COX-2 expression positively correlates with PD-L1 expression in human melanoma cells
- Gerardo Botti1,
- Federica Fratangelo2,
- Margherita Cerrone1,
- Giuseppina Liguori1,
- Monica Cantile1,
- Anna Maria Anniciello1,
- Stefania Scala3,
- Crescenzo D’Alterio3,
- Chiara Trimarco1,
- Angela Ianaro4,
- Giuseppe Cirino4,
- Corrado Caracò5,
- Maria Colombino6,
- Giuseppe Palmieri6,
- Stefano Pepe7,
- Paolo Antonio Ascierto2,
- Francesco Sabbatino†7Email author and
- Giosuè Scognamiglio†1Email author
© The Author(s) 2017
Received: 18 October 2016
Accepted: 17 February 2017
Published: 23 February 2017
The resistance to PD-1/PD-L1 inhibitors for the treatment of melanoma have prompted investigators to implement novel clinical trials which combine immunotherapy with different treatment modalities. Moreover is also important to investigate the mechanisms which regulate the dynamic expression of PD-L1 on tumor cells and PD-1 on T cells in order to identify predictive biomarkers of response. COX-2 is currently investigated as a major player of tumor progression in several type of malignancies including melanoma. In the present study we investigated the potential relationship between COX-2 and PD-L1 expression in melanoma.
Tumor samples obtained from primary melanoma lesions and not matched lymph node metastases were analyzed for both PD-L1 and COX-2 expression by IHC analysis. Status of BRAF and NRAS mutations was analyzed by sequencing and PCR. Co-localization of PD-L1 and COX-2 expression was analyzed by double fluorescence staining. Lastly the BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines were used to evaluate the effect of COX-2 inhibition by celecoxib on expression of PD-L1 in vitro.
BRAFV600E/V600K and NRASQ61R/Q61L were detected in 57.8 and 8.9% of the metastatic lesions, and in 65.9 and 6.8% of the primary tumors, respectively. PD-L1 and COX-2 expression were heterogeneously expressed in both primary melanoma lesions and not matched lymph node metastases. A significantly lower number of PD-L1 negative lesions was found in primary tumors as compared to not matched metastatic lesions (P = 0.002). COX-2 expression significantly correlated with PD-L1 expression in both primary (P = 0.001) and not matched metastatic (P = 0.048) lesions. Furthermore, in melanoma tumors, cancer cells expressing a higher levels of COX-2 also co-expressed a higher level of PD-L1. Lastly, inhibition of COX-2 activity by celecoxib down-regulated the expression of PD-L1 in both BRAFV600E A375 and NRASQ61R SK-MEL-2 melanoma cell lines.
COX-2 expression correlates with and modulates PD-L1 expression in melanoma cells. These findings have clinical relevance since they provide a rationale to implement novel clinical trials to test COX-2 inhibition as a potential treatment to prevent melanoma progression and immune evasion as well as to enhance the anti-tumor activity of PD-1/PD-L1 based immunotherapy for the treatment of melanoma patients with or without BRAF/NRAS mutations.
KeywordsImmune checkpoint molecules PD-L1 COX-2 Celecoxib Melanoma Immunotherapy
The recent introduction of new and more effective therapies, including treatments based on the stimulation of immune response and targeted therapies, has partially changed the prognosis of metastatic melanoma patients . The more effective therapeutic strategies are based on tyrosine kinase inhibitors, such as vemurafenib, dabrafenib and trametinib or inhibitors of the immune checkpoint molecules Cytotoxic T lymphocyte antigen 4 (CTLA-4), Programmed death receptor 1 (PD-1) and its ligand PD-L1 [2–6].
The immune system plays an important role in eradicating melanoma cells. PD-1 is a T cell co-inhibitory receptor with ligand specificity for both PD-L1 and PD-L2. PD-L1 results to be expressed in various types of cells, including placenta, pancreatic islet cells, mesenchymal stem cells and immune cells. However many human cancers, including melanoma, breast, lung, stomach, pancreatic, kidney and ovarian carcinoma are shown to express PD-L1 and in melanoma, its expression correlates with a poor prognosis [7–12]. Interaction of PD-1 with PD-L1 (B7-H1) or PD-L2 (B7-DC) represents one of the major mechanisms of tumor immune escape . It promotes T-cell tolerance and avoids T cell cytolysis of cancer cells.
Several mechanisms have been implicated in the regulation of PD-L1 expression by cancer cells such as activation of mitogenic and pro-survival pathways including MAPK and PI3K/AKT, increased activity of transcriptional factors HIF-1, STAT-3 and NF-κB, and presence of epigenetic modulators including miR-513, miR-570, miR-34a, miR-200 and miR-197 .
Cyclooxygenases (COXs) are enzymes which catalyze the first rate-limiting step in the conversion of arachidonic acid to prostaglandins. Two COX isoenzymes have been identified: COX-1 is constitutively expressed in most tissues and mediates the synthesis of prostaglandins in normal physiological processes, whereas COX-2 is not detectable in most normal tissues but is rapidly induced by various stimuli such as inflammatory reactions . COX-2 is also expressed in various tumor types and its level correlates with invasiveness and prognosis in many tumor entities, suggesting an important role of COX-2 in tumor development and progression . Epidemiological studies showed that prolonged COX-2 inhibition through acetylsalicylic acid or other non-steroidal anti-inflammatory drugs (NSAIDs) might offer some protection against colon cancer and some other malignancies [17–19]. Moreover, a potential role of COX-2 in melanoma development is also not unlikely, since COX-2 is frequently expressed in malignant melanomas  and its inhibition may prevent melanoma progression . Evidences available in the literature have shown that COX-2 can modulate PD-L1 expression in breast cancer cells  and its inhibition by the selective COX-2 inhibitor celecoxib enhances the effects of cytotoxic T lymphocyte (CTL) function by PD-L1 blocking in chronic viral infections .
