SMAD4 Gene Mutation Renders Pancreatic Cancer Resistance to Radiotherapy through Promotion of Autophagy

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant disease of the exocrine pancreas, and is the fourth most common cause of cancer deaths worldwide, leading to estimated 227,000 deaths annually (1, 2). Despite advances in conventional therapies (surgical, chemotherapy, and radiotherapy), little improvement has been observed in the survival rate over the past 30 years (3). The median survival of patients with PDAC is less than 6 months, and the 5-year survival rate is less than 5% (1–3). Since early-stage pancreatic cancer is usually clinically silent, most patients have already developed locally advanced or metastatic disease at diagnosis, and only 10% to 15% of the patients are eligible for surgical resection (4, 5). Most pancreatic cancer patients are treated with chemotherapy in the United States, either alone or in combination with radiotherapy (6–8), whereas chemotherapy is frequently used alone in patients in Europe, based on the European organization for Research and Treatment of Cancer (EORTC) trail (9). However, the U.S. study was criticized for poor patient accrual, early termination, small patient number and suboptimal radiotherapy dose. At the same time, there are some defects in EORTC trial design, including the mixing up of pancreas and peri-ampullary cancers, underpowered analysis for survival benefit, and use of antiquated radiotherapy and chemotherapy techniques. A growing body of evidence showed no survival benefit for adjuvant chemoradiotherapy but revealed a potential benefit for adjuvant chemotherapy (10–13). However, the true benefit of the addition of radiotherapy is still unknown (14). The underlying reason for the striking difference in guidelines of PDAC treatment between these different regions is still unclear. Because many gene mutations affect cell growth and drug responses of cancer cells, we suspect that the difference in the mutational status of some key genes in the pancreatic cancer patients may contribute to resistance to radiotherapy.

Mutations in multiple genes such as SMAD4, KRAS, TP53, and CDKN2A/INK4A (15, 16) have been demonstrated to be important in PDAC progression. Among these, the SMAD4 status is considered to be an important molecular feature which distinguishes two major classes of PDAC. The tumor suppressor gene SMAD4 encoding a common intracellular mediator of the TGFβ superfamily is mutated or deleted in 55% pancreatic cancers (16, 17). This gene is also inactivated at varying frequency in breast, colorectal, and gastric cancer (18, 19). Loss of SMAD4 promotes pancreatic tumor progression and increases metastasis (20, 21). SMAD4 is reported as a negative prognostic factor for overall survival (17, 22–24). Growing evidence showed that the loss of SMAD4 induces resistance to chemotherapy in colorectal, breast, head, and neck cancers (25–27). However, the role of SMAD4 in radioresistance of pancreatic cancer and the underlying molecular mechanism have not been fully elucidated.

In this study, we showed that SMAD4 depletion renders pancreatic cancer cells resistant to ionizing radiation (IR) both in vitro and in vivo. Mechanistically, SMAD4 depletion induces high levels of autophagy and ROS, which appear to contribute to such radioresistance.

The human pancreatic cancer cell lines Panc-1 and MIA PaCa-2 were purchased from the ATCC. Cells were maintained in DMEM medium (GIBCO) supplemented with 10% or 20% FBS and 100 U/mL penicillin (GIBCO). Panc-1 cells transfected with shRNA (Panc-1-shControl and Panc-1-shSMAD4) were maintained in DMEM medium (GIBCO) supplemented with 10% FBS, 100 U/mL penicillin, and 1 μg/mL puromycin (Sigma). All cell lines were cultured in a 37°C incubator with 95% air and 5% CO₂. Each cell line was authenticated and regularly tested for mycoplasma contamination.

Lipofectamine 2000 (Invitrogen) was applied to transfect PcDNA3-Flag-SMAD4 plasmid (Addgene plasmid #80888) into cells by following manufacturers' instruction. N-acetyl-L-cysteine (NAC) and chloroquine (CQ) were purchased from Sigma-Aldrich, and were reconstituted in DMSO and PBS at 100 mmol/L, respectively. Antibodies against SMAD4, Rad51, BRCA-1, LC3, FLAG, and γ-H2AX were purchased from Cell Signaling Technology Inc. A monoclonal anti-β-actin antibody was obtained from Sigma.

