Evaluation of cannabinoid CB1 and CB2 receptors expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patients’ survival Stamatios Theocharis, Constantinos Giaginis, Paraskevi Alexandrou, Jose Rodriguez, Jason Tasoulas, Eugene Danas, Efstratios Patsouris, et al. Tumor Biology Tumor Markers, Tumor Targeting and Translational Cancer Research ISSN 1010-4283 Tumor Biol. DOI 10.1007/s13277-015-4182-8
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Author's personal copy Tumor Biol. DOI 10.1007/s13277-015-4182-8
ORIGINAL ARTICLE
Evaluation of cannabinoid CB1 and CB2 receptors expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patients’ survival Stamatios Theocharis 1,2 & Constantinos Giaginis 1,3 & Paraskevi Alexandrou 1 & Jose Rodriguez 4 & Jason Tasoulas 1 & Eugene Danas 1 & Efstratios Patsouris 1 & Jerzy Klijanienko 2
Received: 7 July 2015 / Accepted: 1 October 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Cannabinoid receptors (CB1R and CB2R) constitute essential members of the endocannabinoid system (ECS) which participates in many different functions indispensable to homeostatic regulation in several tissues, exerting also antitumorigenic effects. The present study aimed to assess the clinical significance of CB1R and CB2R protein expression in mobile tongue squamous cell carcinoma (SCC). CB1R and CB2R expression was assessed immunohistochemically on 28 mobile tongue SCC tissue samples and was analyzed in relation with clinicopathological characteristics and overall and disease-free patients’ survival. CB1R, CB2R, and concomitant CB1R/CB2R expression was significantly increased in older compared to younger mobile tongue SCC patients (p =0.0243, p=0.0079, and p=0.0366, respectively). Enhanced CB2R and concomitant CB1R/CB2R expression was significantly more frequently observed in female compared to male mobile tongue SCC patients (p=0.0025 and p=0.0016, respectively). Elevated CB2R expression was significantly more frequently observed in mobile tongue SCC patients presenting well-defined tumor shape compared to those with diffuse (p=0.0430). Mobile tongue SCC patients presenting enhanced CB1R, CB2R, or concomitant CB1R/CB2R expression showed significantly longer overall
* Stamatios Theocharis
[email protected];
[email protected] 1
First Department of Pathology, Medical School, University of Athens, 75 M. Asias str., Goudi, Athens, Greece GR11527
2
Department of Pathology, Institut Curie, Paris, France
3
Department of Food Science and Nutrition, University of the Aegean, Lemnos, Greece
4
Department of Head and Neck Surgery, Institut Curie, Paris, France
(log-rank test, p=0.004, p=0.011, p=0.018, respectively) and disease-free (log-rank test, p=0.003, p=0.007, p=0.027, respectively) survival times compared to those with low expression. In multivariate analysis, CB1R was identified as an independent prognostic factor for disease-free patients’ survival (Cox-regression analysis, p=0.032). The present study provides evidence that CB1R and CB2R may play a role in the pathophysiological aspects of the mobile tongue SCC and even each molecule may constitute a potential target for the development of novel anti-cancer drugs for this type of malignancy. Keywords Mobile tongue squamous cell carcinoma . Cannabinoid receptors . Clinicopathological parameters . Prognosis . Immunohistochemistry
Introduction Endocannabinoid system (ECS) is an endogenous lipid signal-inducing system, present in various human tissues, that exerts many different and unrelated functions [1]. Substantial studies have indicated the regulatory effects of the ECS on the central and peripheral nervous system, the gastrointestinal tract and the immune system, being involved in multiple processes, such as gastrointestinal motility, mood, pain regulation, memory, and appetite [1]. These functions are triggered by binding of endogenous and exogenous ligands to cannabinoid receptors (CBRs). Besides those well-known functions, ECS also exerts antiproliferative effects through modulation of several signaling pathways [2], while its activation may have prognostic significance for tumor development and progression [3–6].