In this study we tested whether COX-2 expression correlates with PD-L1 expression in primary and not matched metastatic melanoma tumors as well as whether COX-2 activity regulates PD-L1 expression in melanoma cells.
Chemical reagents and antibodies
COX-2-specific mouse monoclonal antibody (mAb) (clone CX-294), mouse IgG2a, rabbit IgG, the peroxidase blocking reagent goat anti-rabbit + horseradish peroxidase (HRP) visualization reagent, DAB substrate buffer, DAB chromogen, bond wash solution, hematoxylin and EnVision FLEX + rabbit linker kitwere purchased from DAKO. PD-L1-specific rabbit mAb (clone SP-142) was purchased from Spring Bioscience. Cell Conditioning Solution was purchased from Ventana medical Systems. Protein serum block, steady plus 3,3′-diaminobenzidine (DAB) kits, goat anti-mouse IgG dylight 488 and goat anti-rabbit IgG dylight 594 were purchased from Abcam. Bond primary antibody diluents and 4′,6′-diamidino-2-phenylindole (DAPI) were purchased from Leica biosystems. MACH 2 DOUBLE STAIN 2 and vulcan fast red chromogen Kit 2 were purchased from Biocare. The COX-2 inhibitor, celecoxib was purchased from Selleck Chemicals LLC. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and trypan blue were purchased from Sigma. GAPDH-, Bcl-2- and β-actin specific mAbs were purchased from Cell Signaling Technology. R-phycoerythrin(PE)-conjugated PD-L1-specific mouse mAb [clone MIH1 (RUO)] and PE-conjugated mouse IgG1 were purchased from BD Biosciences.
Primary melanoma tumor biopsies from treatment naïve patients and not matched lymph node melanoma tumor biopsies from metastatic patients were obtained from the tissue bank at Istituto Nazionale Tumori Fondazione “G. Pascale” (Naples, Italy). All samples were from Caucasian patients. Patients were consented for tissue acquisition per institutional review board (IRB)-approved protocol. Presence of tumor cells in formalin-fixed paraffin embedded (FFPE) tissues was monitored by hematoxylin and eosin (H&E) staining. All samples were reviewed by two experienced pathologists (GB and AA) according to American Joint Committee on Cancer (AJCC) classification criteria, using standard tissue sections and appropriate immunohistochemical (IHC) analyses.
Genotyping of metastatic lymph node melanoma tumors
Genomic DNA was isolated from FFPE tumor tissues, using the QIAamp DNA FFPE tissue kit (QIAGEN Inc; Milan, Italy). The full coding sequences and splice junctions of NRAS (exons 1 and 2) and the entire sequence of the BRAF (exon 15) [24, 25] were screened for mutations. All samples were assessed for the quality of the purified DNA, in order to avoid that discrepant case could arise from insufficient sample quality. Primer sets were designed as described . Sequencing and PCR assay were performed as described .
FFPE tumor tissue sections of 3–4 μm thickness were cut onto adesive slides, baked at 70 °C (dry heat) for 1 hour (h) less than 1 week before use, deparaffinized in four changes of 100% xylene, and rehydrated with a graded ethanol series (100, 70, 40%) to distilled water. COX-2 IHC staining was performed using COX-2-specific mAb (clone CX-294) and EnVision FLEX utilizing the automated DAKO Omnis platform. For PD-L1 staining, prepared slides were incubated for 12 minutes (min) at 110 °C in Cell Conditioning Solution, using a commercial steamer as the heat source (Biocare Medical, Decloaking Chamber DC12). After cooling for 20 min, PD-L1 staining was performed using an automated IHC staining platform (DAKO autostainer Link48). This procedure was carried out at room temperature (RT). Following a 5 min incubation with a peroxidase blocking reagent and a 5 min incubation with a protein serum block (1% goat serum, 4% BSA in PBS, slides were incubated with the PD-L1-specific mAb (clone SP-142) at a concentration of 3.75 μg/mL in the primary antibody diluents for 90 min. The goat anti-rabbit + HRP visualization reagent, which is biotin-independent and reduces the potential for background or nonspecific staining from endogenous biotin, was used for PD-L1-specific antibody detection. The secondary antibody was incubate for 40 min .
For both COX-2 and PD-L1 staining, following an incubation with DAB substrate buffer and DAB chromogen, slides were counterstained on platform with hematoxylin and rinsed in distilled water. Between all incubation steps, slides were extensively washed with bond wash solution. Then the slides were dehydrated out of platform in an ethanol series (30, 70, 100%) and four changes of 100% xylene, and permanently sealed with automatic coverslips (DAKO #CS100). Each staining run contained positive and negative controls.
