% or 11.7% even at 5 Gy or 500 nM. Intriguingly, AZD8055 combined with radiation synergistically induced significant cell apoptosis by 48.2%. Our findings indicate that AZD8055 enhanced ionizing radiation induced cell apoptotic and cell cycle arrest.
Suppression of mTOR activation by AZD8055 enhances antitumor efficacy of radiation in pancreatic cancer xenografts
To evaluate the role of apoptosis in this xenografts model, TUNEL assay was used to detect the tumor tissues and results showed that inhibition of mTOR pathway by AZD8055 significantly enhances apoptosis in pancreatic xenograft tissues (P < 0.01) (Figure 7C).
Pancreatic cancer is the most devastating type of cancer, the 5-year survival rate of patients is less than 5%. Until now, the late diagnosis and persistent resistance to chemo- and radio-therapy are still the leading problems in clinics. Although the current standard gemcitabine therapy and radiotherapy prolong the survival of patients with advanced pancreatic cancer for a few months, the high rate of recurrence still confused the clinical therapy[27, 28].
As we know, radiation has been widely used for pancreatic cancer therapy because it can induce cell death by damaging cell membranes and DNA. However, radiation is also able to stimulate some other important signaling pathways which regulate cell survival, proliferation and apoptosis[30, 31]. Until now, it is unclear about which signaling pathway plays the key role in the radiotherapy for unresectable pancreatic cancer. By exploiting with the patient biopsy samples, we demonstrated that mTOR expression was significantly up-regulated in clinical radiotherapy tissues, suggesting that it may contribute to the clinical radiotherapy resistance. This data provided the direct in vivo clinical evidence supporting that radiation induced mTOR upregulation might in association with pancreatic cancer cell resistance to radiation. From the cell line data, we also observed mTOR over-expression and over-activation after radiotherapy. Considering that miRNAs participated in various physiological and pathological processes by directly regulating target genes expression, we purposely detected various putative miRNAs that may repress mTOR and miR-99b was found to be down-regulated by radiation. Not surprisingly, mTOR was reversely regulated when miR-99b was overexpressed or knocked down under both basal and radiation conditions. In addition, cell sensitivity to radiotherapy was also influenced by miR-99b. Our results not only provide some new clues for mTOR upregulation in radiation-treated pancreatic clinical samples and cell lines, but also demonstrated that miR-99b played important roles in pancreatic cancer radioresistance and maybe a candidate therapeutic target for pancreatic cancer.
Considering mTOR was up-regulated by radiation through miR-99b and mTOR signal pathway plays critical roles in regulating cancer cell survival, proliferation and apoptosis, we wonder whether mTOR inhibition have synergistic effects with radiotherapy. AZD8055, an mTORC1/C2 dual inhibitor, was employed to inhibit mTOR activity and block the feedback activation of AKT. Results demonstrated that AZD8055 treatment significantly potentiates the cytotoxic effects of ionizing radiation in human pancreatic cancer cell lines. Additionally, we also confirmed that the growth inhibition was accompanied by a perturbation of cell cycle with the marked reduction of cells in S phase and an accumulation in G0/G1 phase. Moreover, AZD8055 treatment enhanced radiation induced cell apoptosis. Intriguingly, these events were paralleled by suppressing the expression and function of mTOR, but do not influence the anti-apoptotic family members such as Bcl-2, Bcl-XL and Mcl-1, suggesting that AZD8055 and radiation synergistically induced cell apoptosis through mTOR related signaling pathways but not Bcl-2 family in pancreatic cancer cells.
Similar to in vitro results, the growth of pancreatic cancer xenografts was also inhibited by fractionated radiotherapy or application of AZD8055 in vivo, and surely combination of AZD8055 and radiotherapy suppressed growth of PANC-1 xenografts more effectively than treatment with either therapy alone. On the whole, inhibition of mTOR activity by AZD8055 effectively reversed radio-resistance both in vitro and in vivo. Therefore inhibiting mTOR activity by AZD8055 may be an effective way to overcome radioresistance and potently sensitize pancreatic cancers to radiation.
In summary, our study observed mTOR upregulation in clinically treated biopsy samples and identify a novel mechanism related with mTOR upregulation in pancreatic cancer cells after radiation therapy. miR-99b reduction was involved in mTOR upregulation and therefore affected the radiotherapy sensitivity of pancreatic cancer cells. Blockade of mTOR by AZD8055 represents a new therapeutic strategy to overcome radioresistance in patients with pancreatic cancer.