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Two subtypes of CBRs exist, with different distribution among the human tissues. CB1R is mainly located at the central nervous system, adipocytes, liver, pancreas, skeletal muscle, and T lymphocytes [7]. CB2R is mainly detected not only in immune cells but also in neurons, astrocytes, microglia, as well as in cerebromicrovascular endothelial cells [8]. The activation of CBRs inhibits cAMP formation through its coupling to Gi proteins, resulting in decreased protein kinase A (PKA)-dependent phosphorylation [7, 8]. CBRs also couple to extracellular signal-regulated kinase (ERK) and specifically p42/p44 and p38 [2], participating in phosphatidylinositol 3kinase (PI3K) and ceramide signaling [9]. Other receptors are also attached to the ECS like transient receptor potential cation channel subfamily V member 1 (TRPV-1), peroxisome proliferator-activated receptors (PPARs), and non-CB1/CB2 G-protein-coupled receptors GPR55 [7, 8]. The ECS ligands are the cannabinoids, including the bioactive components of the Cannabis sativa, synthetic CBmimetic compounds, and endogenous CBRs’ ligands [10]. The most important molecule of the first category is Δ9THC, which is well known for its psychoactive traits [10]. The other two categories include synthetic ligands that have currently been developed (CP55940, HU-210, HU-211, abcannabidiol, ajulemic acid, WIN55,212-2) and endocannabinoids that are produced by the human body and are lipid messenger derivatives of arachidonic acid (AA) conjugated with either ethanolamine or glycerol [11]. The most important of these molecules are anandamide (AEA) and 2arachidonoylglycerol (2-AG), but additional substances have also been identified, such as O-arachidonoylethanolamine (OAE, virodhamine), 2-arachidonoylglyceryl ether (2-AGE, noladin ether), N-arachidonoyldopamine (NADA), and palmitoylethanolamide (PEA) [10, 11]. In addition to ECS functions in order to maintain homeostasis, the abovementioned cross-talk between the ECS and the most important oncogenic pathways (MAPK/ERK and PI3K/Akt pathway) has recently gained interest and has highlighted the significance of the ECS in tumorigenesis [12, 13]. Moreover, cannabinoids have been shown to induce apoptosis in cancer cells, inhibit tumor vascularization via VEGF decrease, and suppress cancer cell invasive capacity [12, 13]. Antiproliferative effects prevail and several studies suggest that cannabinoids have potential as antitumoral agents [12, 13]. Mobile tongue squamous cell carcinoma (SCC) is the most common malignancy diagnosed within the oral cavity [14]. It constitutes an aggressive cancer type that presents increased rate of lymph node metastasis [15]. New advanced therapeutic strategies are being applied; however, the 5-year survival rates have not been considerably improved, mainly due to the increased rates of lymph node metastases at diagnosis [15, 16]. Therefore, there is an urgent need for establishing reliable prognostic markers in mobile tongue SCC patients. In light
of above considerations, the present study aimed to assess immunohistochemically the CB1R and CB2R expression levels in 28 mobile tongue SCC samples, in association with clinicopathological parameters, as well as overall and diseasefree patients’ survival.
Materials and methods Patients Medical records and archival histopathological material of 28 mobile tongue SCC patients, initially treated at Institut Curie, Paris, France, within the period 2000 to 2009, were included in this study. The study was approved by the institutional ethical committee of the Medical School of the University of Athens. Informed consent to use their biological samples and clinical data for research purposes was signed by all patients under study. All patients underwent initial partial glossectomy as primary treatment, and 20 patients had elective neck dissections. The surgical specimens were examined at the Department of Pathology of the Institut Curie, according to standard histopathological protocols. Patients with prior radiotherapy, chemotherapy, or surgery for malignancy were not included in the study. Of the total 28 patients, 14 were male and 14 female with male-to-female ratio 1.00. The patients’ age at initial diagnosis ranged between 33 and 94 years (median age 60 years, IQR 53–72 years). Clinical and histopathological parameters, including grade of histological differentiation, tumor thickness, nodal status, perineural invasion, and the presence of lymphovascular emboli, were assessed [17]. Tumor shape was classified as well defined and diffuse dependent on either pushing or infiltrating tumor margins, respectively [18]. The histological grading of tumor differentiation was based on conventional histological criteria which included the assessment of keratinization, cellular and nuclear pleomorphism, and mitotic activity [19]. In