PD-L1 and COX-2 expression were reviewed and enumerated independently and blindly by two experienced pathologists (GB and AA) using a light microscopy. For each sample, at least five fields (inside the tumor and in the peripheral areas) and >500 cells were analysed. Using a semi-quantitative scoring system microscopically and referring to each protein scoring method in other studies, percentage of stained tumor cells in each lesion were evaluated for COX-2 and PD-L1 expression. Variations in the percentage of stained cells were within a 10% range for COX-2 expression. In consideration of the error, we evaluated the percentage of stained tumor cells at 10% intervals. Staining was graded as a semi-quantitative variable ranged from 0 to 100% . For PD-L1 expression variations in the percentage of stained cells were within a 1% range. Results were graded as negative (0+), light positive (1+) and positive (2+) when the PD-L1 score in an entire lesion was 0, 1–5, and >5% respectively [29, 30].
Double-fluorescence staining of PD-L1 and COX-2 were conducted on a total of 12 representative FFPE tissue sections, 6 each from primary and metastatic lesions, including both positive and negative for PD-L1 (clone SP-142) or COX-2 (clone CX-294) at IHC staining. Following their deparaffinization and hydration, prepared slides were incubated with antigen retrieval in a pressure cooker (Biocare) for 10 min at 110 °C. Following an incubation with protein blocking, slides were incubated with a cocktail of PD-L1- and COX-2-specific mAbs at RT. Primary antibodies were detected utilizing a cocktail of goat anti-mouse IgG dylight 488 and goat anti-rabbit IgG dylight 594. After washing for two times, nuclei were stained with DAPI at RT for 10 min, and stored at 4 °C. The slides were examined using a fluorescent microscope (Olympus BX61).
The BRAFV600E A375 and the NRASQ61R SK-MEL-2 melanoma cell lines were purchased from American Type Culture Collection (ATCC). All cell lines were cultured in RPMI 1640 medium (HyClone Laboratories) supplemented with 2 mmol/l l-glutamine (HyClone Laboratories) and 10% fetal bovine serum (FBS) (HyClone Laboratories). All cells were cultured at 37 °C in a 5% CO2 atmosphere.
Cell proliferation and MTT assay
Cells were plated in triplicate in 96-well microtiter plates at the density of 3 × 103 per well in 100 μl of RPMI 1640 medium supplemented with 10% FBS at 5% CO2 atmosphere and treated with celecoxib. Untreated cells were used as a control. Dimethyl sulfoxide (DMSO) (vehicle of the drug) concentration was maintained at 0.02% in all wells. Cell proliferation was evaluated at the indicated time point utilizing the MTT assay which was carried out as reported elsewhere . Data are expressed as percent of proliferation of treated cells as compared to untreated control cells. All experiments were performed three independent times in triplicates. The absorbance value at wavelength of 540 nm was determined using a microplate reader (Bio-Rad Laboratories).
Cell viability assay
Cells were seeded at the density of 4 × 105 in a 75 cm2 tissue culture flask in RPMI 1640 medium supplemented with 10% FBS at 5% CO2 atmosphere and incubated with the indicated doses of celecoxib. Untreated cells were used as a control. The DMSO (vehicle of the drug) concentration was maintained at 0.02% in all flasks. Following a 24 h incubation at 37 °C in a 5% CO2 atmosphere, cell viability was evaluated utilizing the trypan blue assay which was carried out as reported elsewhere .
Western blot analysis
Cells were seeded at the density of 4 × 105 in a 75 cm2 tissue culture flask in RPMI 1640 medium supplemented with 10% FBS at 5% CO2 atmosphere and incubated with the indicated doses of celecoxib. Untreated cells were used as a control. The DMSO (vehicle of the drug) concentration was maintained at 0.02% in all flasks. Following a 24 h incubation at 37 °C in a 5% CO2 atmosphere, cells were collected and lysed in lysis buffer [10 mM Tris-HCl (pH 8.2), 1% NP40, 1 mM EDTA, 0.1% BSA, 150 mM NaCl] containing 1/50 (vol/vol) of protease inhibitor cocktail (Calbiochem). Western blot assay for signaling-related proteins was carried out as described . Immunoreactive bands were quantified using the image analysis tool ImageJ.
Flow cytometry analysis
Cells were seeded at the density of 4 × 105 in a 75 cm2 tissue culture flask in RPMI 1640 medium supplemented with 10% FBS at 5% CO2 atmosphere and incubated with the indicated doses of celecoxib. Untreated cells were used as a control. The DMSO (vehicle of the drug) concentration was maintained at 0.02% in all flasks. Following a 24 h incubation at 37 °C in a 5% CO2 atmosphere, cells were collected and cell surface stained as described . Stained cells were analyzed with a flow cytometer (BD FACSAria II, BD Bioacience). Data were analyzed using Kaluza Flow Cytometry Analysis v1.3 software (Beckman Coulter).
Averages, standard deviations (SD), and unpaired t test were calculated using MS-Excel. Data are shown as mean ± SD of the results obtained in at least three independent experiments. Statistical analysis was performed with the Stata Statistical Software, Release 13 (StataCorp LP, College Station, TX). Correlation of COX-2 and PD-L1 expression was analyzed by Spearman’s rank correlation coefficient. Difference in the expression of COX-2 in according to PD-L1 groups was analyzed using the Kruskal–Wallis rank test. Correlation of PD-L1 expression with pathological features of the tumor samples was analyzed by Fisher exact test. Differences in the expression levels of COX-2 and pathological features of tumors was analyzed using the Mann–Whitney U-test. Difference in COX-2 or PD-L1 expression in primary and not matched metastatic lesion groups were analyzed using the Mann–Whitney U test. P < 0.05 was considered to be statistically significant. All tests used were two-tailed.