In conclusion, the results of this study demonstrate the upregulation of mTOR by radiation via downregulating miR-99b and provide the first evidence of the regulatory effects of radiation on mTOR expression and activation. We propose that mTOR play a critical role in radioresistance and its dual inhibitor AZD8055 can be used in combination with radiation to overcome the radioresistance in pancreatic cancer treatment.
Materials and methods
AZD8055 was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies for mTOR, p-mTOR, Akt, p-Akt (S473), S6 and p-S6 (Ser235/236) were purchased from Cell Signaling Technology (Beverly, MA). Bcl-2, Bcl-XL and Mcl-1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Tumor TACS™ In Situ Apoptosis Detection Kit was purchased from Trevigen, Inc. (Gaithersburg, MD). mTOR shRNA was obtained from Sigma-Aldrich (St. Louis, MO). All other reagents were obtained from stated commercial sources.
Biopsies collection of pancreatic cancer patients
Patients with locally advanced pancreatic cancer were diagnosed by computed tomography (CT) and MRI imaging, and all patients received a comprehensive evaluation and were considered to be unresectable. Eight patients were treated with Intensity-modulated radiation therapy (IMRT) at 50 Gy and responses were evaluated via computed tomography. Five patients who have stable disease (SD) or progressive disease (PD) were resistant to IMRT among total 8 patients. The biopsies were taken by tru-cut needle from these five radiotherapy resistant patients. None of the subjects received other biotherapy or chemotherapy treatments. The study was approved by the ethics committees of the First Hospital of Jilin University and the Fourth Military Medical University. Written informed consents were also obtained from all subjects before study.
Cell culture and sulforhodamine B assay
Human pancreatic cancer cells PANC-1, Capan-2 and BxPC-3 purchased from National Rodent Laboratory Animal Resource (Shanghai, China) were grown as previously described. Briefly, these cell lines were cultured and maintained in exponential growth in Dulbecco’s modified Eagle’s medium (DMEM) containing 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 mM glutamine and 10% heat-inactivated FCS (Atlanta Biologicals, Lawrenceville, GA) in a humidified atmosphere of 5% CO2 at 37°C. For sulforhodamine B (SRB) assay, the exponential growing cells were seeded at 6–8 × 103/well in 96-well plates and cultured overnight. Cells were treated with radiation alone or combined with AZD8055. AZD8055 was added to cultured cells and radiation was applied 4 h later in single doses of 1, 2.5, 5 or 10 Gy. The cells were irradiated using an X-ray machine (X-RAD 320, Precision X-ray) at 320 kV, 10 mA with a 2-mm aluminum filter, and the dose rate was 2 Gy/min. Cells were then cultured at 37°C for 48 h and the surviving fractions were determined using SRB assay as previously described[33, 34]. The absorbance was measured with a spectrophotometer (Bio-Rad Inc) at 510 nm and cell growth inhibition was calculated by using the equation: cell viability (%) = (At/Ac) × 100%, in which At and Ac represent the absorbance in treated and control cultures respectively, as described previously.
Cell lysate and Western blot assay
Cells were lysed in ice-cold EBC buffer (50 mM pH 8.0 Tris, 120 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 μM sodium orthovanadate, 1 × Protease Inhibitors, 1 × Phosphatase Inhibitors) and proteins were quantified and subjected to SDS-PAGE electrophoresis, followed by protein transfer to nitrocellulose membranes. The membranes were incubated with the primary and secondary antibodies, then developed by chemiluminescence.
RNA isolation and quantitative real-time PCR
Total RNA was isolated from cells using Trizol (Invitrogen), 1–10 μg of RNA was used to synthesize cDNA with SuperScript II First-Strand Synthesis System (Invitrogen) or TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). Aliquots of the reaction mixture were used for real-time PCR with Power SYBR Green PCR Master Mix or with the TaqMan® 2 × Universal PCR Master Mix. The reaction conditions: 50°C for 20 s, 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. All real-time PCR experiments were performed in triplicate. A melting curve was obtained to verify the presence of a single amplicon. The primer sequences are as described previously[36–38].
Colony formation assay
PANC-1 cells were seeded in 6-well-plates (1000 /well), and then treated or untreated with radiation and AZD8055, alone or in combination. The medium was replaced with fresh medium containing the reagent and radiation-treatment every three days. After 10 days treatment, the medium was removed and cell colonies were stained with crystal violet (0.1% in 20% methanol). Pictures were taken using a digital camera to record the result as described. To evaluate the colony formation ability of irradiation-resistant cells, PANC-1 irradiation-resistant cell line (PANC-1-RR) was firstly generated by plating PANC-1 cells in 100-mm culture dishes and irradiating with 2 Gy X-ray every three days over a period of 5 months, for a total dose of 100 Gy, and then colony formation assay was used as above mentioned.