this aspect, three typical scale grades were recorded as follows: well, moderate, and poorly differentiated [19]. Tumor thickness was measured from the surface of the tumor to the deepest point of invasion [20, 21]. Chronic inflammatory infiltration was identified as small mononuclear cells in the stroma of the entire tumor. The degree of infiltration was classified as mild, moderate, and intense according to the density of inflammatory cells [17]. Mitotic index in tumor cells was counted at ×400 in ten consecutive randomly chosen fields using hematoxylin and eosin staining [20]. The patients were followed up for a time interval between 2 and 116 months (median 41 months, IQR 14–68 months). Overall survival was defined as the time interval between the date of surgery and the last follow-up or the date of death due to mobile tongue SCC. Disease-free survival was defined
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as the time interval between the date of surgery and the date of detection of recurrence or the date of last follow-up without recurrence for mobile tongue SCC. At the time of the last follow-up, 8 (28.57 %) patients had died from disease, 2 (7.14 %) were alive with disease, and 18 (64.29 %) were alive and disease free. Immunohistochemistry Immunostainings for CB1R and CB2R were performed on formalin-fixed, paraffin-embedded mobile tongue SCC tissue sections using a goat polyclonal CB1R IgG antibody (N-15, sc-10066, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a rabbit polyclonal CB2R IgG antibody (H-60, sc-25494, Santa Cruz Biotechnology). Briefly, 4-μm-thick tissue sections were deparaffinized, rehydrated, immersed in 3 % H2O2 for 30 min, microwaved at 750 W in 0.01 M citrate buffer (pH 6.0) for 15 min, and left to cool down in TBS. Non-specific antibody binding was blocked using Snipper or Eraser, specific blocking reagent for rabbit and goat primary antibodies, respectively (Sniper, Eraser, Biocare Medical, Walnut, Creek, CA, USA) for 5 min. Sections were incubated with primary antibodies for 1 h, at room temperature (37 °C), at a dilution 1:200. After washing three times with PBS, sections were then incubated at room temperature with biotinylated linking reagent (Biocare Medical) for 10 min, followed by incubation with peroxidase-conjugated streptavidin label (Biocare Medical) for 10 min. The resultant immune peroxidase activity was developed using a DAB substrate kit (Vector Laboratories, USA) for 10 min. Sections were counterstained with Harris’ hematoxylin and mounted in Entellan (Merck, Darmstadt, Germany). Appropriate negative controls were performed by omitting the primary antibody and/or substituting it with an irrelevant anti-serum. As positive control, breast carcinoma tissue sections with known CB1R and CB2R expression were used [22]. Evaluation of immunohistochemistry Immunohistochemical evaluation was performed by counting at least 1000 tumor cells in each case by two independent observers (S.T. and J.K.) blinded to the clinical data, with complete observers’ agreement. Specimens were considered Bpositive^ for CB1R and CB2R when the percentage of positively stained tumor cells in the section was more than 5 %. The immunoreactivity of the tumor cells for CB1R and CB2R was scored according to the percentage of CB1R- and CB2Rpositive tumor cells as 0, negative staining, 0–4 % of cells positive; 1, 5–24 % of cells positive; 2, 25–49 % of cells positive; and 3, 50–100 % of cells positive and its intensity as 0, negative staining; 1, mild staining; 2, intermediate staining; and 3, intense staining expression in mobile tongue squamous cell carcinoma [23, 24]. CB1R and CB2R expression
were classified as low, if the total score was 0 or 2, and high, if the total score was ≥3 [23, 24]. In this way, it is ensured that each group has a sufficient and more homogeneous number of cases in order to be comparable with the other groups [23, 24]. Finally, concomitant high CB1R/CB2R expression was defined in cases presenting simultaneously high CB1R and CB2R expression, whereas concomitant low CB1R/CB2R expression was defined in cases presenting low expression in either CB1R or CB2R or both proteins. Statistical analysis Chi-square test was used to assess the associations of CB1R, CB2R, and concomitant CB1R/CB2R expression with clinicopathological variables. Survival curves were constructed using the Kaplan-Meier method and the differences between the curves were compared by the log rank test. A Cox proportional-hazard regression model was developed to evaluate the association between the potential prognostic marker and overall and disease-free survival. Cox regression analysis was conducted at both univariate and multivariate levels. A p value less than 0.05 was considered the limit of statistical significance. SPSS for Windows Software was used for all analyses (SPSS Inc., 2003, Chicago, USA).
