INTIMP-03705; No of Pages 6 International Immunopharmacology xxx (2015) xxx–xxx
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Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma Ana Cristina Castillo-González a,1, Juan Pablo Pelegrín-Hernández b,1, Susana Nieto-Cerón a, Antonio Piñero Madrona c, José Antonio Noguera a, María Fuensanta López-Moreno a, José Neptuno Rodríguez-López d, Cecilio J. Vidal d, Diego Hellín-Meseguer b,⁎, Juan Cabezas-Herrera a,⁎ a
Molecular Therapy and Biomarkers Research Group, Clinical Analysis Service, University Hospital Virgen de la Arrixaca, IMIB-Arrixaca, Ctra Madrid-Cartagena s/n, El Palmar, 30120 Murcia, Spain Otorhinolaryngology Surgical Service, University Hospital Virgen de la Arrixaca IMIB-Arrixaca, Ctra Madrid-Cartagena s/n, El Palmar, 30120 Murcia, Spain Surgery Service of University Hospital Virgen de la Arrixaca IMIB, Ctra Madrid-Cartagena s/n, El Palmar, 30120 Murcia, Spain d Department of Biochemistry and Molecular Biology A, School of Biology, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, 30100 Murcia, Spain b c
a r t i c l e
i n f o
Article history: Received 27 February 2015 Received in revised form 4 May 2015 Accepted 7 May 2015 Available online xxxx Keywords: Cholinergic system Non-neuronal compartment Human airways Larynx cancer
a b s t r a c t Previous reports have demonstrated that a non-neuronal cholinergic system is expressed aberrantly in airways. A proliferative effect is exerted directly by cholinergic agonists through the activation of nicotinic and muscarinic receptors. In cancer, particularly those related with smoking, the mechanism through which tumour cells respond to aberrantly activated cholinergic signalling is a key question. Fifty paired pieces of larynx squamous cell carcinoma and adjacent non-cancerous tissue were compared in terms of their acetylcholinesterase activity (AChE). The AChE activity in non-cancerous tissues (0.248 ± 0.030 milliunits per milligram of wet tissue; mU/mg) demonstrates that upper respiratory tissues express sufficient AChE activity for controlling the level of acetylcholine (ACh). In larynx carcinomas, the AChE activity decreased to 0.157 ± 0.024 mU/mg (p = 0.009). Larynx cancer patients exhibiting low ACh-degrading enzymatic activity had a significantly shorter overall survival (p = 0.031). Differences in the mRNA levels of alternatively spliced AChE isoforms and molecular compositions were noted between glottic and supraglottic cancers. Our results suggest that the low AChE activity observed in larynx squamous cell carcinoma may be useful for predicting the outcome of patients. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Head and neck cancer arise in the mucosal layer of the upper aerodigestive tract (oral cavity, oropharynx, hypopharynx, and larynx). Nearly 90% of head and neck carcinomas are squamous cell carcinomas. Head and neck neoplasia is the sixth most frequent cancer, with more than 600,000 new cases reported worldwide each year [1,2]. Larynx cancer is the second most common type of cancer among all head and neck cancers. At the early stage, patients with larynx carcinoma can be cured with multimodal therapy (surgery, radiation, and/or chemotherapy). Unfortunately, no fully satisfactory treatment has yet been developed, and therefore, the mortality rate of larynx carcinoma patients remains high [3]. Reliable biomarkers for distinguishing patients with poor prognosis or risk of early recurrence and for using personalized therapies are still awaited given the uncertainty of the clinical evolution of larynx carcinoma using the current staging criteria.
⁎ Corresponding authors. Tel.: +34 968 369441. E-mail addresses: [email protected] (D. Hellín-Meseguer), [email protected] (J. Cabezas-Herrera). 1 These authors contributed equally to this work.
An increasing body of evidence notes that several cell types are capable of expressing the range of proteins that form a non-neuronal cholinergic system (NNCS), i.e., the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT), nicotinic (nAChR) and muscarinic (mAChR) receptors, and the ACh-hydrolysing enzymes acetyl(AChE) and butyrylcholinesterase (BChE) [4–7]. A specific cholinergic phenotype depending on the cell type could participate in processes that define a correct tissue physiology. It is worth noting that the results demonstrate that the human respiratory tract epithelium possesses a NNCS engaged in controlling the level of ACh. It appears that this epithelial cholinergic system operates actively to regulate auto/ paracrine actions and thereby reliably controls basic cell functions [8]. The proliferative effects arising from cholinergic overactivation have gained basic and translational significance. Of importance is the susceptibility to lung cancer that confers AChR disorders [9] as well as the nicotine-guided shift in the expression of ACh-related proteins to proliferating/migrating cell phenotypes [10]. It is also worth mentioning that promising therapies based in the blockade or attenuation of cholinergic signalling are under investigation [11–13]. The classical function of AChE is to terminate cholinergic transmission through a very efficient hydrolysis of ACh. There are three main alternative splicing forms of AChE: synaptic or tailed AChE (AChE–T),
http://dx.doi.org/10.1016/j.intimp.2015.05.011 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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erythrocyte or hydrophobic AChE (AChE–H) and read-through AChE (AChE–R). In addition to the hydrolysis of neurotransmitters, AChE has several non-classical roles related with important cell processes such as proliferation, differentiation, apoptosis and cell–cell recognition [14]. The studies on the active involvement of cholinesterases in cell proliferation and differentiation [15] indicate that it is possible that AChE and, to a lower extent, BChE collaborate to tumour development and dissemination. The following examples support this idea: the frequent aberrations in the AChE gene and the structural changes in AChE proteins observed in tumours of diverse origin [8,16–20], the expression of AChE upon apoptosis induction with different stimuli [21–23], and the profitable use of AChE as a prognostic predictor for liver carcinoma and its profitable effects through the suppression of cell growth and the enhancement of chemosensitization [24]. Studies in our laboratory have found that cancer affects the level of AChE activity and/or the content of alternatively spliced mRNAs in the human breast, lymph node, intestine, lung, kidney and prostate. Due to the lack of specific and sensitive biomarkers and tools for early diagnosis, cancer in airways is diagnosed at advanced stages [8,19,25,26]. The aim of this research study was to explore possible changes in the expression of AChE in laryngeal tumours and to test the usefulness of these changes as reliable diagnostic or prognostic markers. 