Open Access

Combined inhibition of JAK1,2/Stat3‑PD‑L1 signaling pathway suppresses the immune escape of castration‑resistant prostate cancer to NK cells in hypoxia

  • Authors:
    • Li‑Jun Xu
    • Qi Ma
    • Jin Zhu
    • Jian Li
    • Bo‑Xin Xue
    • Jie Gao
    • Chuan‑Yang Sun
    • Ya‑Chen Zang
    • Yi‑Bin Zhou
    • Dong‑Rong Yang
    • Yu‑Xi Shan
  • View Affiliations

  • Published online on: April 20, 2018     https://doi.org/10.3892/mmr.2018.8905
  • Pages: 8111-8120
  • Copyright: © Xu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Castration‑resistant prostate cancer (CRPC) is difficult to treat in current clinical practice. Hypoxia is an important feature of the CRPC microenvironment and is closely associated with the progress of CRPC invasion. However, no research has been performed on the immune escape of CRPC from NK cells. The present study focused on this subject. Firstly, when the CRPC cell lines C4‑2 and CWR22Rv1 were induced by hypoxia, the expression of the UL16 binding protein (ULBP) ligand family of natural killer (NK) group 2D (NKG2D; ULBP‑1, ULBP‑2 and ULBP‑3) and MHC class I chain‑related proteins A and B (MICA/MICB) decreased. NKG2D is the main activating receptor of NK cells. Tumor cells were then co‑cultured with NK cells to conduct NK cell‑mediated cytotoxicity experiments, which revealed the decreased immune cytolytic activity of NK cells on hypoxia‑induced CRPC cells. In exploring the mechanism behind this observation, an increase in programmed death‑ligand 1 (PD‑L1) expression in CRPC cells induced by hypoxia was observed, while the addition of PD‑L1 antibody effectively reversed the expression of NKG2D ligand and enhanced the cytotoxic effect of NK cells on CRPC cells. In the process of exploring the upstream regulatory factors of PD‑L1, inhibition of the Janus kinase (JAK)1,2/signal transducer and activator of transcription 3 (Stat3) signaling pathway decreased the expression of PD‑L1 in CRPC cells. Finally, it was observed that combined inhibition of JAK1,2/PD‑L1 or Stat3/PD‑L1 was more effective than inhibition of a single pathway in enhancing the immune cytolytic activity of NK cells. Taking these results together, it is thought that combined inhibition of the JAK1,2/PD‑L1 and Stat3/PD‑L1 signaling pathways may enhance the immune cytolytic activity of NK cells toward hypoxia‑induced CRPC cells, which is expected to provide novel ideas and targets for the immunotherapy of CRPC.

Introduction

With continuous promotion of castration and anti-androgen therapy, clinical treatment of androgen independent protate cancer or castration-resistant prostate cancer (CRPC) has become difficult. It is not uncommon that CRPC develops metastases that chemotherapy and radiotherapy have limited effects on, which seriously affects patients' quality of life. Therefore, research on mechanisms of CRPC progression seems particularly important (13). The tumor microenvironment is essential for tumor genesis and tumor development (4,5), with hypoxia a strong research topic in recent years. Hypoxia can induce vascular formation in tumors, and is also widely involved in tumor formation, development, metastasis and recurrence (68). Hypoxia accelerates epithelial-mesenchymal transition, invasion, and metastasis in prostate cancer. Also, hypoxia may lead to a decreased sensitivity to radiotherapy and chemotherapy in prostate cancer treatment (912). However, there have been scant studies on hypoxia-induced immune evasion in prostate cancer. Therefore, we carried out this study to discover the role of hypoxia in tumor immune regulation.

Hypoxia is involved in immune evasion of a variety of tumors (13) involving many types of immune cells, including T cells, natural killer (NK) cells, macrophages and dendritic cells, that can inhibit or kill tumors (13). Hypoxia may lead to upregulation of the expression of stem cell marker Nanog and transforming growth factor beta 1, resulting in low immune killing capacity of T lymphocytes and macrophages against tumor cells (14). It was discovered in a lung cancer and melanoma study that hypoxia could induce miR-210 expression, which decreased tumor cell susceptibility to antigen-specific cytotoxic T lymphocytes and led to tumor formation and development (15). The NK cell mediated immune response can kill tumor cells directly with no dependence on antibodies or complements, which is a unique advantage in tumor immunity. By improving immune killing ability of NK cells against tumor cells, tumor formation and development can be effectively controlled. Suppression of expression of NK cell activating receptors MICA and MICB on the tumor cell surface by hypoxia can cause immune evasion from NK cells in pancreatic cancer, osteosarcoma, multiple myeloma and other malignant tumors (1619). The role of hypoxia regarding NK cell immune evasion in prostate cancer is rarely reported. In a study of DU145 and PC3 in prostate cancer cells, hypoxia inhibited the expression of NKG2D ligands on the surface of the tumor cells, thereby inhibiting the killing of tumor cells by activated NK cells (20,21).

The mechanism of hypoxia-mediated immune evasion is unknown. Many studies have indicated that programmed cell death ligand 1 (PD-L1) plays an important role in tumor immune evasion (22,23). A study of non-small cell lung cancer showed that tumor cells overexpress PD-L1, thereby binding PD-1 receptors on the surface of T cells and inhibiting T cell immune attack, resulting in immune evasion (24,25). Studies of ovarian cancer, melanoma, bladder cancer, laryngeal squamous cell carcinoma and other malignant tumors have indicated that downregulation of PD-1/PD-L1 improves tumor susceptibility to immune cells (2630). PD-1 is expressed in multiple immune cells, including T cells, B cells, NK cells and dendritic cells (31,32). The effect of PD-L1 in immune evasion of CRPC from NK cells, which is rarely studied, became the research direction of these experiments.