Cells were seeded for 24 hours at a density of 3 × 10⁵ cells/mL prior to IR treatment. The level of intracellular ROS was measured using the fluorescent probe 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) following manufacturer's instruction (Life Technologies). In brief, cells were incubated with 10 μmol/L CM-H₂DCFDA for 30 minutes at 37°C in the dark and then washed in serum-free minimum essential medium without phenol red. The cells were then analyzed in a FACSCanto apparatus (BD Biosciences).

Cells were grown to confluence in 6-cm tissue culture plates, and transfected with one of the four SMAD4-specific siRNA oligos at 50 nmol/L final concentration (si-SMAD4-a: 5′-AGAUGAAUUGGAUUCUUUAdTdT-3′; si-SMAD4-b: 5′-GUGUGCAGUUGGAAUGUAAdTdT-3′; si-SMAD4-c: 5′-GUACUUCAUACCAUGCCGAdTdT-3′; si-SMAD4-d: 5′-CAUCCUAGUAAAUGUGUUAdTdT-3′) using INTERFERin as the transfection reagent according to the manufacturer's instruction. A scrambled siRNA (siScr: 5′-ACGCGUAACGCGGGAAUUUdTdT-3′) was applied as a negative control. Cells were harvested 72 hours later for gene expression analysis by Western blot analysis.

Retroviral vectors carrying a human SMAD4-specific shRNA (target sequence 1: 5′-GGTGTGCAGTTGGAATGTA-3′ for clone #35 and #46. Target sequence 2: 5′-GATTAACACTGCAGAGTAA-3′ for supplementary figures) or a scrambled nontargeting control (sequence: 5′-AAAATGCAGTTGGAATGTA-3) were purchased from Addgene. The shSMAD4 and shControl recombinant viruses were generated by transient transfection with packaging plasmids pMLg/pRRE, pRSV.rev, and pHCMV-G into 293T cells (28). Virus-containing supernatant was collected 72 hours later, and applied to transfect Panc-1 cells, and then selected three individual clones as Panc-1-shSMAD4 #35 and #46 (for target sequence 1) and #2 (for target sequence 2).

For in vitro studies, cells were irradiated at various doses using an XRAD 320 X-ray irradiator (Precision X-Ray, Inc.) at a dose rate of 75 cGy/min. For in vivo IR, mice were anesthetized with ketamine/xylazine (100 mg/kg + 10 mg/kg) and placed in well-ventilated custom jigs (Precision X-Ray), allowing for local delivery of radiation using an XRad 320 X-ray irradiator (Precision X-Ray, Inc.) at a dose rate of 289.8 cGy/min.

Cell survival was determined using a standard clonogenic assay. Cells with or without SMAD4 knockdown were trypsinized to generate a single-cell suspension and seeded into six-well plates in triplicate. After incubation for 16 hours, 800 cells were treated with IR (0–6 Gy). After 7 to 10 incubation, the colonies were fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet. After counting the number of colonies (containing more than 50 cells), the surviving fraction was calculated.

Cells were rinsed twice with PBS and lysed for 2 h with RIPA Lysis buffer (Thermo Scientific) supplemented with a protease inhibitor and a protease inhibitor cocktail. Cell debris was removed by centrifugation at 12,000 rpm for 10 minutes at 4°C. Protein concentration in the supernatant was determined with the BCA Protein Assay Kit (Pierce). The protein samples were separated by electrophoresis using a 4% to 20% Mini-PROTEAN Tetra Cell polyacrylamide gel (Bio-Rad) and then transferred to polyvinylidene fluoride membranes (Bio-Rad). The membrane was blocked with 5% skim milk, and incubated with the indicated primary antibodies, followed by incubation with horseradish peroxidase-labeled secondary antibodies. Antibody-bound proteins were detected using the Enhanced Chemiluminescence (ECL) reagent (GenDEPOT) according to the manufacturer's instructions. The protein levels were quantified using the ImageJ software (NIH) and then normalized by the expression levels in control cells for each group.