2. Material and methods 2.1. Patients and samples A total of 50 human malignant primary larynx squamous cell carcinomas (LSCCs) and their adjacent noncancerous tissues (ANCT) collected through surgeries at Virgen de la Arrixaca Clinical University Hospital in Murcia (Spain) from 2007 to 2012 were included in the current study. Fresh specimens were divided into sections and stored at −80 °C until use. The TNM classification of LSCC specimens was made according to the UICC:TNM Classification of Malignant Tumors. Study approval and the consent procedure were obtained from the Institutional Ethic Committee of our Hospital. All of the patients gave their consent after being appropriately informed. 2.2. Extraction and assay of acetylcholinesterase AChE was extracted from surgical ANCT and LSCC pieces through homogenization (5% w/v) with Tris-saline buffer (TSB; 1 M NaCl, 50 mM MgCl2, 3 mM EDTA, 10 mM Tris, pH 7.0) supplemented with 1% Brij 96 and a fresh mixture of proteinase inhibitors (0.1 mg/ml soybean trypsin inhibitor, 1 mg/ml bacitracin, 0.0022 TIU/ml aprotinin, 10 mg/ml pepstatin A and 20 mg/ml leupeptin). After centrifugation at 30,000 rpm and 4 °C for 1 h with a 70Ti Beckman rotor (Palo Alto, CA, USA), the supernatant with AChE was saved. The AChE activity was determined as described previously [8]. AChE activity is given in milliunits per milligram of wet tissue (mU/mg) indicating that 1 nmol of substrate is hydrolysed per minute and per milligram of wet tissue. 2.3. Sedimentation analysis Possible differences between ANCT and LSCC in the molecular distribution of AChE were tested through sedimentation analysis with sucrose gradients as reported previously [8]. Briefly, the samples and sedimentation markers (bovine liver catalase and intestine alkaline phosphatase) were layered on the top of centrifuge tubes containing 5–20% sucrose gradients in the presence of 0.5% w/v Brij 96 detergent. The gradient tubes were centrifuged at 35,000 rpm and 4 °C for 18 h with a SW41Ti rotor in a Beckman L-80 OPTIMA XP Ultracentrifuge (Fullerton, CA, USA). After centrifugation, fractions of 250 μl were collected from the tube bottom and assayed for AChE activity and enzyme markers.
2.4. mRNA isolation and real-time PCR Differences between ANCT and LSCC specimens in the expression level of AChE mRNAs were studied by RT-PCR. To achieve this, the mRNA from tissues was extracted using the Chemagic mRNA Direct Kit (Chemagen) and reversed transcribed into cDNA by random priming (GeneAmp RNA PCR Kit, Applied Biosystems). A LightCycler thermocycler (Roche Molecular Biochemicals, Mannheim, Germany) was used for RT-PCR. Pairs of primers were designed for quantitative PCR targeting the 3′-alternative mRNAs of AChE with the following sequences: AChE-T, 5′-AACTTTGCCCGCACAGGGGA-3′ (sense) and 5′GCCTCGTCGAGCGTGTCGGT-3′ (antisense); AChE-H, 5′-AACTTTGCCC GCACAGGGGA-3′ (sense) and 5′-GGGAGCCTCCGAGGCGGT-3′ (antisense); AChE-R, 5′-CCCCTGGACCCCTCTCGAAAC-3′ (sense) and 5′ACCTGGCGGGCTCCCACTC-3′ (antisense); and BChE, 5′-TGCAAAATAT GGGAATCCAAA-3′ (sense) and 5′-CCACTCCCATTCTGCTTCAT-3′ (antisense). β-actin mRNA was used as a housekeeper gene with the primers 5′-AGAAAATCTGGCACCACACC-3′ (sense) and 5′-GGGGTGTTGAAGGT CTCAAA-3′ (antisense). The reaction conditions were validated separately for each pair of primers, and a single dissociation curve peak was produced in each reaction run. The buffered medium contained 5 μL of variable dilutions of cDNA, 0.3 μM specific primers, and an appropriate volume of PCR master mix to obtain a final volume of 20 μL. The reactions comprised a first step of 10 min at 95 °C followed by 40 cycles of 10 s to 95 °C, 10 s at 60 °C, and 15 s at 72 °C. A final dissociation stage allowed us to study the melting curves. The relative content of cDNAs with respect to β-actin cDNA was determined by the second derivative method with kinetic PCR efficiency correction. The PCR products were separated in 2% agarose gels and visualized with ethidium bromide to ensure that their lengths coincided with the expected size. Negative controls (without reverse transcriptase) for each primer pair were also prepared. The relative mRNA amounts are given as the numbers of copies per million of the β-actin mRNA copies.
2.5. Statistical analysis The results are given as the means ± SEM from 50 paired ANCT and LSCC samples performed at least in triplicate. The numerical data were analysed for statistical significance using the Wilcoxon signed-rank test. The statistical significance of the differences in the mean values was set to p b 0.05. Kaplan–Meier curves were constructed to estimate the overall survival, and the differences between the curves were assessed using a two-tailed log-rank test. A difference with p b 0.05 was considered to be statistically significant. The data were analysed using the SPSS software, version 11.5 (SPSS Inc., Chicago, IL, USA).
Table 1 Summary of demographic characteristics of LSCC patients and AChE activity. Samples
Total Age b60 N60 Tobacco Non-smoker Former-smoker Active-Smoker Alcohol No Yes Location Glottis Supraglottis
AChE activity (mU/mg tissue) N
ANCT
LSCC
P-value
50
0.248 ± 0.030
0.157 ± 0.024
0.009
15 35
0.299 ± 0.069 0.214 ± 0.029
0.192 ± 0.045 0.170 ± 0.031
0.016 0.066
5 23 18
0.272 ± 0.092 0.158 ± 0.025 0.222 ± 0.059
0.221 ± 0.056 0.204 ± 0.042 0.148 ± 0.040
0.347 0.750 0.002
16 15
0.157 ± 0.025 0.340 ± 0.083
0.193 ± 0.062 0.129 ± 0.030
0.305 0.047
29 21
0.226 ± 0.039 0.275 ± 0.032
0.167 ± 0.029 0.141 ± 0.023
0.020 0.005
LSCC, larynx squamous cell carninoma. ANCT, adjacent non-cancerous tissue. Statistically significant differences are in bold.