NKG2D is an important receptor for activation of NK cells. The upregulation of expression of NKG2D ligands, MHC class I chain-related proteins A and B (MICA and MICB) and UL16-binding proteins ULBP-1, ULBP-2, ULBP-3, can promote immune cytolytic activity of NK cells to tumors (33). This receptor has become the subject of enhancing the anti-tumor immunity of NK cells in this study. Previous work has shown that the combination of PD-1 and PD-L1 constitutes the PD-1-PD-L1 signaling pathway; in addition to this pathway's immunosuppressive effects through T cells (34), it can also inhibit the anti-tumor activity of NK cells by inhibiting the function of NKG2D ligands on the surface of tumor cells (35). Interestingly, based on a review of the literature, we found that the JAK1,2/Stat3 signaling pathway is the upstream regulator of PD-L1, which also includes other widely studied regulators such as Akt, MAPK, MEK, NFκB, and mTOR (3640).

We selected CRPC cell lines C4-2 and CWR22Rv1 as subjects of study. After induction of the cells by hypoxia, we assayed NK cell mediated cytotoxicity and colony formation to determine whether there were any changes in CRPC cell immune susceptibility to NK cells. We investigated the underlying molecular mechanisms of the changes to seek an effective way to enhance the killing ability of NK cells against CRPC cells, with the aim of inhibiting or killing tumors and offering a new approach to CRPC immunotherapy.

Materials and methods

Cell culture

The C4-2 cells used in the assays were generously donated by the China Center for Type Culture Collection (CCTCC). The CWR22Rv1 and NK92 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the cell culture medium was RPMI 1,640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% charcoal stripped fetal bovine serum (Thermo Fisher Scientific, Inc.). We used an anoxic incubator (1%O2, 5%CO2, 94%N2) (Sanyo, Osaka, Japan) and a normoxic incubator (21%O2, 5%CO2, 74%N2) (Sanyo). C4-2 and CWR22Rv1 lines of CRPC cells grown inananoxic incubator for 24 h served as anoxic cells. NK92 cells were cultured in Minimum Essential Medium containing sodium bicarbonate (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), IL-2 (Bio-Techne, Minneapolis, MN, USA), inositol, folic acid, 12.5% horse serum (Sigma-Aldrich; Merck KGaA), 2-mercaptoethanol (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and 12.5% FBS (HyClone; GE Healthcare Life Sciences, Logan, UT, USA).

NK cell mediated cytotoxicity assay

Tumor cells were seeded at 2,000 cells/well in a 96-well plate and cultured overnight. After aspirating all the medium, NK cells were added at a ratio of tumor cells to NK cells of 1:1, 1:5, or 1:15; the inhibitors PD-L1 Ab (329710; BioLegend, Inc., San Diego, CA, USA), JAK inhibitor 1 (CAS457081-03-7; EMD Millipore, Billerica, MA, USA), and Stattic were added at a ratio of 1:1,000 at the same time. Cells were cultured for 4 h, then a 50 µl aliquot of medium was used in a LDH cytotoxic assay using the LDH cytotoxic assay kit (88954; Thermo Fisher Scientific, Inc.). The experimental release was corrected by subtracting the amount released spontaneously in cells at corresponding dilutions. The percentage cytotoxicity was expressed as

Experimental value-Effector cells spontaneous control-Target cells spontaneous controlTarget cell maximum control-Target cells spontaneous control×arg

and used to calculate the immune killing ability of NK cells against tumor cells.

Colony formation assay

Using the gradient dilution cell-count method, we seeded 200 cells in 6 cm dishes (REF353002; Corning Incorporated, Corning, NY, USA). After overnight culture, tumor cells and NK cells were incubated for 4 h at 1:1, 1:5 and 1:15 ratios. After aspirating all the medium and removing NK cells, tumor cells were continuously cultured for 14 days after replacing the conventional culture medium. Then the target cells were fixed with formaldehyde for 15 min, stained with crystal violet for 30 min, and observed and photographed under the microscope. The protocol was repeated three times in each group and the results were averaged.

Total RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

When the adherent tumor cells had grown over about 70% of the surface, PD-L1 Ab, JAK inhibitor 1, Stattic, LY294002, SB203580, and U0126 were added separately at a ratio of 1:1,000, then cells were cultured for 6 h, and total RNA was extracted. We used Superscript III transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.) to reverse-transcribe total RNA (1 µg). Following primer setup, (the primer solutions included RT buffer, dNTPs, RT random primers, MultiScribe reverse transcriptase and RNase-free water), we conducted qPCR with a Bio-Rad CFX96 reaction system (reactions included cDNA, RNAase-free water, SYBR Green and primers; Table I). Using GAPDH as a reference, the expression of target gene mRNA was measured by the intensity of green fluorescence.

Table I.

Primer sequences used for reverse transcription-quantitative polymerase chain reaction.

Table I.

Primer sequences used for reverse transcription-quantitative polymerase chain reaction.