Cells were seeded onto four-well LabTek II chamber slides (NUNC) for 24 hours, and then treated with IR at 2, 4 or 6 Gy. After incubation for 2 hours, cells were fixed with 4% paraformaldehyde for 10 minutes, washed three times with PBS and blocked with 3% BSA and 0.1% Triton-X100 in PBS for 30 minutes at room temperature. After removing the blocking buffer, cells were incubated with the primary antibodies (rabbit γ-H2AX antibody, LC3 antibody and mouse SMAD4 antibody) overnight at 4°C. After being washed three times with PBS, cells were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 594-conjugated anti-mouse IgG secondary antibodies (Invitrogen) for 2 hours in the dark. The cells were then washed and mounted with mounting medium containing DAPI (VECTOR Laboratories, Burlingame, CA, USA). Finally, a Nikon A1 confocal microscope was applied to examine and quantitate γ-H2AX, LC3, and SMAD4 signals. At least 300 nuclei were evaluated for each data point.

All mouse studies were performed in compliance with the guidelines of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals following protocols approved by the Institutional Animal Care and Use Committee (IACUC). Six-week-old athymic nude mice were purchased from Charles River Laboratories (Boston, MA). Mice (8 per group) were injected with 2 × 10⁶ Panc-1-shControl or Panc-1-shSMAD4 cells suspended in 100 μL medium with 5% Matrigel directly into the pancreas for orthotopic tumor development. Three weeks after inoculation, the mice received 4 Gy radiation for three times with 2-day intervals between irradiations. The mice were sacrificed at 3 weeks after the last treatment, and the tumors were removed and weighed.

IHC staining was performed as previously described (29). Briefly, tumor sections were deparaffinized, rehydrated, and treated with 10 mmol/L citrate buffer (pH 6.0) at 95°C to retrieve antigens. After quenching endogenous peroxidase activity with H₂O₂ and blocking with 10% horse serum, the sections were incubated sequentially with the primary antibodies mouse anti-SMAD4 (1:100; Cell Signaling Technology Inc.), rabbit anti-LC3 (1:100; Cell Signaling Technology Inc.), rabbit anti-γ-H2AX (1:100; Cell Signaling Technology Inc.), biotinylated secondary antibodies, and the Avidin-Biotin Complexes (ABC) reagent (Gene Tech). The immunostaining was visualized with 3.3-diaminobenzidine (Gene Tech). The sections were then counterstained with hematoxylin. Negative controls were performed in each case by replacing the primary antibody with PBS.

All experiments were performed in triplicate. The data were expressed as means ± SD. Statistical analyses were performed using Student t test and ANOVA. Differences across groups selected to be compared were detected via repeated measures ANOVA. Values of P < 0.05 were considered statistical significant, and all the statistical test were two sides. All statistical analyses were conducted in R program (R core team, version 3.4.2).

In order to examine whether SMAD4 was involved in response to IR, we knocked down SMAD4 gene expression in Panc-1 cells with retroviral shRNA constructs (sh-SMAD4 #35 and sh-SMAD4 #46) or siRNA oligos (si-SMAD4-1 and si-SMAD4-2) targeting the SMAD4 gene. To exclude the off-target effect of shRNA, we also established cell lines stably expressing another shRNA construct which targeted different sites in SMAD4. Western blot analysis showed that SMAD4 was depleted by siRNA or shRNA (Fig. 1A and B; Supplementary Fig. S1). The same siRNA oligos were effective in suppressing SMAD4 expression in MIA PaCa-2 cells (Fig. 1C). The cancer cells were treated with various doses of IR, and then seeded on cell culture plates for colony formation. Interestingly, Panc-1 cells infected with the SMAD4 gene-specific shRNA retrovirus were more resistant to IR than both the untreated cells and those treated with retrovirus carrying a control scramble shRNA (Fig. 1D). Similar results were obtained when Pan-1 cells treated with siRNA oligos (Fig. 1E). Consistent with Panc-1 cells, MIA-PaCa-2 cells treated with SMAD4 siRNA oligos were more resistant to IR than those treated with the control siRNA oligos (Fig. 1F). We also found that SMAD4 wild-type (WT) pancreatic cancer cell lines (Panc-1, MIA PaCa-2) were more sensitive to IR than SMAD4 mutated cell lines (Bxpc-3, Capan-1; Fig. 1G). Furthermore, we found that the Bxpc-3 cells transfected with Flag-SMAD4 plasmid were more sensitive to IR than control cells with empty vector (Fig. 1H). These results indicate that suppression of SMAD4 expression increases radioresistance in pancreatic cancer cells.