Tumor pathologic characteristics of primary melanomas
2.92 ± 2.25 mm
PD-L1 expression in primary and not matched metastatic melanoma tissues and its correlation with the pathological features of tumors analyzed
No correlation was identified between PD-L1 expression on melanoma cells and the presence of ulceration and the T stage of the primary melanoma lesions. In addition no correlation was identified between PD-L1 expression on melanoma cells and the BRAF/NRAS genotype of the lesions analyzed.
COX-2 expression in primary and not matched metastatic melanoma tissues and its correlation with the pathological features of tumors analyzed
PD-L1 and COX-2 correlation in primary and metastatic melanoma tissues
PD-L1 and COX-2 co-localize in primary and not matched metastatic melanoma tissues
COX-2 inhibition down-regulates PD-L1 expression in both BRAF and NRAS mutant melanoma cell lines
Targeting the immune checkpoint molecules CTLA-4, PD-1 and PD-L1 is completely revolutionizing the treatment of advanced melanoma. Several clinical trials have convincingly shown that immune checkpoint inhibitors increase the overall survival of metastatic melanoma patients with or without BRAF/NRAS mutations [2–6]. However the response rate of these novel immunotherapeutic approaches is about 40–60%. A growing body of evidence indicates that a major obstacle to the success of immunotherapy is represented by the development of escape mechanisms utilized by tumor cells to avoid recognition and destruction by the host’s immune system. The PD-1/PD-L1 axis represents one of the major mechanisms of tumor immune escape since expression of PD-L1 on cancer cells through the interaction with PD-1 on T cells inhibits the recognition and destruction of cancer cells by the host immune response.
So far PD-L1 expression by cancer cells correlates with melanoma progression. Furthermore PD-L1 expression by cancer cells is currently investigated as a potential biomarker in order to predict clinical responses to anti-PD-1 and -PD-L1 based immunotherapy in several malignancies . In addition a higher expression of PD-L1 in tumors appears to predict for a higher clinical response rate in patients treated with anti-PD-1/PD-L1 based immunotherapy as compared to patients which carry tumors that did not express PD-L1. Nevertheless, some patients not expressing PD-L1 in their tumors also benefit from PD-1/PD-L1 based immunotherapy . These findings might reflect the dynamic expression of PD-1/PD-L1 by both cancer cells and immune cells. Indeed PD-1/PD-L1 expression can be induced on immune cells and cancer cells by the presence of soluble factors or cytokines present in tumor microenvironment. The latter are induced by the interaction of cancer cells with host immune cells during treatment with anti-PD-1/PD-L1 mAbs. In addition PD-1/PD-L1 expression can be induced by pro-tumorigenic pathways which are activated in cancer cells during cancer progression . In this study we showed a relationship between COX-2 and PD-L1 expression in melanoma cells. COX-2 is an important key survival molecule, currently investigated as a molecular marker and a potential tumor therapeutic target. In fact, the prophylactic use of COX-2 inhibitors such as acetylsalicylic acid has been shown to decrease the incidence of several cancers. In particular, its synergistic activity with other anti-tumoral treatments, such as chemotherapy and radiotherapy [37–39] or with other molecular targets related to tumor growth and proliferation, including VEGFR or aromatase inhibitors [40–42] has been often highlighted. In their recent published work Markosyan et al.  describe a cause effect relationship between COX-2 and PD-L1 expression in breast cancer cells. In addition Prima et al. describe that COX-2 and the PGE2 pathway regulates PD-L1 expression in tumor-associated macrophages and myeloid-derived suppressor cells . Our data in melanoma cells are in line with these findings since COX-2 is significantly associated with PD-L1 in melanoma tumors and its inhibition by the COX-2 inhibitor celecoxib down-regulates the expression of PD-L1 in vitro. There is no clear evidence in the literature of the mechanisms which might underlie the effect on PD-L1 by COX-2. Some works reported a relationship between COX-2 and activation of AKT/STAT3 or NF-κB pathways [44, 45] which are known to regulate PD-L1 expression [46–49]. However further studies are warranted to determine the underlying mechanisms. Future studies addressing the molecular signals that regulate PD-L1 surface expression by COX-2 in cancer cells might shed light on developing novel therapeutic agents to modulate cancer cell immune responses.
COX-2 isoform is shown to be up-regulated during both inflammation and cancer [16, 19]. In melanoma COX-2 is shown to be expressed in both primary and metastatic lesions and its expression correlates with pathological features of primary tumors and patient’s prognosis [28, 50, 51]. Our data are in line with previous reports since COX-2 by cancer cells in melanoma tumors is found to be highly expressed in both primary and not matched metastatic melanoma lesions although no significant correlation between COX-2 and the pathological features of tumor analyzed was found. These findings may reflect the low number of lesions analyzed as well as the selection of the tumors.