GeneDirectionSequence
GAPDHForward 5′-AACGGATTTGGTCGTATTGGG-3′
Reverse 5′-CCTGGAAGATGGTGATGGGAT-3′
PD-L1Forward 5′-GCTATGGTGGTGCCGACTAC-3′
Reverse 5′-TTGGTGGTGGTGGTCTTACC-3′
MICAForward 5′-ACTGCTTGAGCCGCTGAGA-3′
Reverse 5′-GAGGTGCAAAAGGGAAGATGC-3′
MICBForward 5′-GGGGCGCAGGTGACTAAAT-3′
Reverse 5′-CCTACGTCGCCACCTTCTCA-3′
ULBP-1Forward 5′-CAGCAGACGATGAGGACATT-3′
Reverse 5′-GACAGAAAGTGGCAGAAGGTG-3′
ULBP-2Forward 5′-CATTACTTCTCAATGGGAGACTGT-3′
Reverse 5′-TGTGCCTGAGGACATGGCGA-3′
ULBP-3Forward5′- ATTCTTCCGTACCTGCTATT-3′
Reverse 5′-GCTATCCTTCTCCCACTTCT-3′

[i] PD-L1, programmed death-ligand 1; MICA, MHC class I chain-related protein A; MICB, MHC class I chain-related protein B; ULBP, UL16 binding protein.

Western blot analysis

Tumor cells were collected after centrifugation and the supernatant removed. Then cell lysate was added, the protein concentration measured, sodium dodecyl sulphate-polyacrylamide gel electrophoresis performed, and the protein transferred to polyvinylidene difluoride membranes (EMD Millipore). After adding blocking solution and incubating with primary antibodies (1:1,000), the membranes were incubated with horseradish peroxidase labeled secondary antibody (1:5,000), followed by capture of images with an ECL imaging system (Thermo Fisher Scientific, Inc.). Primary antibodies used in the assay included p-HIF-1α (EP1215Y; ab210073; Abcam, Cambridge, UK), p-PD-L1 (MAB1086; R&D Systems, Inc., Minneapolis, MN, USA), JAK1 (pY1022, A7125; Assay Biotech, Fremont, CA, USA), p-JAK2 (pY1007 + 1008, 601–670; Abbomax, Inc., San Jose, CA, USA), p-MAPK (9101S; Cell Signaling Technology, Inc., Danvers, MA, USA), p-MEK (Ser 217/221, 9121; Cell Signaling Technology, Inc.), p-Akt (S473, 9271; Cell Signaling Technology, Inc.), p-Stat3 (Y705, ab76315; Abcam), p-NFκB (S536, ab86299; Abcam), and GAPDH (2118S; Cell Signaling Technology, Inc.).

Signaling pathway inhibitors

We added appropriately diluted inhibitors of various cell signaling pathways, JAK inhibitor 1 (5 µM) (CAS457081-03-7; EMD Millipore), Stattic (10 µM) (CAS19983-44-9; EMD Millipore), PD-L1 Ab (329710; BioLegend, Inc.), LY294002, SB203580 (both Sigma-Aldrich; Merck KGaA) and U0126 (Cell Signaling Technology, Inc.), which respectively inhibit JAK1, JAK2, Stat3, Akt, MAPK and MEK, to hypoxia-treated cells before co-incubation with tumor cells.

Statistical analysis

All data were reported as the mean ± standard deviation of 3 experimental repeats., and were analyzed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). The differences between two groups were analyzed by a two-tailed Student's t-test. One-way analysis of variance followed by Fisher's least significant difference post hoc test were used for comparisons among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Hypoxia-induced C4-2 and CWR22Rv1 cells showed changes in shape and increased expression of hypoxia-inducible factor-1α (HIF-1α)

After culture of C4-2 and CWR22Rv1 lines of CRPC cells in a hypoxic incubator for 24 h, the expected changes in cell morphology, such as to spindle-shaped and polygonal cells, and an increase in cell refractive index were observed (Fig. 1A). Normoxic cultured cells were the control group. We observed significantly upregulated expression of HIF-1α in tumor cells exposed to hypoxia. While HIF-1α can be easily degraded by the intracellular oxygen-dependent ubiquitin-proteasome pathway under normoxic conditions, under hypoxic conditions, its expression is stable (33), suggesting that the assayed C4-2 and CWR22Rv1 cells were effectively induced by hypoxia (Fig. 1B).

Hypoxia reduced expression of NKG2D activating ligandsin C4-2 and CWR22Rv1 cell lines and led to increased tolerance to immune killing of NK cells

We also detected changes in expression of genes encoding NKG2D ligands in hypoxia-induced C4-2 and CWR22Rv1 cells. Gene expression of these ligands (ULBP-1, ULBP-2, ULBP-3, MICA and MICB, all found on the surface of target cells) significantly decreased (Fig. 2A). In hypoxia-induced CRPC cells co-cultured for 4 h with various ratios of NK cells, lactate dehydrogenase release showed that the killing ability of NK cells significantly decreased against hypoxia induced target cells (Fig. 2B). The colony formation assay suggested that as the ratio of co-cultured NK cells to tumor cells increased, the clone-forming ability of the hypoxia group showed no obvious changes, while a significant decrease was observed in the normoxia group. These results indicated that hypoxia increased survival of target cells co-cultured with NK cells, which was consistent with the findings in the NK cell mediated cytotoxicity assay (Fig. 2C). In conclusion, hypoxia can lead to CRPC cell immune tolerance to NK cells.