Radiation kills cancer cells by inducing DNA damage which triggers DNA damage response (DDR), including genomic instability. To investigate whether SMAD4 loss affects genomic stability, we examined several DDR endpoints. Knockdown of SMAD4 expression activated phosphorylation of the H2AX histone (γ-H2AX), a molecular marker for DNA double-strand breaks (DSB; ref. 30), in Panc-1 cells without IR (Fig. 2A). After IR treatment, the γ-H2AX level increased in both control and SMAD4 depleted cells (Fig. 2A; Supplementary Fig. S2). Interestingly, the levels of Brca1 and Rad51, two key proteins involved in DNA DSB repair, were reduced in cells with suppressed SMAD4 expression (Fig. 2A; Supplementary Fig. S2). Immunostaining revealed a similar pattern of γ-H2AX levels in Panc-1 cells (Fig. 2B and C). There was a larger number of γ-H2AX foci in Panc-1 cells treated with si-SMAD4 before IR treatment, and massive staining of γ-H2AX foci were readily visible within 2 hours of IR in the nuclei of Panc-1 cells treated with either the control siRNA or SMAD4 siRNA (Fig. 2B and C). These results indicate that the SMAD4 protein is required for maintaining a proper DDR machinery in cancer cells.

It has been well documented that autophagy occurs in response to IR, which rescues cells from IR-induced cellular damage, thereby contributing to radioresistance (31–33). Inhibition of autophagy has been shown to sensitize tumor cells to the cytotoxic effects of both chemotherapy and radiotherapy (34, 35). In order to determine whether autophagy played a role in SMAD4-dependent resistance to IR, we knocked down SMAD4 expression in Panc-1 and MIA Paca-2 cells, and measured cellular levels of the autophagy marker protein LC3-II, a lipidated LC3 protein that is associated with autophagosomes (36). Western blot analysis revealed that knockdown of SMAD4 promoted the conversion of LC3-I to LC3-II in Panc-1 (1.45-fold increase) and MIA PaCa-2 (2.15-fold increase) cells (Fig. 3A–C). Upon IR (6 Gy) treatment, LC3-II levels increased steadily in a time-dependent manner in both control cells and SMAD4-depleted cells. The peak reached at the 24-hour time point in both cell lines. However, LC3-II increase was more profound in cells with suppressed SMAD4 expression compared with that in control cells. We also observed similar results in the Panc-1 cells stable expressing SMAD4 shRNA (Supplementary Fig. S2A). During authophagy, autophagic flux could be easily visualized by fluorescence microscopy using LC3-II antibody, which is tightly bound to the membrane of autophagosome. Immunofluorescent imaging analysis (Fig. 3D and E; Supplementary Fig. S2B) showed that, in SMAD4-depleted cells, LC3 signals shifted from a diffuse cytoplasmic pattern to a punctate membrane pattern. In addition, SMAD4-depleted cells displayed a higher level of membrane LC3 signal, indicating autophagosome formation (Fig. 3D and E; Supplementary Fig. S2B). Collectively, our results suggest that radioresistance mediated by SMAD4 depletion is accompanied by an increase in autophagic flux in pancreatic cancer cells.

It has been previously reported that autophagy occurs in response to stress, such as ROS (37, 38) and nutrient starvation (39). IR induce intracellular ROS production, and ROS accumulation is necessary for the initiation of autophagy (40–43). We next examined ROS levels in SMAD4-depleted pancreatic cancer cells based on CM-H₂DCFDA fluorescence staining (Fig. 4; Supplementary Fig. S3). ROS levels were increased within 2 to 6 hours after IR treatment in Panc-1 and MIA PaCa-2 cell lines (Fig. 4C and D). The basal levels of ROS were elevated in both cell lines with SMAD4 depletion, and radiation-induced ROS levels were higher in SMAD4-depleted cells than those in control cells within 2 to 6 hours after IR treatment (Fig. 4C and D; Supplementary Fig. S3B). The result indicates that induction of autophagy was accompanied by accumulation of ROS in SMAD4-depleted pancreatic cancer cells.