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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3. Results 3.1. Characteristics of patients Fifty patients participated in this research study (Table 1). They were grouped according to age, lifestyle risk (tobacco exposure and alcohol consumption) and anatomical tumour location. The age of the patients ranged from 24 to 89 years with a mean ± SD of 60.02 ± 10.20. Most of the patients were male (46 out of 50). Current or former smokers constituted 82% of the patients. The percentage of patients with glottic carcinomas was 58% (29 out of 50), and the remaining patients (42%; 21 out of 50) had supraglottic tumours. 3.2. AChE activity was decreased in larynx squamous cell carcinomas The reports of the presence and pathophysiological importance of non-neuronal cholinergic components in human airways [7] prompted us to examine the activity and expression of AChE in human larynx tissues. Because AChE is the main enzyme for regulating the ACh levels and therefore for controlling cholinergic signalling, the AChE activity levels in ANCT and LSCC samples were compared. The determination of AChE activity (0.248 ± 0.030 mU/mg) in non-cancerous tissues (ANCT) demonstrated that the upper respiratory tract expressed sufficient ACh-degrading enzymatic activity to regulate its availability. In cancerous tissues (LSCC), the activity of AChE decreased to 0.157 ± 0.045 mU/mg (p = 0.009; Table 1). The results showed that the AChE activity in LSCC was significantly lower relative to that in ANCT in the smokers group (p = 0.002) and alcohol drinking group (p = 0.047) (Table 1). The AChE activity was analysed according to the anatomical location of the tumours. The glottic cancer tissues (glottic squamous cell carcinoma; GSCC) expressed 0.167 ± 0.029 mU/mg which is significantly lower than control tissue (0.226 ± 0.039 mU/mg of AChE in glottic ANCT), and the cancerous supraglottic tissues (supraglottic squamous cell carcinoma; SGSCC) contained 0.141 ± 0.023 mU/mg vs. 0.275 ± 0.032 mU/mg of AChE in supraglottic ANCT. The AChE activity in both cancerous tissues decreased compared with their non-cancerous adjacent tissues (p = 0.020 and p = 0.005 for GSCC and SGSCC, respectively) (Table 1). 3.3. Molecular distribution of AChE in laryngeal epithelium Because the AChE molecular forms are frequently altered under pathological conditions, the AChE molecules in the human larynx were analysed to assess the existence of any cancer-specific change in the AChE molecular pattern. Sedimentation analysis revealed abundant ≈4.4 S AChE forms and fewer ≈9.7 S and ≈3.0 S components in the ANCT and LSCC samples (Fig. 1). According to previous data [25], the major 4.4S forms were assigned to amphiphilic AChE dimers (G2A), which most likely consist of GPI-linked AChE and arise from AChE–H mRNA. The 3.0S forms were attributed to GPI-bound G1A AChE, and the 9.7S forms were attributed to PRiMA bound tetramers (G4A), consisting of four AChE–T subunits bonded to PRiMA. The distribution of AChE forms was similar in ANCT samples with the exception of G4A AChE, which was very predominantly found in supraglottic tissues. Neither the glottic or supraglottic tumours analysed contained AChE tetramers (Fig. 1). 3.4. Gene expression of AChE in laryngeal carcinomas A downregulation of the AChE gene produced by the downregulation of either the three alternative AChE mRNAs or only one of them could explain the lower AChE activity found in LSCC. This possibility was assessed by measuring the AChE mRNA levels in unaffected and cancerous samples. The data showed that healthy larynx samples contained mainly AChE–T mRNAs and less AChE–H mRNA. AChE-R mRNA was very scarce with levels comparable to those found for
Fig. 1. Sedimentation profiles depicting the AChE components in the larynx epithelium. Molecular forms of AChE in unaffected (circles) and cancerous tissues (squares) in glottis (GSCC) and supraglottis (SGSCC) areas.
BChE mRNA. The levels of the three AChE mRNA variants were studied according to the anatomical location. As shown in Fig. 2, glottic cancer tissues underwent a marked decrease in the AChE–T mRNA level, whereas the cancerous supraglottic tissues exhibited unchanged or increased amounts of AChE–T mRNA (Fig. 2). It is worth noting the increased level of BChE mRNA was found in supraglottic cancer tissues relative to ANCT or glottic carcinomas (Fig. 2). 3.5. Survival and cholinesterase activity To test whether AChE activity may be of clinical use as a prognostic marker, the overall (OS) survival rate was studied. The median followup time was 36.60 months (range 6–60 months), and a cut-off value of the 50th percentile of AChE (0.087 mU/mg tissue) was set for the comparisons of the activity values in tumours with OS rates. The results indicated that larynx tumours with AChE activity below 0.087 mU/mg were statistically associated with poorer OS (p = 0.031; Fig. 3A). Despite the early mortality found in patients with both glottic and supraglottic cancers and lower tumoural AChE activity, no significant differences in the OS were found. This lack of statistically significant differences is likely due to the small number of patients with tumours in different anatomical locations included in the study. 4. Discussion Our study provides evidence that the human upper airway truck epithelium expresses active AChE. First evidence about the epithelial non-neuronal cholinergic system in the airways was presented by Reinheimer et al. [4,5]. These and more recent observations [6,7] lend
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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Fig. 2. Histograms showing differences between unaffected and cancerous samples in the levels of the distinct AChE mRNA variants and the single BChE transcript. The mRNA from adjacent non-cancerous tissues (ANCT) and larynx squamous cell carcinomas (LSCCs) was extracted, retro-transcribed and amplified using the primers indicated in Materials and Methods. β-actin mRNA was used as the housekeeping marker. AChE–T, AChE–H, and AChE–R stand for tailed (synaptic) AChE, hydrophobic (erythrocytic) AChE, and readthrough AChE, respectively. AChE and BChE mRNA levels in the larynx (LSCC) and noncancerous adjacent tissues (ANCT) pieces (A) and in tumours located in glottis (GSCC, B) or supraglottis areas (SGSCC, C). Note the lower AChE T mRNA level (p = 0.046) and higher BChE mRNA level (p = 0.029) in LSCC compared with ANCT samples (A) and the opposite changes in the AChE–T mRNA level found in glottis (GSCC, B) and supraglottic (SGSCC, C) tumours.