A body of evidence indicates a role for COX-2 in the development/modulation of different steps of cancer progression and several mechanisms may underlie this pro-tumorigenic activity of COX-2 including (i) production of reactive oxygen species responsible for DNA damage, (ii) aberrant activation of intracellular pathways such as MAPK and the PI3 K/AKT pathways, (iii) activation of STAT3, (iv) induction of Bcl-2 family members and (v) production of growth factors including epidermal growth factor (EGF) and fibroblast growth factor (FGF) . These in turn favor the increase rate of mutations, the proliferation, pro-survival signals and initiated tumor cells by determining an imbalance between cell proliferation and cell death stimuli. Our data demonstrated that COX-2 is involved in the proliferation of melanoma cells since treatment of melanoma cells with or without BRAF/NRAS mutations with the COX-2 inhibitor celecoxib inhibits their proliferation and induces cell death. This effect are in line with previous work [21, 52, 53], providing COX-2 inhibition as an additional valid option in order to inhibit melanoma cell progression. The molecular mechanisms underlying this anti-tumor activity of celecoxib in melanoma cells was not fully investigated in the present study. In A375 cell line inhibition of cell proliferation and/or induction of cell death by celecoxib was associated with down-regulation of Bcl-2. These data are line with previous works which have reported that Bcl-2 down-regulation by COX-2 inhibition is mediated by NF-kB . In contrast in SK-MEL-2 cell line treatment with celecoxib did not change the expression of Bcl-2 which results to be not expressed (Additional file 1: Figure S2). Further studies are needed to clarify the molecules which are involved in inducing cell death and/or preventing cell proliferation following treatment with COX-2 inhibitor in this cell line.
Lastly COX-2 is shown to contribute to immune evasion and resistance to cancer immunotherapy in several cancer types including breast, lung, colon cancer and melanoma [22, 52, 54–57]. COX-2 has been involved in suppressing the activity of immune cells including dendritic cells, natural killer and T cells as well as in promoting type-2 immunity which causes tumor immune evasion. In the present work the activity of COX-2 and its inhibition on immune cell activation/proliferation and/or on the interaction between immune cells and melanoma cells was not investigated. However the novel information we show is that in melanoma cells COX-2 activity causes tumor immune escape by modulating PD-L1 expression. Indeed COX-2 is found to be co-expressed to PD-L1 in both primary and not matched metastatic lesions and inhibition of COX-2 by celecoxib down-regulates PD-L1 in melanoma cells. The latter plays a major role in inhibiting the host immune response.
Over the past years the possibility to combine different therapies, such as biological therapy and immunotherapy, has contributed to completely change the setting of treatment plans . The limited duration of the response obtained with BRAF and MEK inhibition in BRAFV600 melanoma and the impressive durability but the relatively low response rate obtained with the block of the immune checkpoint molecules proved to be surprising. Several clinical trials are examining the combinations of different immune checkpoint inhibitors as well as the combination of immune checkpoint inhibitors with the small molecule inhibitors [59–62]. In this view our data shown that COX-2 expression correlates with and modulates PD-L1 expression in melanoma cells. Besides modulating PD-L1 expression, COX-2 is also shown to be involved in melanoma progression since its inhibition induces cell death and prevents cell proliferation. These findings have clinical relevance since they provide a rationale to implement novel clinical trials to test COX-2 inhibition as a potential treatment to prevent melanoma progression and immune evasion as well as to enhance the anti-tumor activity of PD-1/PD-L1 based immunotherapy for the treatment of melanoma patients with or without BRAF/NRAS mutations.
American Joint Committee on Cancer
American Type Culture Collection
cytotoxic T lymphocytes
cytotoxic T lymphocyte antigen 4
fetal bovine serum
formalin-fixed paraffin embedded
hematoxylin and eosin
institutional review board
non-steroidal anti-inflammatory drugs
programmed death receptor 1
programmed death receptor ligand 1
GS conceived work. GS and FS designed the work. GS, FS and MCa wrote the manuscript. GB, FF, MCe, CC, GS, CT, MCo acquired the samples, performed the experiments and acquired the data. GB, AMA, GP, GS and FS analyzed the data. SS, CD, AI, GC, GL, PAA, and SP discussed results and implications of findings. GS supervised the work. All authors read and approved the final manuscript.
We would like to thank the “The Italian Melanoma Intergroup (IMI)” for his support.
PAA has a consultant/advisory role for Bristol Myers Squibb, Roche-Genentech, Merck Sharp & Dohme, Ventana, Novartis, Amgen, and Array. He received research grant from Roche-Genentech, Bristol Myers Squibb, Ventana and Array.
Availability of data and materials
The dataset used and/or analyzed the current study is available from the corresponding authors on reasonable request.