Results Clinical significance of CB1R expression in mobile tongue SCC Twenty-three (82.1 %) out of 28 mobile tongue SCC patients were found positive for CB1R. High CB1R expression was noted in 15 (53.6 %) and out of 28 mobile tongue SCC cases. The subcellular pattern of CB1R distribution was predominantly cytoplasmic and occasionally membranous. Normal surrounding areas adjacent to tumor were found negative for CB1 receptor. A representative CB1R immunostaining is depicted in Fig. 1a. In crosstabulation, high CB1R expression was significantly associated with older patients’ age (Table 1, p=0.0243) and borderline with female gender (Table 1, p=0.0581). A marginal association between high CB1R expression and presence of muscular invasion was also reported (Table 1, p= 0.0967). CB1 receptor expression was positively associated with mitotic index, at a no significant level though (Table 1, p=0.1355). Kaplan-Meier survival curves indicated that mobile tongue SCC patients with high CB1R expression presented significantly longer overall and disease-free survival times compared to those with low expression (Fig. 2a, b, Table 2, log-rank test, p=0.004 and p=0.003, respectively). In multivariate analysis, CB1R expression but not patients’ age, gender and depth of
Author's personal copy Tumor Biol. Fig. 1 Representative immunostainings for a CB1R and b CB2R expression in mobile tongue SCC. Streptavidin-biotinperoxidase, DAB chromogen, Harris hematoxylin counterstain (original magnification ×400)
Table 1
Associations of CBRs expression in tumor cells with clinicopathological parameters in 28 mobile tongue squamous cell carcinoma patients
Clinicopathological Characteristics
N=28 Age (mean±SD; years) ≤62.7±14.6 years >62.7±14.6 years Gender
CB1R expression Low (%)
High (%)
13 (46.4)
15 (53.6) 4 (14.3) 11 (39.3)
9 (32.1)
5 (17.9)
Female Histopathological grade
4 (14.3)
10 (35.7)
I II Stromal inflammatory reaction
8 (28.6) 5 (17.9)
11 (39.3) 4 (14.3)
4 (14.3) 9 (32.1)
3 (10.7) 12 (42.9)
Mild/moderate Dense Muscular invasion Yes No Shape Diffuse Well defined Vascular invasion Yes No Perineural invasion Yes No Depth of invasion I+II III Lymph node metastases Yes No Mitotic index ≤median value >median value
p value
Low (%)
High (%)
14 (50.0)
14 (50.0)
0.0243 9 (32.1) 4 (14.3)
Male
CB2R expression
10 (35.7) 4 (14.3)
3 (10.7) 11 (39.3)
11 (39.3)
3 (10.7)
3 (10.7)
11 (39.3)
8 (28.6) 6 (21.4)
11 (39.3) 3 (10.7)
5 (17.9) 9 (32.1)
2 (7.1) 12 (42.9)
11 (39.3) 3 (10.7)
12 (42.9) 2 (7.1)
12 (42.9) 2 (7.1)
7 (25.0) 7 (25.0)
6 (21.4) 7 (25.0)
4 (14.3) 11 (39.3)
9 (32.1) 4 (14.3)
12 (42.9) 3 (10.7)
6 (21.4) 7 (25.0)
5 (17.9) 10 (35.7)
8 (28.6) 5 (17.9)
5 (17.9) 10 (35.7)
2 (7.1) 8 (28.6)
13 (46.4)
1 (3.6)
5 (17.9)
9 (32.1)
4 (14.3) 10 (35.7)
2 (7.1) 12 (42.9)
5 (17.9) 9 (32.1)
5 (17.9) 9 (32.1)
0.0016
0.3051 11 (39.3) 7 (25.0)
8 (28.6) 2 (7.1)
6 (21.4) 12 (42.9)
1 (3.6) 9 (32.1)
0.1718
0.0659 13 (46.4) 5 (17.9)
10 (35.7) 0 (0.0)
13 (46.4) 5 (17.9)
6 (21.4) 4 (14.3)
0.5069
9 (32.1) 5 (17.9)
12 (42.9) 2 (7.1)
7 (25.0) 7 (25.0)
4 (14.3) 10 (35.7)
7 (25.0) 7 (25.0)
6 (21.4) 8 (28.6)
0.2719 5 (17.9) 13 (46.4)
1 (3.6) 9 (32.1)
7 (25.0) 11 (39.3)
3 (10.7) 7 (25.0)
13 (46.4) 5 (17.9)
8 (28.6) 2 (7.1)
8 (28.6) 10 (35.7)
3 (10.7) 7 (25.0)
9 (32.1) 9 (32.1)
4 (14.3) 6 (21.4)
1.0000
0.5116
0.6381
0.1904
0.4886
0.6488
0.2457
0.1355
p value
0.0366
0.3569
0.2831
Significant data are depicted with italic
11 (39.3) 7 (25.0)
0.0430
0.2621 2 (7.1) 13 (46.4)
10 (35.7)
0.6217
0.3389
4 (14.3) 9 (32.1)
18 (64.3)
0.1904
0.0967
9 (32.1) 6 (21.4)
High (%)
0.2248
0.5116
10 (35.7) 3 (10.7)
Low (%)
0.0025
0.5051
14 (50.0) 1 (3.6)
p value
0.0079
0.0581
9 (32.1) 4 (14.3)
Concomitant CB1R and CB2R expression
0.4533
0.7047
0.6111
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Fig. 2 Kaplan-Meier survival analysis stratified according to CB1R and CB2R expression in mobile tongue SCC patients. a CB1R expression and overall patients’ survival. b CB1R expression and disease-free patients’
survival. c CB2R expression and overall patients’ survival. d CB2R and disease-free patients’ survival
invasion was identified as independent prognostic factor of disease-free patients’ survival (Table 3, Cox regression analysis, p=0.032). CB1R expression, patients’ age, gender, and tumor shape and grade did not remain significant in multivariate analysis as far as concern overall patients’ survival (Table 3, p>0.1).