strong support to the presence of a physiologically active NNCS in the larynx epithelium. This NNCS is expected to be crucial for the precise and reliable control of the intensity and duration of cholinergic inputs and downstream events, including cell growth and proliferation. AChE is involved in at least two key processes of tumour progression. One refers to the required control of the ACh level to prevent augmented
Fig. 3. Kaplan–Meier estimated overall survival (OS) rates according to the AChE activity values. The tumours (n = 50) were split into those that exhibited higher or lower values than the 50th percentile for AChE activity in cancerous tissues (0.087 mU/mg wet tissue; A). Low tumour AChE activity was found to be statistically associated with adverse OS rates (p = 0.031). Despite the early mortality of patients with lower tumoural AChE activity obtained when the tumours were analysed according to location, the OS rates for both glottis (0.082 mU/mg tissue as 50th percentile; B) and supraglottis areas (0.129 mU/mg tissue as 50th percentile; C) failed to reach statistical significance.
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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cellular proliferation linked to the overstimulation of ACh receptors [6]. In contrast, the AChE enzyme enhances apoptosis [21] in a process regulated by miR-212 [27]. In spite of the abundant information on cancer-associated changes in the AChE gene, AChE activity, assembly of AChE subunits, and processing of their linked oligoglycans in human breast, lymph node, gut, lung, prostate and kidney tumours [8,23,28], conclusive proofs of a causal relationship between tumour progression and AChE changes are still lacking. Nevertheless, several observations have lent weight to the possible role of AChE in tumour biology. One of them refers to the relationship between human astrocytoma aggressiveness and altered alternative splicing-derived AChE variants [29]. At this respect, it is interesting to note the opposite results for the association between AChE immunostaining and survival rate of patients with ovarian cancer [30] and non-small cell lung cancer [27], and the shorter disease-free and overall survival rates of patients with low AChE-activity-expressing liver carcinomas [24]. The AChE activity in human non-cancerous larynx epithelium is lower than that found in lymph nodes [28] and brain tumours [20], roughly the same as that in the lung [8] and greater than that in the breast [31], prostate [26], colon [32] and kidney [25]. Tissues with high AChE activity such as the lung [8] and bladder (unpublished results) are those that exhibit greater cancer-related decreases in AChE activity and a higher smoking-related risk for cancer [33,34]. There are no conclusive proofs of a causal relationship of AChE changes with tumour behaviour, but this possibility cannot be ruled out. The significant AChE activity found in healthy larynx tissue (Table 1) demonstrate that the tissue is able to regulate the available ACh and, therefore, cholinergic signalling. Moreover, the decrease in AChE activity with cancer and the differences in other important ACh-related proteins [8] between non-cancerous and malignant epithelia lend strong support to the notion that alterations in the NNCS may modulate cancer biology. The heretofore unnoticed observation that patients carrying larynx cancer expressing low AChE have a poorer survival rate (Fig. 3) is very similar to the case of patients with hepatocellular carcinoma for whom a low level of AChE activity is correlated with an increased risk of post-operative recurrence [24] and patients with non-small cell lung cancer for whom a low level of AChE protein is correlated with a decreased survival rate [27]. These results strengthen the idea of a link between low ACh-hydrolysing activity and/or AChE protein and tumour growth in some cancers. Because of the heterogeneity in AChE levels between different tumours, the role of AChE should be specific for each type of cancer. In addition, the results shown in Table 1 suggest a probable causal relationship between cancer-promoting lifestyles (smoking or alcohol intake) and decreases in AChE activity. Our results support the notion that a lower AChE activity in LSCC is associated with poorer prognosis, likely due to cholinergic overactivation arising from an increased level of ACh in the neighbourhood of cancerous cells. The function of BChE remains a puzzle. It has been suggested that BChE acts as a scavenging enzyme in the detoxification of natural compounds [35]. BuChE is also able to hydrolyse ACh but much less efficiently than AChE. Therefore, BChE could partially compensate for the drop of AChE in tumour tissues as occurs in AChE knockout mice [36]. The presence in both unaffected and malignant respiratory tract epithelia of principal amphiphilic AChE dimers (G2A), most likely consisting of GPI-linked AChE (Fig. 1), agrees with the molecular profiles observed in studies of unaffected and cancerous epithelial tissues [8,25,31]. The predominance in epithelial tissues and blood cells of GPI-linked G2A AChE, which arises from AChE–H mRNA, reinforces the hypothesis that the AChE–H mRNA variant is the principal source (if not the unique) of AChE activity in the airway epithelium and other non-nervous tissues [37]. Interestingly, the AChE gene expression was found to vary in larynx cancer according to the anatomical location of the tumours. Thus, whilst the supraglottic tumours exhibited either unchanged or even increased