Hypoxia-induced C4-2, and CWR22Rv1 CRPC cells had increased PD-L1 expression, resulting in immune escape from NK cells

In further study on the mechanisms affecting expression of PD-L1, both PD-L1 gene and protein expression in C4-2 and CWR22Rv1 cells were higher after hypoxia induction than under normoxic conditions (Fig. 3A). To determine whether hypoxia induces CRPC cells to evade NK cell immunity, we added PD-L1 antibodies to hypoxia-induced CRPC cells and detected upregulation of NKG2D ligand expression (Fig. 3B). We assayed NK cell mediated cytotoxicity, and discovered that addition of PD-L1 antibodies significantly increased the susceptibility of hypoxia-induced CRPC cells to NK cell immunity (Fig. 3C), which confirmed that PD-L1 protein is involved in hypoxia-induced CRPC cell immune evasion.

PD-L1 expression is inhibited by blocking the JAK1,2/Stat3 signaling pathway

To explore the mechanism behind PD-L1 regulation of CRPC cells against NK cell immunity, we selected well-studied signaling pathways of PD-L1 regulation, including JAK1, JAK2, Stat3, PI3K/Akt, MAPK, MEK, and NFκB (3439). We first detected differences in expression of these molecular markers in both normoxia and hypoxia groups by western blotting, and found that the expression of JAK1, JAK2, Stat3, Akt, MAPK, MEK in hypoxia group was increased compared with the normoxia group (Fig. 4A); thus, we think that these proteins may be involved in the regulation of PD-L1 under hypoxic conditions in the tumor. To observe their regulation of PD-L1, we added inhibitors of these proteins' corresponding signaling pathways JAK inhibitor 1 (which can simultaneously inhibit the expression of JAK 1 and JAK 2), Stattic, LY294002, SB203580 and U0126 at a ratio of 1:1,000 to hypoxic tumor cells. After 6 h, the level of PD-L1 was detected by RT-qPCR. The untreated group served as a control. The results showed that JAK inhibitor 1 and Stattic significantly down-regulate PD-L1 expression. PI3k inhibitor LY294002 could also significantly reduce the expression of PD-L1, but JAK inhibitor 1 and Stattic inhibited PD-L1 more. So we chose JAK inhibitor 1 and Stattic as the research subjects. Of course, given this result, PI3k inhibitor LY294002 will also be our future research direction (Fig. 4B).

JAK1,2/Stat3 signaling pathway inhibition increases the susceptibility of CRPC cells to NK cell immunity, with a combination of PD-L1 antibodies and JAK inhibitor 1/Stattic more effective either alone. CRPC cells in the hypoxia group were used as the study subject, and the JAK1,2 inhibitor JAK inhibitor 1 or the Stat3 inhibitor Stattic were added. The untreated group was used as the control. RT-qPCR showed increased expression of NKG2D ligands (Fig. 5A), suggesting that inhibition of the JAK1,2/Stat3 signaling pathway may improve the immune cytolytic activity of NK cells toward CRPC cells under hypoxic conditions.

Since blocking the JAK1,2/Stat3 signaling pathway can inhibit PD-L1 expression in hypoxic CRPC cells and may upregulate expression of NKG2D ligands of CRPC cells, to test whether this also enhances the immune killing function of NK cells against tumors by down-regulating PD-L1, we used hypoxic CRPC cells as targets and added the same JAK1,2/Stat3 signaling pathway inhibitors to an NK cell mediated cytotoxicity assay. Both JAK inhibitor 1 and Stattic significantly increased susceptibility of CRPC cells to NK cell immunity compared to an untreated group (Fig. 5B). Finally, we added combinations of PD-L1 antibody/JAK inhibitor 1 and PD-L1 antibody/Stattic to the NK cell mediated cytotoxicity assay and discovered that combined treatments were superior to each single application. Combined treatments achieved better results in enhancing NK cell immune killing function against CRPC cells, which could provide new approaches to CRPC targeted therapy (Fig. 5C).

Discussion

In clinical practice, the incidence of both local progression and distant metastasis in CRPC patients has significantly increased, seriously affecting patients' quality of life (40,41). Although the mechanisms behind this increase are not yet fully understood, hypoxia, as an important feature of the tumor microenvironment, plays an essential role (42,43). In this study, CRPC cell lines C4-2 and CWR22Rv1 were grown in an incubator under conditions of hypoxia. We observed changes in the morphology of these cells to polygonal and spindle shapes, and high expression of HIF-1α that was dependent on the oxygen concentration, consistent with hypoxia-induced changes in the cells, which may provide fundamental data for further exploration of the effects of hypoxia on tumor immunity. In the hypoxic microenvironment of prostate cancer, HIF-1α and related proteins present a major research direction. Our focus on the JAK1,2/Stat3-PD-L1 signaling pathway has not been studies before, and our results suggest this pathway was upregulated in a hypoxic microenvironment, resulting in immune escape of CRPC from NK cells.

The occurrence and development of tumors are closely related to the immunity of the human body, in which the direct killing function of NK cells against tumors has a unique advantage; however, there are scant studies on the mechanisms of NK cell immunity against CRPC. Our study took this as a starting point to explore the mechanisms of NK cell immunity against CRPC in a hypoxic microenvironment. Firstly we examined the expression of ligands of NKG2D, a major activating receptor of NK cells, which include the ULBP family (ULBP-1, ULBP-2 and ULBP-3) and MICA/MICB (44). Expression of all these markers showed a downward trend, consistent with NKG2D playing an important role in tumor immunity of NK cells. CRPC cells also had stronger immune tolerance to NK cells in a hypoxic microenvironment, confirming a stronger immune evasion function under these conditions.