CB2R expression was significantly more frequently observed in mobile tongue SCC patients presenting well defined tumor shape compared to those with diffuse tumor shape (Table 1, p=0.0430). An increased incidence of high CB2R expression was also observed in mobile tongue SCC patients presenting low histopathological grade, dense stromal inflammatory reaction, earlier depth of invasion, and absence of lymph node metastases, at a non-significant level though (Table 1, p>0.1). Kaplan-Meier survival curves indicated that mobile tongue SCC patients with high CB2R expression presented significantly longer overall and disease-free survival times compared to those with low expression (Fig. 2c and d, Table 2, log-rank test, p=0.011 and p=0.007, respectively). In multivariate analysis, neither clinicopathological parameter nor CB2R expression remain significant (Table 4, p>0.1).
Clinical significance of CB2R expression in mobile tongue SCC Twenty-eight (100.0 %) out of 28 mobile tongue SCC patients were found positive for CB2R. High CB2R expression was noted in 14 (50.0 %) and out of 28 mobile tongue SCC cases. The subcellular pattern of CB2R distribution was predominantly cytoplasmic and occasionally membranous. Benign tongue epithelium cells of the basal line were CB2R positive. Additionally, lymphoid infiltrating cells were occasionally CB2R positive. A representative CB2R immunostaining are depicted in Fig. 1b. In crosstabulation, high CB2R expression was significantly associated with older patients’ age and female gender (Table 1, p = 0.0079 and p = 0.0025, respectively). High
Clinical significance of concomitant CB1R/CB2R expression in mobile tongue SCC Ten (35.7 %) out of 28 mobile tongue SCC patients presented concomitant high CB1R/CB2R expression, whereas the remaining 18 (64.3 %) mobile tongue SCC patients showed
Author's personal copy Tumor Biol. Table 2 Association of clinicopathological parameters, CB1R, CB2R, and concomitant CB1R/CB2R expression in tumor cells with patients’ survival: univariate analysis Mean overall survival (95 % CI)
p value
Mean disease-free survival (95 % CI)
p value
median value
108.7 (87.5–130.0)
Low
53.0 (40.9–65.1)
High
152.9 (133.7–172.1)
Low
78.1 (48.2–108.0)
High
153.2 (134.8–171.5)
Low
54.2 (34.3–68.7)
High
154.6 (130.6–173.1)
138.1 (112.9–163.2) 0.038
73.8 (47.0–100.7)
0.031
140.7 (113.0–168.4) 0.226
127.7 (101.4–154.0)
0.048
61.0 (33.2–88.9) 0.779
83.5 (51.1–127.7)
0.666
115.2 (86.9–143.5) 0.927
115.8 (89.5–142.1)
0.529
68.6 (31.6–105.7) 0.111
77.2 (53.4–100.9)
0.031
151.0 (128.8–173.2) 0.280
87.5 (52.5–122.5)
0.694
116.5 (88.5–144.4) 0.966
95.2 (72.6–117.8)
0.457
106.3 (74.3–138.4) 0.026
123.1 (96.4–149.7)
0.171
71.3 (34.9–107.6) 0.329
102.0 (62.7–141.3)
0.473
102.3 (78.6–126.0) 0.358
95.0 (57.1–132.9)
0.198
103.0 (80.3–125.6) 0.004
47.8 (35.9–59.6)
0.003
144.6 (120.8–168.4) 0.011
67.6 (40.0–95.1)
0.007
144.4 (120.9–167.9) 0.018
76.9 (52.5–101.3)
0.027
150.2 (126.6–173.8)
Significant data are depicted with italic Table 3 Association of clinicopathological parameters and CB1R expression with patients’ survival: Multivariate analysis Clinicopathological parameters
HR (95 % CI)
p value
Table 4 Association of clinicopathological parameters and CB2R expression with patients’ survival: multivariate analysis Clinicopathological parameters
Age (≤62.7/> 62.7±14.6 years)
Overall survival 3.909 (0.297–51.387) 1.667 (0.128–21.783) 0.487 (0.094–2.526) 4.043 (0.296–55.251) Disease-free survival 0.779 (0.163–3.713)