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levels of AChE mRNA, the three alternatively spliced mRNAs of AChE tended to be decreased in glottic tumours (Fig. 2). In addition, the decreased AChE–T mRNA level in glottis-located tumours and its increased level in supraglottis tumours indicated that AChE–T mRNA is not likely the leading transcript for AChE activity in unaffected and cancerous pieces. Instead, the decreased AChE–H mRNA levels in glottis and supraglottis tumours strongly support the proposal of AChE–H mRNA as the principal source of enzyme activity in non neural peripheral tissues [37]. Moreover, the opposite changes in the AChE–T mRNA levels in glottis and supraglottis tumours suggest that the particular environment surrounding tumour cells may determine the transcriptional and post-transcriptional events in the biology of AChE. Post-translational events, including conversion of catalytically incompetent into competent subunits, oligomerization, and rapid secretion of AChE–T made oligomers may explain the lack of correlation between the discrepant levels of AChE–T mRNA and of AChE hydrolyzing activity found in supraglottis tumours (Table 1). If AChE–T mRNA is the source of a catalytically incompetent AChE protein needed for apoptosis, the lack of correlation between the discrepant levels of AChE–T mRNA and AChE hydrolysing activity may be explained. Several studies hint at a causal relationship of cholinergic activation with tumour growth in different cancers (review in [4]). Interestingly, the differences between smokers and non-smokers in the gene expression of nicotinic receptor subunits in the airway epithelium [10] firmly support the possibility that the tobacco components drive phenotypic changes favouring proliferation and tumour progression. Thus, it is tempting to speculate that daily and persistent nicotine/nitrosamine exposure may lead to changes in the expression of ACh-related proteins in favour of the formation of a pro-tumorigenic non-neuronal cholinergic system in airway epithelia. The feeding of the system by endogenous ACh would explain the high risk of tumourigenesis that former smokers show long after smoking cessation. The combination of increased nicotine-induced ACh synthesis and decreased degradation due to down-expressed ChEs can result in an increase in available ACh in the tumour and in an increase in proliferative stimuli to both ‘tumoral’ mAChR and nAChR. If this hypothesis is proven, it may be especially relevant for the carcinogenesis of smoking-related cancer. To summarize, the fact that larynx carcinomas expressing low AChE activity exhibit a poor prognosis raises the possibility of using the AChE activity measurement as a reliable prognosis predictor. Any new biomarker for the assessments of smoking-related cancer risk as well as for targeted therapy must be a priority in cancer research. Abbreviations LSCC larynx squamous cell carcinoma GSCC glottic squamous cell carcinoma SGSCC supraglottic squamous cell carcinoma ANCT adjacent non-cancerous tissue ACh acetylcholine AChE acetylcholinesterase BChE butyrylcholinesterase nAChR nicotinic acetylcholine receptor mAChR muscarinic acetylcholine receptor Competing interests The authors declare that they have no competing interests. Acknowledgements This work was supported in part by grants from FIS (Project 01/3025), MINECO (Projects SAF2006-070040-C02-01 and SAF2006-070040-C0202) and Fundación Séneca de la Región de Murcia (Project 10/15265). A.C.C-G and S.N-C were granted by FFIS (Murcia). J.N.R-L. was supported by a grant from Ministerio de Economia y Competitividad (MINECO) (SAF2013-48375-C2-1-R).
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma Ana Cristina Castillo-González a,1, Juan Pablo Pelegrín-Hernández b,1, Susana Nieto-Cerón a, Antonio Piñero Madrona c, José Antonio Noguera a, María Fuensanta López-Moreno a, José Neptuno Rodríguez-López d, Cecilio J. Vidal d, Diego Hellín-Meseguer b,⁎, Juan Cabezas-Herrera a,⁎ a
Molecular Therapy and Biomarkers Research Group, Clinical Analysis Service, University Hospital Virgen de la Arrixaca, IMIB-Arrixaca, Ctra Madrid-Cartagena s/n, El Palmar, 30120 Murcia, Spain Otorhinolaryngology Surgical Service, University Hospital Virgen de la Arrixaca IMIB-Arrixaca, Ctra Madrid-Cartagena s/n, El Palmar, 30120 Murcia, Spain Surgery Service of University Hospital Virgen de la Arrixaca IMIB, Ctra Madrid-Cartagena s/n, El Palmar, 30120 Murcia, Spain d Department of Biochemistry and Molecular Biology A, School of Biology, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, 30100 Murcia, Spain b c
a r t i c l e
i n f o
Article history: Received 27 February 2015 Received in revised form 4 May 2015 Accepted 7 May 2015 Available online xxxx Keywords: Cholinergic system Non-neuronal compartment Human airways Larynx cancer
a b s t r a c t Previous reports have demonstrated that a non-neuronal cholinergic system is expressed aberrantly in airways. A proliferative effect is exerted directly by cholinergic agonists through the activation of nicotinic and muscarinic receptors. In cancer, particularly those related with smoking, the mechanism through which tumour cells respond to aberrantly activated cholinergic signalling is a key question. Fifty paired pieces of larynx squamous cell carcinoma and adjacent non-cancerous tissue were compared in terms of their acetylcholinesterase activity (AChE). The AChE activity in non-cancerous tissues (0.248 ± 0.030 milliunits per milligram of wet tissue; mU/mg) demonstrates that upper respiratory tissues express sufficient AChE activity for controlling the level of acetylcholine (ACh). In larynx carcinomas, the AChE activity decreased to 0.157 ± 0.024 mU/mg (p = 0.009). Larynx cancer patients exhibiting low ACh-degrading enzymatic activity had a significantly shorter overall survival (p = 0.031). Differences in the mRNA levels of alternatively spliced AChE isoforms and molecular compositions were noted between glottic and supraglottic cancers. Our results suggest that the low AChE activity observed in larynx squamous cell carcinoma may be useful for predicting the outcome of patients. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Head and neck cancer arise in the mucosal layer of the upper aerodigestive tract (oral cavity, oropharynx, hypopharynx, and larynx). Nearly 90% of head and neck carcinomas are squamous cell carcinomas. Head and neck neoplasia is the sixth most frequent cancer, with more than 600,000 new cases reported worldwide each year [1,2]. Larynx cancer is the second most common type of cancer among all head and neck cancers. At the early stage, patients with larynx carcinoma can be cured with multimodal therapy (surgery, radiation, and/or chemotherapy). Unfortunately, no fully satisfactory treatment has yet been developed, and therefore, the mortality rate of larynx carcinoma patients remains high [3]. Reliable biomarkers for distinguishing patients with poor prognosis or risk of early recurrence and for using personalized therapies are still awaited given the uncertainty of the clinical evolution of larynx carcinoma using the current staging criteria.