To explore the mechanisms of immune evasion, we selected PD-L1, a relatively well-studied protein in tumor targeted therapy (45,46), as a research subject. PD-L1 expression of hypoxia-induced CRPC cells significantly increased, and by adding PD-L1 antibodies, the expression of NKG2D ligands was reversed, while the immune killing ability of NK cells against tumor cells was significantly enhanced. The combination of PD-1 and PD-L1 constitutes the PD-1-PD-L1 signaling pathway, thus affecting immune cytolytic activity of immune cells to tumor cells, which is mainly reflected in the following aspects: (1) Inhibition of function of TIL cells by (a) inhibiting the activation of TIL cells, (b) influencing Th cell differentiation, (c) inhibiting the production of effector cytokines, (d) promoting the secretion of suppressive cytokines, and (e) increasing TIL apoptosis, thus resulting in tumor immune escape (34); (2) Inhibiting the function of NK cells, thereby reducing their anti-tumor effect (35); (3) Expression of PD-L1 on TAM, which can cause synergistic stimulation, inhibiting the immune susceptibility of tumor cells (47).

During examination of PD-L1 upstream regulatory factors, we found that the JAK1,2/Stat3 signaling pathways played an important role, and by adding appropriate antibodies (JAK inhibitor 1 or PD-L1 antibody), PD-L1 expression of CRPC cells was inhibited under hypoxic conditions. The JAK1,2/Stat3 signaling pathway is widely involved in proliferation, differentiation, migration and other processes of cells (48). It plays an important role in occurrence and development of many tumors including prostate cancer (4953). The results of our study also indicated that in a hypoxic microenvironment, the JAK1,2/Stat3 signaling pathway promotes immune evasion of NK cells by CRPC cells. The current targeted immunotherapy of PD-1/PD-L1 against tumors has achieved good results, effectively reducing tumor size and prolonging the survival of patients. However, clinical observation shows that there is still some degree of immunosuppression, which may be related to the complex immune escape mechanism of the tumor itself. Combined administration of drugs through multiple ways may be an effective choice for tumor targeted immunotherapy (54). Thus, we studied the combined inhibition of JAK1,2/PD-L1 and Stat3/PD-L1, and observed that a combined inhibition was more effective in enhancing NK cell cytotoxicity toward hypoxia-induced CRPC cells. Given these results, we may develop a combination inhibitor of JAK1,2/Stat3 and PD-L1, and conduct related animal studies and clinical trials, thereby providing a more effective means of clinical immunotherapy of CRPC.

Acknowledgements

The authors would like to thank Dr. Soo Ok Lee (Department of Urology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China) for assisting with the preparation of the manuscript.

Funding

The present study was supported by the Pre Research Fund Project of The Second Affiliated Hospital of Soochow University (grant no. SDFEYBS1707), The National Natural Science Foundation of China (grant nos. 81472776 and 81773221), and by Preponderant Discipline Construction Funding of the Second Affiliated Hospital of Soochow University (grant no. XKQ2015008).

Availability of data and materials

The analyzed datasets generated during the study are available from the corresponding author on reasonable request.

Authors' contributions

LJX, QM and JZ performed the experiments and statistical analyses, and created the figures. JL and BXX contributed to the generation of the knockdown cell lines. JG and CYS provided and performed the staining of human tissues. YCZ and YBZ assisted with the interpretation of data and reviewed the manuscript. DRY and YXS conceived the idea and wrote the manuscript. All authors reviewed and agreed to the information in this manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Cornford P, Bellmunt J, Bolla M, Briers E, De Santis M, Gross T, Henry AM, Joniau S, Lam TB, Mason MD, et al: EAU-ESTRO-SIOG Guidelines on prostate cancer. Part II: Treatment of relapsing, metastatic, and castration-resistant prostate cancer. Eur Urol. 71:630–642. 2017. View Article : Google Scholar : PubMed/NCBI

2 

Lowrance WT, Roth BJ, Kirkby E, Murad MH and Cookson MS: Castration-resistant prostate cancer: AUA Guideline Amendment 2015. J Urol. 195:1444–1452. 2016. View Article : Google Scholar : PubMed/NCBI

3 

Crawford ED, Higano CS, Shore ND, Hussain M and Petrylak DP: Treating patients with metastatic castration resistant prostate cancer: A comprehensive review of available therapies. J Urol. 194:1537–1547. 2015. View Article : Google Scholar : PubMed/NCBI

4 

Qiu J, Jiang W, Yang Y, Feng C, Chen Z, Guan G, Zhuo S and Chen J: Monitoring changes of tumor microenvironment in colorectal submucosa using multiphoton microscopy. Scanning. 37:17–22. 2015. View Article : Google Scholar : PubMed/NCBI

5 

Casey SC, Amedei A, Aquilano K, Azmi AS, Benencia F, Bhakta D, Bilsland AE, Boosani CS, Chen S, Ciriolo MR, et al: Cancer prevention and therapy through the modulation of the tumor microenvironment. Semin Cancer Biol. 35 Suppl:S199–S223. 2015. View Article : Google Scholar : PubMed/NCBI

6 

Wolff M, Kosyna FK, Dunst J, Jelkmann W and Depping R: Impact of hypoxia inducible factors on estrogen receptor expression in breast cancer cells. Arch Biochem Biophys. 613:23–30. 2017. View Article : Google Scholar : PubMed/NCBI