PANC-1 cells were suspended in DMEM supplemented with 10% FBS and seed in 6-well plates (1 × 106/well) and transfected with miR-99b precursor or inhibitor (Ambion) with Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s instruction. After 48 h of transfection, cells were treated by radiation at 5 Gy, then harvested and lysed for Western blot assay. For mTOR interfering, mTOR shRNA with the sequence of CCGGGCTGTGCTAC ACTACAAACATCTCGAGATGTTTGTAGTGTAGCACAGCTTTTTG was used to transfect PANC-1 cells.
Annexin V/PI Apoptosis Detection kit (Clontech Laboratories) was used for quantification of apoptosis. Cells were seeded in 6-well plates in the absence or presence of AZD8055 (500 nM), then radiation was applied 4 h later. After cultured for 24 h, 0.5-1 × 106 cells were collected into each tube and gently washed with PBS. Cell pellets were suspended in 1 × binding buffer and stained with Annexin V and PI. After incubated for 15 min at RT in the dark, the apoptosis analysis was carried out using a FACScan (BD Biosciences) and analyzed using FlowJo software (Tree Star Inc).
Cell cycle analysis
Cells were synchronized by growing in serum free medium for 48 h and then released into the cell cycle by adding 10% FBS to the medium. The cells were treated with radiation in the absence or presence of AZD8055 (500 nM) for 24 h, harvested, fixed with 70% ethanol, and stained with PI. Data were acquired using flow cytometry and analyzed using FlowJo software.
Pancreatic cancer xenografts and treatments
Animal experiments were careful to follow the protocols approved by Jilin University and the Fourth Military Medical University Institutional Animal Care and Use Committees. PANC-1 cells (7 × 106) were resuspended in HBSS and injected subcutaneously into the flank region of 6-week-old female athymic (nu/nu) mice (Shanghai, China). The tumors were allowed to grow to average volume of 200 mm3 prior to initiation of therapy as described. Then mice were assigned randomly to four groups (n =10) as following: (1) vehicle control (5% DMSO, 100 μl/d p.o.); (2) 8 Gy fractionated radiotherapy (2 Gy for every three days); the radiation was performed using the same X-ray machine with a different filter (1.5 mm aluminum, 0.8 mm tin, and 0.25 mm copper), at a dose rate of 1 Gy/min; (3) AZD8055 (20 mg/kg/d), AZD8055 was dissolved in DMSO and administered by oral gavage (0.1 ml/10 g of body weight); (4) Combination of AZD8055 (20 mg/kg/d) and 8 Gy (2Gy × 4) fractionated radiotherapy. Tumor volumes were measured with a caliper every other day and calculated based on the formula: V = 4/3 × π(length/2 × (width/2)2). After 21 days treatment, mice were sacrificed and the tumors were removed and submerged in 10% neutrally buffered formalin for immunohistochemistry analysis.
Four-μm thick paraffin sections were deparaffinised, rehydrated and stained using the R.T.U.Vectastain kit following the manufacturer’s standard protocol (Vector Laboratories). The sections were incubated with anti-mTOR antibody (1:50) overnight at 4°C, then stained with secondary antibody. Thereafter, the slides were exposed to DAB chromogen for 5 min, then hematoxylin counter stained, dehydrated, and treated with xylene following the approach as earlier reported. Finally all slides were examined and representative pictures were taken using an Olympus BX41 microscope.
TUNEL staining was performed by using Tumor TACS™ In Situ Apoptosis Detection Kit (Trevigen), the specimens were deparaffinised and labeled following the procedure provided by the manufacturer. Finally, DAB staining were visualized under microscopy. For TUNEL assay, ten fields were randomly selected from each slide for measurement, the images were analyzed by MetaMorph software and presented as a percentage of the total number of cells.
Levels of significance were determined by different methods, two-sided unpaired student’s t-test and one-factor ANOVA were used in the comparison between groups, and LSD-t tests was used in multiple comparisons. Results were considered statistically significant at P values < 0.05.
Feng Wei and Yan Liu are joint first authors.
This work was supported by the Grants from National Natural Science Foundation of China (81201711, 30900537 and 31000406).
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