⁎ Corresponding authors. Tel.: +34 968 369441. E-mail addresses: [email protected] (D. Hellín-Meseguer), [email protected] (J. Cabezas-Herrera). 1 These authors contributed equally to this work.
An increasing body of evidence notes that several cell types are capable of expressing the range of proteins that form a non-neuronal cholinergic system (NNCS), i.e., the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT), nicotinic (nAChR) and muscarinic (mAChR) receptors, and the ACh-hydrolysing enzymes acetyl(AChE) and butyrylcholinesterase (BChE) [4–7]. A specific cholinergic phenotype depending on the cell type could participate in processes that define a correct tissue physiology. It is worth noting that the results demonstrate that the human respiratory tract epithelium possesses a NNCS engaged in controlling the level of ACh. It appears that this epithelial cholinergic system operates actively to regulate auto/ paracrine actions and thereby reliably controls basic cell functions [8]. The proliferative effects arising from cholinergic overactivation have gained basic and translational significance. Of importance is the susceptibility to lung cancer that confers AChR disorders [9] as well as the nicotine-guided shift in the expression of ACh-related proteins to proliferating/migrating cell phenotypes [10]. It is also worth mentioning that promising therapies based in the blockade or attenuation of cholinergic signalling are under investigation [11–13]. The classical function of AChE is to terminate cholinergic transmission through a very efficient hydrolysis of ACh. There are three main alternative splicing forms of AChE: synaptic or tailed AChE (AChE–T),
http://dx.doi.org/10.1016/j.intimp.2015.05.011 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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erythrocyte or hydrophobic AChE (AChE–H) and read-through AChE (AChE–R). In addition to the hydrolysis of neurotransmitters, AChE has several non-classical roles related with important cell processes such as proliferation, differentiation, apoptosis and cell–cell recognition [14]. The studies on the active involvement of cholinesterases in cell proliferation and differentiation [15] indicate that it is possible that AChE and, to a lower extent, BChE collaborate to tumour development and dissemination. The following examples support this idea: the frequent aberrations in the AChE gene and the structural changes in AChE proteins observed in tumours of diverse origin [8,16–20], the expression of AChE upon apoptosis induction with different stimuli [21–23], and the profitable use of AChE as a prognostic predictor for liver carcinoma and its profitable effects through the suppression of cell growth and the enhancement of chemosensitization [24]. Studies in our laboratory have found that cancer affects the level of AChE activity and/or the content of alternatively spliced mRNAs in the human breast, lymph node, intestine, lung, kidney and prostate. Due to the lack of specific and sensitive biomarkers and tools for early diagnosis, cancer in airways is diagnosed at advanced stages [8,19,25,26]. The aim of this research study was to explore possible changes in the expression of AChE in laryngeal tumours and to test the usefulness of these changes as reliable diagnostic or prognostic markers. 2. Material and methods 2.1. Patients and samples A total of 50 human malignant primary larynx squamous cell carcinomas (LSCCs) and their adjacent noncancerous tissues (ANCT) collected through surgeries at Virgen de la Arrixaca Clinical University Hospital in Murcia (Spain) from 2007 to 2012 were included in the current study. Fresh specimens were divided into sections and stored at −80 °C until use. The TNM classification of LSCC specimens was made according to the UICC:TNM Classification of Malignant Tumors. Study approval and the consent procedure were obtained from the Institutional Ethic Committee of our Hospital. All of the patients gave their consent after being appropriately informed. 2.2. Extraction and assay of acetylcholinesterase AChE was extracted from surgical ANCT and LSCC pieces through homogenization (5% w/v) with Tris-saline buffer (TSB; 1 M NaCl, 50 mM MgCl2, 3 mM EDTA, 10 mM Tris, pH 7.0) supplemented with 1% Brij 96 and a fresh mixture of proteinase inhibitors (0.1 mg/ml soybean trypsin inhibitor, 1 mg/ml bacitracin, 0.0022 TIU/ml aprotinin, 10 mg/ml pepstatin A and 20 mg/ml leupeptin). After centrifugation at 30,000 rpm and 4 °C for 1 h with a 70Ti Beckman rotor (Palo Alto, CA, USA), the supernatant with AChE was saved. The AChE activity was determined as described previously [8]. AChE activity is given in milliunits per milligram of wet tissue (mU/mg) indicating that 1 nmol of substrate is hydrolysed per minute and per milligram of wet tissue. 2.3. Sedimentation analysis Possible differences between ANCT and LSCC in the molecular distribution of AChE were tested through sedimentation analysis with sucrose gradients as reported previously [8]. Briefly, the samples and sedimentation markers (bovine liver catalase and intestine alkaline phosphatase) were layered on the top of centrifuge tubes containing 5–20% sucrose gradients in the presence of 0.5% w/v Brij 96 detergent. The gradient tubes were centrifuged at 35,000 rpm and 4 °C for 18 h with a SW41Ti rotor in a Beckman L-80 OPTIMA XP Ultracentrifuge (Fullerton, CA, USA). After centrifugation, fractions of 250 μl were collected from the tube bottom and assayed for AChE activity and enzyme markers.