7 

Wu X, Qiao B, Liu Q and Zhang W: Upregulation of extracellular matrix metalloproteinase inducer promotes hypoxia-induced epithelial-mesenchymal transition in esophageal cancer. Mol Med Rep. 12:7419–7424. 2015. View Article : Google Scholar : PubMed/NCBI

8 

Clavo B, Robaina F, Fiuza D, Ruiz A, Lloret M, Rey-Baltar D, Llontop P, Riveros A, Rivero J, Castañeda F, et al: Predictive value of hypoxia in advanced head and neck cancer after treatment with hyperfractionated radio-chemotherapy and hypoxia modification. Clin Transl Oncol. 19:419–424. 2017. View Article : Google Scholar : PubMed/NCBI

9 

Li W, Dong Y, Zhang B, Kang Y, Yang X and Wang H: PEBP4 silencing inhibits hypoxia-induced epithelial-to-mesenchymal transition in prostate cancer cells. Biomed Pharmacother. 81:1–6. 2016. View Article : Google Scholar : PubMed/NCBI

10 

Li M, Wang YX, Luo Y, Zhao J, Li Q, Zhang J and Jiang Y: Hypoxia inducible factor-1α-dependent epithelial to mesenchymal transition under hypoxic conditions in prostate cancer cells. Oncol Rep. 36:521–527. 2016. View Article : Google Scholar : PubMed/NCBI

11 

Wang W, Liu M, Guan Y and Wu Q: Hypoxia-responsive Mir-301a and Mir-301b promote radioresistance of prostate cancer cells via downregulating NDRG2. Med Sci Monit. 22:2126–2132. 2016. View Article : Google Scholar : PubMed/NCBI

12 

Nomura T, Yamasaki M, Hirai K, Inoue T, Sato R, Matsuura K, Moriyama M, Sato F and Mimata H: Targeting the Vav3 oncogene enhances docetaxel-induced apoptosis through the inhibition of androgen receptor phosphorylation in LNCaP prostate cancer cells under chronic hypoxia. Mol Cancer. 12:272013. View Article : Google Scholar : PubMed/NCBI

13 

Barsoum IB, Koti M, Siemens DR and Graham CH: Mechanisms of hypoxia-mediated immune escape in cancer. Cancer Res. 74:7185–7190. 2014. View Article : Google Scholar : PubMed/NCBI

14 

Hasmim M, Noman MZ, Messai Y, Bordereaux D, Gros G, Baud V and Chouaib S: Cutting edge: Hypoxia-induced Nanog favors the intratumoral infiltration of regulatory T cells and macrophages via direct regulation of TGF-β1. J Immunol. 191:5802–5806. 2013. View Article : Google Scholar : PubMed/NCBI

15 

Noman MZ, Buart S, Romero P, Ketari S, Janji B, Mari B, Mami-Chouaib F and Chouaib S: Hypoxia-inducible miR-210 regulates the susceptibility of tumor cells to lysis by cytotoxic T cells. Cancer Res. 72:4629–4641. 2012. View Article : Google Scholar : PubMed/NCBI

16 

Lu Y, Hu J, Sun W, Duan X and Chen X: Hypoxia-mediated immune evasion of pancreatic carcinoma cells. Mol Med Rep. 11:3666–3672. 2015. View Article : Google Scholar : PubMed/NCBI

17 

Yamada N, Yamanegi K, Ohyama H, Hata M, Nakasho K, Futani H, Okamura H and Terada N: Hypoxia downregulates the expression of cell surface MICA without increasing soluble MICA in osteosarcoma cells in a HIF-1α-dependent manner. Int J Oncol. 41:2005–2012. 2012. View Article : Google Scholar : PubMed/NCBI

18 

Sarkar S, Germeraad WT, Rouschop KM, Steeghs EM, van Gelder M, Bos GM and Wieten L: Hypoxia induced impairment of NK cell cytotoxicity against multiple myeloma can be overcome by IL-2 activation of the NK cells. PLoS One. 8:e648352013. View Article : Google Scholar : PubMed/NCBI

19 

Labiano S, Palazon A and Melero I: Immune response regulation in the tumor microenvironment by hypoxia. Semin Oncol. 42:378–386. 2015. View Article : Google Scholar : PubMed/NCBI

20 

Hamilton TK, Hu N, Kolomitro K, Bell EN, Maurice DH, Graham CH and Siemens DR: Potential therapeutic applications of phosphodiesterase inhibition in prostate cancer. World J Urol. 31:325–330. 2013. View Article : Google Scholar : PubMed/NCBI

21 

Siemens DR, Hu N, Sheikhi AK, Chung E, Frederiksen LJ, Pross H and Graham CH: Hypoxia increases tumor cell shedding of MHC class I chain-related molecule: Role of nitric oxide. Cancer Res. 68:4746–4753. 2008. View Article : Google Scholar : PubMed/NCBI

22 

Barsoum IB, Smallwood CA, Siemens DR and Graham CH: A mechanism of hypoxia-mediated escape from adaptive immunity in cancer cells. Cancer Res. 74:665–674. 2014. View Article : Google Scholar : PubMed/NCBI

23 

Chen J, Jiang CC, Jin L and Zhang XD: Regulation of PD-L1: A novel role of pro-survival signalling in cancer. Ann Oncol. 27:409–416. 2016. View Article : Google Scholar : PubMed/NCBI