Gender (male/female) Tumor grade (I/II) Tumor shape (diffuse/well defined) CB1R expression (low/high)
2.650 (0.450–15.094) 0.261 (0.057–1.194) 8.348 (0.790–88.239) 8.217 (1.204–56.064)
Age (≤62.7/> 62.7±14.6 years) Gender (male/female) Depth of invasion (I+II/III) CB1R expression (low/high)
0.300 0.697 0.392 0.295 0.754 0.285 0.083 0.078 0.032
A Cox proportional-hazard regression model was developed to evaluate whether the prognostic markers found in univariate analysis remained significant at multivariate level Significant data are depicted with italic
Age (≤62.7/> 62.7±14.6 years) Gender (male/female) Depth of invasion (I+II/III) CB2R expression (low/high)
HR (95 % CI) Overall survival 4.894 (0.416–57.631)
1.622 (0.114–23.073) 0.475 (0.089–2.531) 3.166 (0.209–47.969) Disease-free survival Age (≤62.7/> 62.7±14.6 years) 0.797 (0.139–4.556) Gender (male/female) 5.379 (0.354–81.607) Tumor grade (I/II) 0.538 (0.139–2.078) Tumor shape (diffuse/well defined) 7.683 (0.467–126.356) CB2R expression (low/high) 1.278 (0.092–17.841)
p value
0.207 0.721 0.383 0.406 0.799 0.225 0.368 0.154 0.855
A Cox proportional-hazard regression model was developed to evaluate whether the prognostic markers found in univariate analysis remained significant at multivariate level
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concomitant low expression. CB1R expression was borderline positively associated with CB2R expression, at nonsignificant level though (Spearman’s rho=0.358, p=0.061). In crosstabulation, concomitant high CB1R/CB2R expression was significantly associated with older patients’ age and female gender (Table 1, p = 0.0366 and p = 0.0016, respectively). A borderline association between concomitant high CB1R/CB2R expression and presence of muscular invasion was noted (Table 1, p=0.0659). An increased incidence of concomitant high CB1R/CB2R expression was also observed in mobile tongue SCC patients presenting dense stromal inflammatory reaction, at a non-significant level though (Table 1, p>0.1), while non-associations with the other clinicopathological parameters were recorded (Table 1, p>0.1). Kaplan-Meier survival curves indicated that mobile tongue SCC patients with concomitant high CB1R/CB2R expression presented significantly longer overall and disease-free survival times compared to those with concomitant low expression (Fig. 3a, b, Table 2, log-rank test, p=0.018 and p=0.027, respectively). In multivariate analysis, neither clinicopathological parameter nor concomitant CB1R/CB2R expression remains significant (Table 5, p>0.05).
show any association with PPAR-γ expression (Spearman’s rho=0.142, p=0.472). CB1R expression was negatively associated with pErk expression, at a non significant level though (Spearman’s rho= −0.358, p=0.061). Moreover, CB1R expression was marginally positively associated with cytoplasmic Erk1 expression (Spearman’s rho=0.351, p=0.067), whereas non association with nuclear Erk1 expression was noted (Spearman’s rho= 0.113, p=0.566). CB2R expression was not associated with pErk, nuclear and cytoplasmic Ekr1 expression (Spearman’s rho=−0.286; p=0.141, Spearman’s rho=−0.158; p=0.422 and Spearman’s rho=0.144; p=0.464, respectively). Concomitant CB1R/CB2R expression was negatively associated with pErk expression, at a non-significant level though (Spearman’s rho=−0.298, p=0.123). Concomitant CB1R/CB2R expression was not associated with nuclear Ekr1 expression (Spearman’s rho=0.24, p=0.905), whereas a marginally positive association with cytoplasmic Erk1 expression was noted (Spearman’s rho=0.344, p=0.073).