Because SMAD4 depletion caused higher level of autophagy and ROS production in pancreatic cancer cells, we set up experiments to test whether autophagy and ROS play a critical role on IR resistance induced by SMAD4 depletion in PDAC. We transfected Panc-1 cells with a control scramble siRNA or SMAD4-specific siRNA (Fig. 5A), and also treated cells with chloroquine (CQ), an autophagy inhibitor. These cells were subjected to increasing doses of IR treatment, and then seeded for colony formation. While only marginal difference was observed in the control Panc-1 cells with or without CQ treatment, cells transfected with SMAD4 siRNA were significantly more resistant to IR at 4 and 6 Gy doses compared with control cells, and CQ treatment partially reduced such resistance (Fig. 5B). In a separate experiment, Panc-1 cells transfected with a control or SMAD4-specific siRNA were treated with N-acetyl-l-cysteine (NAC), a ROS inhibitor, before IR. Similar to the result of CQ cotreatment, pretreatment with NAC re-sensitized cells to IR in SMAD4-depleted cells, but not in control cells (Fig. 5C). Similar phenomena were observed in the Panc-1 cells expressing SMAD4 shRNA after NAC and CQ treatment (Supplementary Fig. S4). Furthermore, we found that SMAD4-induced autophagy is partially rescued by ROS scavenger (Supplementary Fig. S5A) and NAC or CQ did not affect Rad 51 and Brca-1 protein levels (Supplementary Fig. S5B). Taken together, our data show that the autophagy and ROS inhibitors sensitized pancreatic cancer cells to IR, indicating that SMAD4 loss induced IR resistance is at least partially due to increased level of ROS and autophagy.

To validate whether the above cell-based findings were applicable in vivo, we generated orthotopic pancreatic cancer murine models by intra-pancreatic inoculation of Panc-1 cells, and treated tumor-bearing mice with IR (Fig. 6A). Although growth of the control Panc-1 tumors was significantly inhibited by IR treatment (P = 0.038), SMAD4 depletion largely abolished such radio-sensitivity of Panc-1 tumors (P = 0.819; Fig. 6B and C). Consistent with the cell-based results, SMAD4 depletion promoted basal LC3II and γ-H2AX expression in tumor tissues (Fig. 6D). We also found that γ-H2AX expression level was higher in tumor sections derived from SMAD4 shRNA-expressing cells than those from control shRNA cells. Similar to the cell-based study (Fig. 2), LC3-II and γ-H2AX expression levels were further increased after IR (Fig. 6D). These results suggest that SMAD4 is essential for pancreatic cancer radiosensitivity.

Chemotherapy alone or chemotherapy combined with radiotherapy is used both after surgical resection (adjuvant therapy) and prior to the tumor resection (neoadjuvant therapy) of PDAC in North America (6–8). However, many patients with PDAC are either intrinsically resistant or develop acquired resistance to radiotherapy (44). It is crucial to understand the molecular mechanism of such radioresistance, so as, to develop effective targeted therapies for pancreatic cancer. In this study, we identified a critical role of SMAD4 in mediating pancreatic cancer cells' response to radiotherapy. Our in vitro and in vivo studies demonstrated that SMAD4 depletion led to radioresistance through induction of autophagy and ROS.

It was reported that 55% of PDAC showed SMAD4 inactivation (16, 17). Therefore, the SMAD4 status is considered to be a molecular biomarker for PDAC. However, how SMAD4 mutations contribute to the efficacy of radiotherapy of PDAC remains elusive. Homozygous deletion of SMAD4 gene or its inactivating mutation was observed in several types of cancer including pancreatic cancer (15) and colorectal cancer (45, 46). Multiple clinical studies showed that the PDAC patients with intact SMAD4 more commonly had local dominant pattern of tumor progression, whereas the patients with SMAD4 loss commonly had distant disease progression (17, 22–24). Recent research reported that the combination of Runx3 expression levels and SMAD4 status coordinately modulate the balance between dissemination and cell division in PDAC (47). However, very few studies have reported the correlation between SMAD4 and radioresistance in pancreatic cancer. Here we evaluated the effect of SMAD4 loss on the radioresistance of pancreatic cancer cells and in the animal model. We discovered that SMAD4 mutant cell lines were more resistant to IR than SMAD4 WT cell lines. SMAD4 depletion significantly increased IR resistance, and SMAD4 restoration significantly increased IR sensitivity of pancreatic cancer cells in vitro. Consistently, depletion of SMAD4 decreases the IR radiosensitivity of pancreatic tumor in vivo. Mechanistically, we found the SMAD4 depletion-induced IR resistance occurred via elevated levels of ROS and autophagy in pancreatic cancer cells. Pretreatment of autophagy inhibitor CQ or ROS scavenger NAC significantly increased radio-sensitivity of SMAD4-depleted pancreatic cancer cells.