24 

Shi MH, Xing YF, Zhang ZL, Huang JA and Chen YJ: Effect of soluble PD-L1 released by lung cancer cells in regulating the function of T lymphocytes. Zhonghua Zhong Liu Za Zhi. 35:85–88. 2013.(In Chinese). PubMed/NCBI

25 

Akbay EA, Koyama S, Carretero J, Altabef A, Tchaicha JH, Christensen CL, Mikse OR, Cherniack AD, Beauchamp EM, Pugh TJ, et al: Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discov. 3:1355–1363. 2013. View Article : Google Scholar : PubMed/NCBI

26 

Mahoney KM, Freeman GJ and McDermott DF: The next immune-checkpoint inhibitors: PD-1/PD-L1 blockade in melanoma. Clin Ther. 37:764–782. 2015. View Article : Google Scholar : PubMed/NCBI

27 

Mandai M, Hamanishi J, Abiko K, Matsumura N, Baba T and Konishi I: Anti-PD-L1/PD-1 immune therapies in ovarian cancer: Basic mechanism and future clinical application. Int J Clin Oncol. 21:456–461. 2016. View Article : Google Scholar : PubMed/NCBI

28 

Bardoli AD, Afshar M, Viney R, Foster M, Porfiri E, Zarkar A, Stevenson R, James ND, Bryan RT and Patel P: The PD-1/PD-L1 axis in the pathogenesis of urothelial bladder cancer and evaluating its potential as a therapeutic target. Future Oncol. 12:595–600. 2016. View Article : Google Scholar : PubMed/NCBI

29 

Brower V: Anti-PD-L1 antibody active in metastatic bladder cancer. Lancet Oncol. 16:e112015. View Article : Google Scholar : PubMed/NCBI

30 

Vassilakopoulou M, Avgeris M, Velcheti V, Kotoula V, Rampias T, Chatzopoulos K, Perisanidis C, Kontos CK, Giotakis AI, Scorilas A, et al: Evaluation of PD-L1 expression and associated tumor-infiltrating lymphocytes in laryngeal squamous cell carcinoma. Clin Cancer Res. 22:704–713. 2016. View Article : Google Scholar : PubMed/NCBI

31 

Yao S and Chen L: PD-1 as an immune modulatory receptor. Cancer J. 20:262–264. 2014. View Article : Google Scholar : PubMed/NCBI

32 

Huang BY, Zhan YP, Zong WJ, Yu CJ, Li JF, Qu YM and Han S: The PD-1/B7-H1 pathway modulates the natural killer cells versus mouse glioma stem cells. PLoS One. 10:e01347152015. View Article : Google Scholar : PubMed/NCBI

33 

Joo HY, Yun M, Jeong J, Park ER, Shin HJ, Woo SR, Jung JK, Kim YM, Park JJ, Kim J and Lee KH: SIRT1 deacetylates and stabilizes hypoxia-inducible factor-1α (HIF-1α) via direct interactions during hypoxia. Biochem Biophys Res Commun. 462:294–300. 2015. View Article : Google Scholar : PubMed/NCBI

34 

Doi T, Ishikawa T, Okayama T, Oka K, Mizushima K, Yasuda T, Sakamoto N, Katada K, Kamada K, Uchiyama K, et al: The JAK/STAT pathway is involved in the upregulation of PD-L1 expression in pancreatic cancer cell lines. Oncol Rep. 37:1545–1554. 2017. View Article : Google Scholar : PubMed/NCBI

35 

Bellucci R, Martin A, Bommarito D, Wang K, Hansen SH, Freeman GJ and Ritz J: Interferon-γ-induced activation of JAK1 and JAK2 suppresses tumor cell susceptibility to NK cells through upregulation of PD-L1 expression. Oncoimmunology. 4:e10088242015. View Article : Google Scholar : PubMed/NCBI

36 

Atefi M, Avramis E, Lassen A, Wong DJ, Robert L, Foulad D, Cerniglia M, Titz B, Chodon T, Graeber TG, et al: Effects of MAPK and PI3K pathways on PD-L1 expression in melanoma. Clin Cancer Res. 20:3446–3457. 2014. View Article : Google Scholar : PubMed/NCBI

37 

Yang L, Huang F, Mei J, Wang X, Zhang Q, Wang H, Xi M and You Z: Posttranscriptional control of PD-L1 expression by 17β-estradiol via PI3K/Akt signaling pathway in ERα-positive cancer cell lines. Int J Gynecol Cancer. 27:196–205. 2017. View Article : Google Scholar : PubMed/NCBI

38 

Jiang X, Zhou J, Giobbie-Hurder A, Wargo J and Hodi FS: The activation of MAPK in melanoma cells resistant to BRAF inhibition promotes PD-L1 expression that is reversible by MEK and PI3K inhibition. Clin Cancer Res. 19:598–609. 2013. View Article : Google Scholar : PubMed/NCBI

39 

Gowrishankar K, Gunatilake D, Gallagher SJ, Tiffen J, Rizos H and Hersey P: Inducible but not constitutive expression of PD-L1 in human melanoma cells is dependent on activation of NF-κB. PLoS One. 10:e01234102015. View Article : Google Scholar : PubMed/NCBI

40 

Hernandez RK, Cetin K, Pirolli M, Quigley J, Quach D, Smith P, Stryker S and Liede A: Estimating high-risk castration resistant prostate cancer (CRPC) using electronic health records. Can J Urol. 22:7858–7864. 2015.PubMed/NCBI