2.4. mRNA isolation and real-time PCR Differences between ANCT and LSCC specimens in the expression level of AChE mRNAs were studied by RT-PCR. To achieve this, the mRNA from tissues was extracted using the Chemagic mRNA Direct Kit (Chemagen) and reversed transcribed into cDNA by random priming (GeneAmp RNA PCR Kit, Applied Biosystems). A LightCycler thermocycler (Roche Molecular Biochemicals, Mannheim, Germany) was used for RT-PCR. Pairs of primers were designed for quantitative PCR targeting the 3′-alternative mRNAs of AChE with the following sequences: AChE-T, 5′-AACTTTGCCCGCACAGGGGA-3′ (sense) and 5′GCCTCGTCGAGCGTGTCGGT-3′ (antisense); AChE-H, 5′-AACTTTGCCC GCACAGGGGA-3′ (sense) and 5′-GGGAGCCTCCGAGGCGGT-3′ (antisense); AChE-R, 5′-CCCCTGGACCCCTCTCGAAAC-3′ (sense) and 5′ACCTGGCGGGCTCCCACTC-3′ (antisense); and BChE, 5′-TGCAAAATAT GGGAATCCAAA-3′ (sense) and 5′-CCACTCCCATTCTGCTTCAT-3′ (antisense). β-actin mRNA was used as a housekeeper gene with the primers 5′-AGAAAATCTGGCACCACACC-3′ (sense) and 5′-GGGGTGTTGAAGGT CTCAAA-3′ (antisense). The reaction conditions were validated separately for each pair of primers, and a single dissociation curve peak was produced in each reaction run. The buffered medium contained 5 μL of variable dilutions of cDNA, 0.3 μM specific primers, and an appropriate volume of PCR master mix to obtain a final volume of 20 μL. The reactions comprised a first step of 10 min at 95 °C followed by 40 cycles of 10 s to 95 °C, 10 s at 60 °C, and 15 s at 72 °C. A final dissociation stage allowed us to study the melting curves. The relative content of cDNAs with respect to β-actin cDNA was determined by the second derivative method with kinetic PCR efficiency correction. The PCR products were separated in 2% agarose gels and visualized with ethidium bromide to ensure that their lengths coincided with the expected size. Negative controls (without reverse transcriptase) for each primer pair were also prepared. The relative mRNA amounts are given as the numbers of copies per million of the β-actin mRNA copies.
2.5. Statistical analysis The results are given as the means ± SEM from 50 paired ANCT and LSCC samples performed at least in triplicate. The numerical data were analysed for statistical significance using the Wilcoxon signed-rank test. The statistical significance of the differences in the mean values was set to p b 0.05. Kaplan–Meier curves were constructed to estimate the overall survival, and the differences between the curves were assessed using a two-tailed log-rank test. A difference with p b 0.05 was considered to be statistically significant. The data were analysed using the SPSS software, version 11.5 (SPSS Inc., Chicago, IL, USA).
Table 1 Summary of demographic characteristics of LSCC patients and AChE activity. Samples
Total Age b60 N60 Tobacco Non-smoker Former-smoker Active-Smoker Alcohol No Yes Location Glottis Supraglottis
AChE activity (mU/mg tissue) N
ANCT
LSCC
P-value
50
0.248 ± 0.030
0.157 ± 0.024
0.009
15 35
0.299 ± 0.069 0.214 ± 0.029
0.192 ± 0.045 0.170 ± 0.031
0.016 0.066
5 23 18
0.272 ± 0.092 0.158 ± 0.025 0.222 ± 0.059
0.221 ± 0.056 0.204 ± 0.042 0.148 ± 0.040
0.347 0.750 0.002
16 15
0.157 ± 0.025 0.340 ± 0.083
0.193 ± 0.062 0.129 ± 0.030
0.305 0.047
29 21
0.226 ± 0.039 0.275 ± 0.032
0.167 ± 0.029 0.141 ± 0.023
0.020 0.005
LSCC, larynx squamous cell carninoma. ANCT, adjacent non-cancerous tissue. Statistically significant differences are in bold.
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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3. Results 3.1. Characteristics of patients Fifty patients participated in this research study (Table 1). They were grouped according to age, lifestyle risk (tobacco exposure and alcohol consumption) and anatomical tumour location. The age of the patients ranged from 24 to 89 years with a mean ± SD of 60.02 ± 10.20. Most of the patients were male (46 out of 50). Current or former smokers constituted 82% of the patients. The percentage of patients with glottic carcinomas was 58% (29 out of 50), and the remaining patients (42%; 21 out of 50) had supraglottic tumours. 3.2. AChE activity was decreased in larynx squamous cell carcinomas The reports of the presence and pathophysiological importance of non-neuronal cholinergic components in human airways [7] prompted us to examine the activity and expression of AChE in human larynx tissues. Because AChE is the main enzyme for regulating the ACh levels and therefore for controlling cholinergic signalling, the AChE activity levels in ANCT and LSCC samples were compared. The determination of AChE activity (0.248 ± 0.030 mU/mg) in non-cancerous tissues (ANCT) demonstrated that the upper respiratory tract expressed sufficient ACh-degrading enzymatic activity to regulate its availability. In cancerous tissues (LSCC), the activity of AChE decreased to 0.157 ± 0.045 mU/mg (p = 0.009; Table 1). The results showed that the AChE activity in LSCC was significantly lower relative to that in ANCT in the smokers group (p = 0.002) and alcohol drinking group (p = 0.047) (Table 1). The AChE activity was analysed according to the anatomical location of the tumours. The glottic cancer tissues (glottic squamous cell carcinoma; GSCC) expressed 0.167 ± 0.029 mU/mg which is significantly lower than control tissue (0.226 ± 0.039 mU/mg of AChE in glottic ANCT), and the cancerous supraglottic tissues (supraglottic squamous cell carcinoma; SGSCC) contained 0.141 ± 0.023 mU/mg vs. 0.275 ± 0.032 mU/mg of AChE in supraglottic ANCT. The AChE activity in both cancerous tissues decreased compared with their non-cancerous adjacent tissues (p = 0.020 and p = 0.005 for GSCC and SGSCC, respectively) (Table 1). 3.3. Molecular distribution of AChE in laryngeal epithelium Because the AChE molecular forms are frequently altered under pathological conditions, the AChE molecules in the human larynx were analysed to assess the existence of any cancer-specific change in the AChE molecular pattern. Sedimentation analysis revealed abundant ≈4.4 S AChE forms and fewer ≈9.7 S and ≈3.0 S components in the ANCT and LSCC samples (Fig. 1). According to previous data [25], the major 4.4S forms were assigned to amphiphilic AChE dimers (G2A), which most likely consist of GPI-linked AChE and arise from AChE–H mRNA. The 3.0S forms were attributed to GPI-bound G1A AChE, and the 9.7S forms were attributed to PRiMA bound tetramers (G4A), consisting of four AChE–T subunits bonded to PRiMA. The distribution of AChE forms was similar in ANCT samples with the exception of G4A AChE, which was very predominantly found in supraglottic tissues. Neither the glottic or supraglottic tumours analysed contained AChE tetramers (Fig. 1). 3.4. Gene expression of AChE in laryngeal carcinomas A downregulation of the AChE gene produced by the downregulation of either the three alternative AChE mRNAs or only one of them could explain the lower AChE activity found in LSCC. This possibility was assessed by measuring the AChE mRNA levels in unaffected and cancerous samples. The data showed that healthy larynx samples contained mainly AChE–T mRNAs and less AChE–H mRNA. AChE-R mRNA was very scarce with levels comparable to those found for
Fig. 1. Sedimentation profiles depicting the AChE components in the larynx epithelium. Molecular forms of AChE in unaffected (circles) and cancerous tissues (squares) in glottis (GSCC) and supraglottis (SGSCC) areas.