Associations of CB1R and CB2R with related molecules
In the last few years, several studies have demonstrated that the ECS is altered in different tumor types. However, little is known about the precise role of the ECS in tumor progression. Certain recent studies have examined the clinical significance of CB1R or CB2R expression in hepatocellular, pancreatic, prostate, oral squamous cell, and colorectal carcinoma [3–6, 27, 28]. On the other hand, non-available data concerning mobile tongue SCC have currently existed. In this aspect, the present study evaluated for the first time the clinical significance of CB1R and CB2R expression in mobile tongue SCC. An increased incidence of CB1R (82.1 %) and CB2R (100 %) positivity was recorded, while approximately half of the examined cases presented increased
Potential associations of CB1R, CB2R and concomitant CB1R/CB2R expression with PPAR-γ, cytoplasmic and nuclear Erk1 and phosphorylated Erk (pErk) (data not shown) that have immunohistochemically been assessed in our previous works were examined [25, 26]. An increased incidence of high CB1R expression was observed in mobile SCC cases presenting high PPAR-γ expression, at a non-significant level though (Spearman’s rho= 0.278, p=0.153), whereas non association between CB2R and PPAR-γ expression was noted (Spearman’s rho=0.073, p=0.712). Concomitant CB1R/CB2R expression also did not
Discussion
Fig. 3 Kaplan-Meier survival analysis stratified according to concomitant CB1R/CB2R expression in mobile tongue SCC patients. a Concomitant CB1R/CB2R expression and overall patients’ survival. b Concomitant CB1R/CB2R expression and disease-free patients’ survival
Author's personal copy Tumor Biol. Table 5 Association of clinicopathological parameters and concomitant CB1R/CB2R expression with patients’ survival: multivariate analysis
HR (95 % CI)
p value
Age (≤62.7/> 62.7±14.6 years)
Overall survival 4.347 (0.305–61.974)
0.278
Gender (male/female)
0.783 (0.071–8.634)
0.841
Depth of invasion (I+II/III) Concomitant CB1R/CB2R expression (low/high)
0.385 (0.068–2.183) 1.008 (0.047–128.574)
0.281 0.956
Clinicopathological parameters
Disease-free survival Age (≤62.7/> 62.7±14.6 years) Gender (male/female)
0.801 (0.140–4.587) 3.143 (0.336–29.410)
0.803 0.316
Tumor grade (I/II) Tumor shape (diffuse/well defined)
0.534 (0.140–2.037) 7.683 (0.467–126.356)
0.358 0.094
Concomitant CB1R/CB2R expression (low/high)
3.291 (0.231–46.817)
0.379
A Cox proportional-hazard regression model was developed to evaluate whether the prognostic markers found in univariate analysis remained significant at multivariate level
incidence of high CB1R and CB2R expression (53.6 and 50.0 %, respectively). Positive and negative controls were used to speculate the accuracy of the experimental procedure. The elevated expression levels of CB1R and CB2R in mobile tongue SCC may reinforce the therapeutic utility of CBRs targeting in mobile tongue SCC chemoprevention, taking into consideration the anti-cancer properties of ECS ligands in cell proliferation, differentiation, and apoptosis [12, 13]. Both CB1R and CB2R expression was significantly associated with patients’ age and gender, and further CB2R expression showed an association with tumor shape. Increased incidence of high CB1R expression was noted in mobile tongue SCC patients with presence of muscular invasion and absence of vascular and perineural invasion at a non-significant level though. In addition, an increased incidence of high CB2R expression was noted in mobile tongue SCC patients with low histopathological grade, dense stromal inflammatory reaction, and earlier depth of invasion at a non-significant level though. Most importantly, both CB1R and CB2R expression was significantly associated with overall and disease-free patients’ survival. Notably, CB1R expression was proved as a better prognostic marker compared to CB2R and concomitant CB1R/CB2R expression, since it remained significant at both univariate and multivariate survival analysis. In accordance with the present study, CBRs were upregulated in certain human malignancies, including oral squamous cell carcinoma, pancreatic, hepatocellular, prostatic, and colorectal carcinoma, whereas they were not expressed in normal tissues of these organs [3–6, 28]. On the other hand, CB1R was downregulated in colorectal carcinoma in contrast to adjacent normal tissues, pointing to the different roles of the ECS in various organs and tumors, indicating also the multiple interactions between the ECS and the mechanisms that control cell growth and proliferation [27]. In accordance with the present findings, elevated CBRs’ expression was significantly associated with improved prognosis and longer disease-free