Ethic approval and consent to participate
Patients were consented for tissue acquisition per institutional review board (IRB)-approved protocol.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Ugurel S, Rohmel J, Ascierto PA, Flaherty KT, Grob JJ, Hauschild A, Larkin J, Long GV, Lorigan P, McArthur GA, et al. Survival of patients with advanced metastatic melanoma: the impact of novel therapies. Eur J Cancer. 2016;53:125–34.View ArticlePubMedGoogle Scholar
- McArthur GA, Chapman PB, Robert C, Larkin J, Haanen JB, Dummer R, Ribas A, Hogg D, Hamid O, Ascierto PA, et al. Safety and efficacy of vemurafenib in BRAF(V600E) and BRAF(V600K) mutation-positive melanoma (BRIM-3): extended follow-up of a phase 3, randomised, open-label study. Lancet Oncol. 2014;15:323–32.View ArticlePubMedPubMed CentralGoogle Scholar
- Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, Rutkowski P, Blank CU, Miller WH Jr, Kaempgen E, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2012;380:358–65.View ArticlePubMedGoogle Scholar
- Flaherty KT, Infante JR, Daud A, Gonzalez R, Kefford RF, Sosman J, Hamid O, Schuchter L, Cebon J, Ibrahim N, et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N Engl J Med. 2012;367:1694–703.View ArticlePubMedPubMed CentralGoogle Scholar
- Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R, Robert C, Schadendorf D, Hassel JC, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med. 2010;363:711–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Robert C, Thomas L, Bondarenko I, O’Day S, Weber J, Garbe C, Lebbe C, Baurain JF, Testori A, Grob JJ, et al. Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med. 2011;364:2517–26.View ArticlePubMedGoogle Scholar
- Choueiri TK, Fay AP, Gray KP, Callea M, Ho TH, Albiges L, Bellmunt J, Song J, Carvo I, Lampron M, et al. PD-L1 expression in nonclear-cell renal cell carcinoma. Ann Oncol. 2014;25:2178–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Harshman LC, Choueiri TK, Drake C, Stephen Hodi F Jr. Subverting the B7-H1/PD-1 pathway in advanced melanoma and kidney cancer. Cancer J. 2014;20:272–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Soliman H, Khalil F, Antonia S. PD-L1 expression is increased in a subset of basal type breast cancer cells. PLoS ONE. 2014;9:e88557.View ArticlePubMedPubMed CentralGoogle Scholar
- Darb-Esfahani S, Kunze CA, Kulbe H, Sehouli J, Wienert S, Lindner J, Budczies J, Bockmayr M, Dietel M, Denkert C, et al. Prognostic impact of programmed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor-infiltrating lymphocytes in ovarian high grade serous carcinoma. Oncotarget. 2016;7:1486–99.PubMedGoogle Scholar
- Hino R, Kabashima K, Kato Y, Yagi H, Nakamura M, Honjo T, Okazaki T, Tokura Y. Tumor cell expression of programmed cell death-1 ligand 1 is a prognostic factor for malignant melanoma. Cancer. 2010;116:1757–66.View ArticlePubMedGoogle Scholar
- Zhang Y, Wang L, Li Y, Pan Y, Wang R, Hu H, Li H, Luo X, Ye T, Sun Y, Chen H. Protein expression of programmed death 1 ligand 1 and ligand 2 independently predict poor prognosis in surgically resected lung adenocarcinoma. Onco Targets Ther. 2014;7:567–73.View ArticlePubMedPubMed CentralGoogle Scholar
- Okazaki T, Honjo T. PD-1 and PD-1 ligands: from discovery to clinical application. Int Immunol. 2007;19:813–24.View ArticlePubMedGoogle Scholar
- Chen J, Jiang CC, Jin L, Zhang XD. Regulation of PD-L1: a novel role of pro-survival signalling in cancer. Ann Oncol. 2016;27:409–16.View ArticlePubMedGoogle Scholar
- Hla T, Neilson K. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci USA. 1992;89:7384–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Sobolewski C, Cerella C, Dicato M, Ghibelli L, Diederich M. The role of cyclooxygenase-2 in cell proliferation and cell death in human malignancies. Int J Cell Biol. 2010;2010:215158.View ArticlePubMedPubMed CentralGoogle Scholar
- Chan AT, Ogino S, Fuchs CS. Aspirin and the risk of colorectal cancer in relation to the expression of COX-2. N Engl J Med. 2007;356:2131–42.View ArticlePubMedGoogle Scholar
- Bucher C, Jordan P, Nickeleit V, Torhorst J, Mihatsch MJ. Relative risk of malignant tumors in analgesic abusers. Effects of long-term intake of aspirin. Clin Nephrol. 1999;51:67–72.PubMedGoogle Scholar
- Thun MJ, Henley SJ, Gansler T. Inflammation and cancer: an epidemiological perspective. Novartis Found Symp. 2004;256:6–21.View ArticlePubMedGoogle Scholar
- Lee C, Ramirez JA, Guitart J, Diaz LK. Expression of cyclooxygenase-2 and peroxisome proliferator-activated receptor gamma during malignant melanoma progression. J Cutan Pathol. 2008;35:989–94.View ArticlePubMedGoogle Scholar
- Elmets CA, Ledet JJ, Athar M. Cyclooxygenases: mediators of UV-induced skin cancer and potential targets for prevention. J Invest Dermatol. 2014;134:2497–502.View ArticlePubMedPubMed CentralGoogle Scholar