Ionizing radiation generates large amount of intracellular free radicals and ROS (37, 38), and excessive production of ROS could induce autophagy (40, 41). Moderate autophagy, in turn, assists in the clearance of excessive ROS to protect cells from oxidative damage, which may reflect the balance of either cell survival or death (41, 43). There has been compelling evidence that clinically anticancer therapeutics, including DNA-damaging agents and IR, induce autophagy in cell culture and animal models (48, 49). Autophagy plays a cytoprotective role during anticancer therapy, and its inhibition can sensitize cancer cells to these therapies (31–33, 50). Activation of DDR may evoke autophagy as an early adaptive response, which can be considered as the DDR mechanisms of DNA damage tolerance. It is therefore expected that dysregulated autophagy may protect pancreatic cancer cells from IR-induced cellular damage, thereby enhancing radioresistance.

Indeed, we observed elevated level of autophagy in SMAD4-depleted pancreatic cancer cells after IR, and correlates with reduced radiosensitivity. Consistent with above reports, our results suggest that autophagy may function as a cytoprotective mechanism during radiotherapy in pancreatic cancer. Given that the process of autophagy may be used by cells to escape or delay apoptotic death in response to IR-induced DNA damage, molecules regulating autophagy and autophagic cell death may serve as viable targets for eliminating resistance to pancreatic cancer treatment.

Resistance to IR in pancreatic cancer has contributed to treatment challenges (9–13). Thus, understanding the molecular mechanisms underlying radioresistance is an important pursuit. In this study, we discovered SMAD4 loss-induced radioresistance, and elucidated the underlying mechanism, which may explain the phenomenon of which have been reported in previously clinical studies (23, 24). Our findings suggested that SMAD4 expression level correlated with radio-sensitivity of pancreatic cancer cells via regulating ROS and autophagy pathway. Thus, assessment of SMAD4 status may represent a vital screening test before radiotherapy. However, further clinical investigations are warranted to evaluate the relationship of SMAD4 status and survival advantages of chemoradiotherapy in patients with pancreatic cancer, which would be helpful to guide the administration of targeted therapies in the adjuvant setting based on SMAD4 status.

In conclusion, we have shown that SMAD4 depletion induced radioresistance in pancreatic cancer cells both in vitro and in vivo through elevated levels of ROS and autophagy under both basal and after IR treatment. However, the special mechanism by which SMAD4 participates into or regulates autophagy need to be confirmed on patient tumor tissues. Our investigation of the role of SMAD4 in regulating autophagy and radiosensitivity revealed a novel function for this frequently mutated gene in PDAC, which may have significant implication in deciding on the proper treatment plan for PDAC patients.

No potential conflicts of interest were disclosed.

Conception and design: F. Wang, X. Xia, C. Yang, E.J. Koay, H. Shen

Development of methodology: X. Xia, J. Mai, Y. Kang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): F. Wang, X. Xia, J. Shen, J. Mai, D. Kirui, J.B. Fleming, E.J. Koay

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Wang, X. Xia, C. Yang

Writing, review, and/or revision of the manuscript: F. Wang, X. Xia, C. Yang, J. Shen, J.B. Fleming, E.J. Koay, H. Shen

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Yang, H.-C. Kim, D. Kirui

Study supervision: S. Mitra, M. Ferrari, H. Shen

The authors acknowledge financial support from the following sources: NIH grants (Nos. 1R01CA193880, U54CA143837, and U54CA210181), Department of Defense grants (No. W81XWH-12-1-0414), National Natural Science Foundation of China (No. 31771497, 81472578), and the Ernest Cockrell Jr. Presidential Distinguished Chair.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.