survival in hepatocellular carcinoma patients [3]. Such findings were also combined with the histopathological tumor characteristics, as high CBRs’ levels were observed in cases presenting well differentiation and limited portal tract involvement [3]. On the other hand, CB2R immunoreactivity was associated with shorter disease-free survival in head and neck squamous cell carcinoma [6]. Concerning CB1R, its overexpression was also associated with poor patients’ outcome in colorectal, prostatic, and pancreatic carcinoma [4, 5, 28]. Notably, enhanced CB1R expression in stage IV colorectal carcinoma patients was independently correlated with poor prognosis [28]. Increased CB1R expression was also associated with aggressive prostatic adenocarcinoma presenting higher Gleason score, larger tumor size, and increased cell proliferation rate, as well as presence of metastasis at diagnosis [5]. Moreover, low CB1R expression was correlated with longer survival rate and higher pain status [4]. Similar but not statistically significant results for CB2R were also recorded [4]. In addition, CB2R expression was associated with luminal molecular subtype carcinoma, positively correlated with intermediate disease stage and estrogen receptor expression in invasive breast carcinoma [22]. Taking into account the abovementioned data, the currently available studies on CBR levels and their associations with prognosis in various tumors seem contradictory and may be closely related to the extent of ECS participation in tumorigenesis. The antitumor effects of ECS have also been depicted in numerous in vitro studies. In colorectal cancer, endocannabinoids and synthetic cannabinoids were able to induce apoptosis and inhibit carcinogenesis by mechanisms involving both CBRs, TRPV1 channels, and PPARγpathway [29, 30]. Similar results have occurred in studies conducted on pancreatic, lung and breast cancer, cholangiocarcinoma, and hepatocellular carcinoma cell lines [31–35]. In fact, cannabinoids lead to apoptosis of pancreatic tumor cells via a CB2R and de novo synthesized ceramide-dependent
Author's personal copy Tumor Biol.
upregulation of p8 and the endoplasmic reticulum stressrelated genes ATF-4 and TRB3 [31]. In non-small cell lung carcinoma cell lines, both CB1R and CB2R agonists inhibited phosphorylation of AKT, a key signaling molecule controlling cell survival, migration, and apoptosis, and reduced matrix metalloproteinase (MMP)-9 expression and activity [32]. CB1R agonists also inhibited tumor cell invasion and metastasis by modulating FAK phosphorylation in breast cancer cell lines [33]. The synthetic cannabinoid WIN55,212-2 sensitized hepatocellular carcinoma cells to tumor necrosis factor related apoptosis-inducing ligand (TRAIL)-induced apoptosis by activating p8/CCAAT/enhancer binding protein homologous protein (CHOP)/death receptor 5 (DR5) axis [35]. Synergistic effects of cannabinoids with conventional antitumor chemotherapy in human colorectal carcinoma cells have also been reported [36].
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Conclusions The present study supported evidence that CB1R and CB2R expression is elevated in mobile tongue SCC patients. Significant associations with patients’ age and gender and indicative associations with histopathological characteristics that affect disease progression were recorded. Both CB1R and CB2R expression was also associated with patients’ prognosis. Moreover, CB1R expression was proved as a better prognostic marker compared to CB2R and concomitant CB1R/CB2R expression concerning either overall or disease-free patients’ survival. However, larger cohort studies are strongly recommended in order to validate CBRs’ implication in malignant transformation in mobile tongue SCC and its possible use as potential therapeutic targets in this type of neoplasia.
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15. Acknowledgments We would like to thank Ms Dimitra Lambiri and Ms Eugenie Ventouri for their excellent technical assistant. All authors verify that they have not accepted any funding or support from an organization that may in any way gain or lose financially from the results of the present study. All authors verify that they have not been employed by an organization that may in any way gain or lose financially from the results of the present study. Compliance with ethical standards
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Conflicts of interest None 19. 20.
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