- Markosyan N, Chen EP, Evans RA, Ndong V, Vonderheide RH, Smyth EM. Mammary carcinoma cell derived cyclooxygenase 2 suppresses tumor immune surveillance by enhancing intratumoral immune checkpoint activity. Breast Cancer Res. 2013;15:R75.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen JH, Perry CJ, Tsui YC, Staron MM, Parish IA, Dominguez CX, Rosenberg DW, Kaech SM. Prostaglandin E2 and programmed cell death 1 signaling coordinately impair CTL function and survival during chronic viral infection. Nat Med. 2015;21:327–34.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee JH, Choi JW, Kim YS. Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis. Br J Dermatol. 2011;164:776–84.View ArticlePubMedGoogle Scholar
- Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–54.View ArticlePubMedGoogle Scholar
- Colombino M, Capone M, Lissia A, Cossu A, Rubino C, De Giorgi V, Massi D, Fonsatti E, Staibano S, Nappi O, et al. BRAF/NRAS mutation frequencies among primary tumors and metastases in patients with melanoma. J Clin Oncol. 2012;30:2522–9.View ArticlePubMedGoogle Scholar
- Scognamiglio G, De Chiara A, Di Bonito M, Tatangelo F, Losito NS, Anniciello A, De Cecio R, D’Alterio C, Scala S, Cantile M, Botti G. Variability in immunohistochemical detection of programmed death ligand 1 (PD-L1) in cancer tissue types. Int J Mol Sci. 2016;17:790.View ArticlePubMed CentralGoogle Scholar
- Panza E, De Cicco P, Ercolano G, Armogida C, Scognamiglio G, Anniciello AM, Botti G, Cirino G, Ianaro A. Differential expression of cyclooxygenase-2 in metastatic melanoma affects progression free survival. Oncotarget. 2016;7:57077.PubMedPubMed CentralGoogle Scholar
- Combaz-Lair C, Galateau-Salle F, McLeer-Florin A, Le Stang N, David-Boudet L, Duruisseaux M, Ferretti GR, Brambilla E, Lebecque S, Lantuejoul S. Immune biomarkers PD-1/PD-L1 and TLR3 in malignant pleural mesotheliomas. Hum Pathol. 2016;52:9–18.View ArticlePubMedGoogle Scholar
- Powles T, Eder JP, Fine GD, Braiteh FS, Loriot Y, Cruz C, Bellmunt J, Burris HA, Petrylak DP, Teng SL, et al. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature. 2014;515:558–62.View ArticlePubMedGoogle Scholar
- Yu L, Favoino E, Wang Y, Ma Y, Deng X, Wang X. The CSPG4-specific monoclonal antibody enhances and prolongs the effects of the BRAF inhibitor in melanoma cells. Immunol Res. 2011;50:294–302.View ArticlePubMedGoogle Scholar
- Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol. 2001, Appendix 3:Appendix 3B.
- Wang X, Osada T, Wang Y, Yu L, Sakakura K, Katayama A, McCarthy JB, Brufsky A, Chivukula M, Khoury T, et al. CSPG4 protein as a new target for the antibody-based immunotherapy of triple-negative breast cancer. J Natl Cancer Inst. 2010;102:1496–512.View ArticlePubMedPubMed CentralGoogle Scholar
- Sabbatino F, Wang Y, Scognamiglio G, Favoino E, Feldman SA, Villani V, Flaherty KT, Nota S, Giannarelli D, Simeone E, et al. Antitumor activity of BRAF inhibitor and IFNalpha combination in BRAF-mutant melanoma. J Natl Cancer Inst. 2016;108:435.View ArticleGoogle Scholar
- Festino L, Botti G, Lorigan P, Masucci GV, Hipp JD, Horak CE, Melero I, Ascierto PA. Cancer treatment with anti-PD-1/PD-L1 agents: is PD-L1 expression a biomarker for patient selection? Drugs. 2016;76:925–45.View ArticlePubMedGoogle Scholar
- Fusi A, Festino L, Botti G, Masucci G, Melero I, Lorigan P, Ascierto PA. PD-L1 expression as a potential predictive biomarker. Lancet Oncol. 2015;16:1285–7.View ArticlePubMedGoogle Scholar
- Reckamp KL, Krysan K, Morrow JD, Milne GL, Newman RA, Tucker C, Elashoff RM, Dubinett SM, Figlin RA. A phase I trial to determine the optimal biological dose of celecoxib when combined with erlotinib in advanced non-small cell lung cancer. Clin Cancer Res. 2006;12:3381–8.View ArticlePubMedGoogle Scholar
- Liao Z, Komaki R, Milas L, Yuan C, Kies M, Chang JY, Jeter M, Guerrero T, Blumenschien G, Smith CM, et al. A phase I clinical trial of thoracic radiotherapy and concurrent celecoxib for patients with unfavorable performance status inoperable/unresectable non-small cell lung cancer. Clin Cancer Res. 2005;11:3342–8.View ArticlePubMedGoogle Scholar
- Penas-Prado M, Hess KR, Fisch MJ, Lagrone LW, Groves MD, Levin VA, De Groot JF, Puduvalli VK, Colman H, Volas-Redd G, et al. Randomized phase II adjuvant factorial study of dose-dense temozolomide alone and in combination with isotretinoin, celecoxib, and/or thalidomide for glioblastoma. Neuro Oncol. 2015;17:266–73.View ArticlePubMedGoogle Scholar
- Zhang H, Li Z, Wang K. Combining sorafenib with celecoxib synergistically inhibits tumor growth of non-small cell lung cancer cells in vitro and in vivo. Oncol Rep. 2014;31:1954–60.PubMedGoogle Scholar
- Ahmed M, Hussain AR, Siraj AK, Uddin S, Al-Sanea N, Al-Dayel F, Al-Assiri M, Beg S, Al-Kuraya KS. Co-targeting of cyclooxygenase-2 and FoxM1 is a viable strategy in inducing anticancer effects in colorectal cancer cells. Mol Cancer. 2015;14:131.View ArticlePubMedPubMed CentralGoogle Scholar