41 

Kamoto T: Evaluation and diagnosis for castration resistant prostate cancer: CRPC. Nihon Rinsho. 72:2103–2107. 2014.(In Japanese). PubMed/NCBI

42 

Walsh JC, Lebedev A, Aten E, Madsen K, Marciano L and Kolb HC: The clinical importance of assessing tumor hypoxia: Relationship of tumor hypoxia to prognosis and therapeutic opportunities. Antioxid Redox Signal. 21:1516–1554. 2014. View Article : Google Scholar : PubMed/NCBI

43 

Jensen LD: The circadian clock and hypoxia in tumor cell de-differentiation and metastasis. Biochim Biophys Acta. 1850:1633–1641. 2015. View Article : Google Scholar : PubMed/NCBI

44 

Lanier LL: NKG2D receptor and its ligands in host defense. Cancer Immunol Res. 3:575–582. 2015. View Article : Google Scholar : PubMed/NCBI

45 

Swaika A, Hammond WA and Joseph RW: Current state of anti-PD-L1 and anti-PD-1 agents in cancer therapy. Mol Immunol. 67:4–17. 2015. View Article : Google Scholar : PubMed/NCBI

46 

Ohaegbulam KC, Assal A, Lazar-Molnar E, Yao Y and Zang X: Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway. Trends Mol Med. 21:24–33. 2015. View Article : Google Scholar : PubMed/NCBI

47 

Cai B, Cai JP, Luo YL, Chen C and Zhang S: The specific roles of JAK/STAT signaling pathway in sepsis. Inflammation. 38:1599–1608. 2015. View Article : Google Scholar : PubMed/NCBI

48 

Teng Y, Ross JL and Cowell JK: The involvement of JAK-STAT3 in cell motility, invasion, and metastasis. JAKSTAT. 3:e280862014.PubMed/NCBI

49 

O'Shea JJ, Holland SM and Staudt LM: JAKs and STATs in immunity, immunodeficiency, and cancer. N Engl J Med. 368:161–170. 2013. View Article : Google Scholar : PubMed/NCBI

50 

Wang SW and Sun YM: The IL-6/JAK/STAT3 pathway: Potential therapeutic strategies in treating colorectal cancer (Review). Int J Oncol. 44:1032–1040. 2014. View Article : Google Scholar : PubMed/NCBI

51 

Liu RY, Zeng Y, Lei Z, Wang L, Yang H, Liu Z, Zhao J and Zhang HT: JAK/STAT3 signaling is required for TGF-β-induced epithelial-mesenchymal transition in lung cancer cells. Int J Oncol. 44:1643–1651. 2014. View Article : Google Scholar : PubMed/NCBI

52 

Lapeire L, Hendrix A, Lambein K, Van Bockstal M, Braems G, Van Den Broecke R, Limame R, Mestdagh P, Vandesompele J, Vanhove C, et al: Cancer-associated adipose tissue promotes breast cancer progression by paracrine oncostatin M and Jak/STAT3 signaling. Cancer Res. 74:6806–6819. 2014. View Article : Google Scholar : PubMed/NCBI

53 

Duzagac F, Inan S, Simsek Ela F, Acikgoz E, Guven U, Khan SA, Rouhrazi H, Oltulu F, Aktug H, Erol A and Oktem G: JAK/STAT pathway interacts with intercellular cell adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) while prostate cancer stem cells form tumor spheroids. J BUON. 20:1250–1257. 2015.PubMed/NCBI

54 

Kim JM and Chen DS: Immune escape to PD-L1/PD-1 blockade: Seven steps to success (or failure). Ann Oncol. 27:1492–1504. 2016. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

June-2018
Volume 17 Issue 6

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Xu LJ, Ma Q, Zhu J, Li J, Xue BX, Gao J, Sun CY, Zang YC, Zhou YB, Yang DR, Yang DR, et al: Combined inhibition of JAK1,2/Stat3‑PD‑L1 signaling pathway suppresses the immune escape of castration‑resistant prostate cancer to NK cells in hypoxia. Mol Med Rep 17: 8111-8120, 2018
APA
Xu, L., Ma, Q., Zhu, J., Li, J., Xue, B., Gao, J. ... Shan, Y. (2018). Combined inhibition of JAK1,2/Stat3‑PD‑L1 signaling pathway suppresses the immune escape of castration‑resistant prostate cancer to NK cells in hypoxia. Molecular Medicine Reports, 17, 8111-8120. https://doi.org/10.3892/mmr.2018.8905
MLA
Xu, L., Ma, Q., Zhu, J., Li, J., Xue, B., Gao, J., Sun, C., Zang, Y., Zhou, Y., Yang, D., Shan, Y."Combined inhibition of JAK1,2/Stat3‑PD‑L1 signaling pathway suppresses the immune escape of castration‑resistant prostate cancer to NK cells in hypoxia". Molecular Medicine Reports 17.6 (2018): 8111-8120.
Chicago
Xu, L., Ma, Q., Zhu, J., Li, J., Xue, B., Gao, J., Sun, C., Zang, Y., Zhou, Y., Yang, D., Shan, Y."Combined inhibition of JAK1,2/Stat3‑PD‑L1 signaling pathway suppresses the immune escape of castration‑resistant prostate cancer to NK cells in hypoxia". Molecular Medicine Reports 17, no. 6 (2018): 8111-8120. https://doi.org/10.3892/mmr.2018.8905