- Lustberg MB, Povoski SP, Zhao W, Ziegler RM, Sugimoto Y, Ruppert AS, Lehman AM, Shiels DR, Mrozek E, Ramaswamy B, et al. Phase II trial of neoadjuvant exemestane in combination with celecoxib in postmenopausal women who have breast cancer. Clin Breast Cancer. 2011;11:221–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Prima V, Kaliberova LN, Kaliberov S, Curiel DT, Kusmartsev S. COX2/mPGES1/PGE2 pathway regulates PD-L1 expression in tumor-associated macrophages and myeloid-derived suppressor cells. Proc Natl Acad Sci USA. 2017;114:1117–22.View ArticlePubMedGoogle Scholar
- Yang MY, Lee HT, Chen CM, Shen CC, Ma HI. Celecoxib suppresses the phosphorylation of STAT3 protein and can enhance the radiosensitivity of medulloblastoma-derived cancer stem-like cells. Int J Mol Sci. 2014;15:11013–29.View ArticlePubMedPubMed CentralGoogle Scholar
- De Cicco P, Panza E, Ercolano G, Armogida C, Sessa G, Pirozzi G, Cirino G, Wallace JL, Ianaro A. ATB-346, a novel hydrogen sulfide-releasing anti-inflammatory drug, induces apoptosis of human melanoma cells and inhibits melanoma development in vivo. Pharmacol Res. 2016;114:67–73.View ArticlePubMedGoogle Scholar
- Jiang X, Zhou J, Giobbie-Hurder A, Wargo J, 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. 2013;19:598–609.View ArticlePubMedGoogle Scholar
- Lienlaf M, Perez-Villarroel P, Knox T, Pabon M, Sahakian E, Powers J, Woan KV, Lee C, Cheng F, Deng S, et al. Essential role of HDAC6 in the regulation of PD-L1 in melanoma. Mol Oncol. 2016;10:735–50.View ArticlePubMedGoogle Scholar
- Gowrishankar K, Gunatilake D, Gallagher SJ, Tiffen J, Rizos H, Hersey P. Inducible but not constitutive expression of PD-L1 in human melanoma cells is dependent on activation of NF-kappaB. PLoS ONE. 2015;10:e0123410.View ArticlePubMedPubMed CentralGoogle Scholar
- Kondo A, Yamashita T, Tamura H, Zhao W, Tsuji T, Shimizu M, Shinya E, Takahashi H, Tamada K, Chen L, et al. Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes. Blood. 2010;116:1124–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Goulet AC, Einsphar JG, Alberts DS, Beas A, Burk C, Bhattacharyya A, Bangert J, Harmon JM, Fujiwara H, Koki A, Nelson MA. Analysis of cyclooxygenase 2 (COX-2) expression during malignant melanoma progression. Cancer Biol Ther. 2003;2:713–8.View ArticlePubMedGoogle Scholar
- Denkert C, Kobel M, Berger S, Siegert A, Leclere A, Trefzer U, Hauptmann S. Expression of cyclooxygenase 2 in human malignant melanoma. Cancer Res. 2001;61:303–8.PubMedGoogle Scholar
- Zelenay S, van der Veen AG, Bottcher JP, Snelgrove KJ, Rogers N, Acton SE, Chakravarty P, Girotti MR, Marais R, Quezada SA, et al. Cyclooxygenase-dependent tumor growth through evasion of immunity. Cell. 2015;162:1257–70.View ArticlePubMedPubMed CentralGoogle Scholar
- Gowda R, Madhunapantula SV, Desai D, Amin S, Robertson GP. Simultaneous targeting of COX-2 and AKT using selenocoxib-1-GSH to inhibit melanoma. Mol Cancer Ther. 2013;12:3–15.View ArticlePubMedGoogle Scholar
- Bronger H, Kraeft S, Schwarz-Boeger U, Cerny C, Stockel A, Avril S, Kiechle M, Schmitt M. Modulation of CXCR3 ligand secretion by prostaglandin E2 and cyclooxygenase inhibitors in human breast cancer. Breast Cancer Res. 2012;14:R30.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu B, Qu L, Yan S. Cyclooxygenase-2 promotes tumor growth and suppresses tumor immunity. Cancer Cell Int. 2015;15:106.View ArticlePubMedPubMed CentralGoogle Scholar
- Gobel C, Breitenbuecher F, Kalkavan H, Hahnel PS, Kasper S, Hoffarth S, Merches K, Schild H, Lang KS, Schuler M. Functional expression cloning identifies COX-2 as a suppressor of antigen-specific cancer immunity. Cell Death Dis. 2014;5:e1568.View ArticlePubMedPubMed CentralGoogle Scholar
- Whiteside TL. Disarming suppressor cells to improve immunotherapy. Cancer Immunol Immunother. 2012;61:283–8.View ArticlePubMedGoogle Scholar
- Ascierto PA, Marincola FM, Atkins MB. What’s new in melanoma? Combination! J Transl Med. 2015;13:213.View ArticlePubMedPubMed CentralGoogle Scholar
- Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, Schadendorf D, Dummer R, Smylie M, Rutkowski P, et al. Combined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015;373:23–34.View ArticlePubMedGoogle Scholar
- Postow MA, Chesney J, Pavlick AC, Robert C, Grossmann K, McDermott D, Linette GP, Meyer N, Giguere JK, Agarwala SS, et al. Nivolumab and ipilimumab versus ipilimumab in untreated melanoma. N Engl J Med. 2015;372:2006–17.View ArticlePubMedGoogle Scholar
- Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, Segal NH, Ariyan CE, Gordon RA, Reed K, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013;369:122–33.View ArticlePubMedGoogle Scholar
- Adams JL, Smothers J, Srinivasan R, Hoos A. Big opportunities for small molecules in immuno-oncology. Nat Rev Drug Discov. 2015;14:603–22.View ArticlePubMedGoogle Scholar