BChE mRNA. The levels of the three AChE mRNA variants were studied according to the anatomical location. As shown in Fig. 2, glottic cancer tissues underwent a marked decrease in the AChE–T mRNA level, whereas the cancerous supraglottic tissues exhibited unchanged or increased amounts of AChE–T mRNA (Fig. 2). It is worth noting the increased level of BChE mRNA was found in supraglottic cancer tissues relative to ANCT or glottic carcinomas (Fig. 2). 3.5. Survival and cholinesterase activity To test whether AChE activity may be of clinical use as a prognostic marker, the overall (OS) survival rate was studied. The median followup time was 36.60 months (range 6–60 months), and a cut-off value of the 50th percentile of AChE (0.087 mU/mg tissue) was set for the comparisons of the activity values in tumours with OS rates. The results indicated that larynx tumours with AChE activity below 0.087 mU/mg were statistically associated with poorer OS (p = 0.031; Fig. 3A). Despite the early mortality found in patients with both glottic and supraglottic cancers and lower tumoural AChE activity, no significant differences in the OS were found. This lack of statistically significant differences is likely due to the small number of patients with tumours in different anatomical locations included in the study. 4. Discussion Our study provides evidence that the human upper airway truck epithelium expresses active AChE. First evidence about the epithelial non-neuronal cholinergic system in the airways was presented by Reinheimer et al. [4,5]. These and more recent observations [6,7] lend
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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Fig. 2. Histograms showing differences between unaffected and cancerous samples in the levels of the distinct AChE mRNA variants and the single BChE transcript. The mRNA from adjacent non-cancerous tissues (ANCT) and larynx squamous cell carcinomas (LSCCs) was extracted, retro-transcribed and amplified using the primers indicated in Materials and Methods. β-actin mRNA was used as the housekeeping marker. AChE–T, AChE–H, and AChE–R stand for tailed (synaptic) AChE, hydrophobic (erythrocytic) AChE, and readthrough AChE, respectively. AChE and BChE mRNA levels in the larynx (LSCC) and noncancerous adjacent tissues (ANCT) pieces (A) and in tumours located in glottis (GSCC, B) or supraglottis areas (SGSCC, C). Note the lower AChE T mRNA level (p = 0.046) and higher BChE mRNA level (p = 0.029) in LSCC compared with ANCT samples (A) and the opposite changes in the AChE–T mRNA level found in glottis (GSCC, B) and supraglottic (SGSCC, C) tumours.
strong support to the presence of a physiologically active NNCS in the larynx epithelium. This NNCS is expected to be crucial for the precise and reliable control of the intensity and duration of cholinergic inputs and downstream events, including cell growth and proliferation. AChE is involved in at least two key processes of tumour progression. One refers to the required control of the ACh level to prevent augmented
Fig. 3. Kaplan–Meier estimated overall survival (OS) rates according to the AChE activity values. The tumours (n = 50) were split into those that exhibited higher or lower values than the 50th percentile for AChE activity in cancerous tissues (0.087 mU/mg wet tissue; A). Low tumour AChE activity was found to be statistically associated with adverse OS rates (p = 0.031). Despite the early mortality of patients with lower tumoural AChE activity obtained when the tumours were analysed according to location, the OS rates for both glottis (0.082 mU/mg tissue as 50th percentile; B) and supraglottis areas (0.129 mU/mg tissue as 50th percentile; C) failed to reach statistical significance.
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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cellular proliferation linked to the overstimulation of ACh receptors [6]. In contrast, the AChE enzyme enhances apoptosis [21] in a process regulated by miR-212 [27]. In spite of the abundant information on cancer-associated changes in the AChE gene, AChE activity, assembly of AChE subunits, and processing of their linked oligoglycans in human breast, lymph node, gut, lung, prostate and kidney tumours [8,23,28], conclusive proofs of a causal relationship between tumour progression and AChE changes are still lacking. Nevertheless, several observations have lent weight to the possible role of AChE in tumour biology. One of them refers to the relationship between human astrocytoma aggressiveness and altered alternative splicing-derived AChE variants [29]. At this respect, it is interesting to note the opposite results for the association between AChE immunostaining and survival rate of patients with ovarian cancer [30] and non-small cell lung cancer [27], and the shorter disease-free and overall survival rates of patients with low AChE-activity-expressing liver carcinomas [24]. The AChE activity in human non-cancerous larynx epithelium is lower than that found in lymph nodes [28] and brain tumours [20], roughly the same as that in the lung [8] and greater than that in the breast [31], prostate [26], colon [32] and kidney [25]. Tissues with high AChE activity such as the lung [8] and bladder (unpublished results) are those that exhibit greater cancer-related decreases in AChE activity and a higher smoking-related risk for cancer [33,34]. There are no conclusive proofs of a causal relationship of AChE changes with tumour behaviour, but this possibility cannot be ruled out. The significant AChE activity found in healthy larynx tissue (Table 1) demonstrate that the tissue is able to regulate the available ACh and, therefore, cholinergic signalling. Moreover, the decrease in AChE activity with cancer and the differences in other important ACh-related proteins [8] between non-cancerous and malignant epithelia lend strong support to the notion that alterations in the NNCS may modulate cancer biology. The heretofore unnoticed observation that patients carrying larynx cancer expressing low AChE have a poorer survival rate (Fig. 3) is very similar to the case of patients with hepatocellular carcinoma for whom a low level of AChE activity is correlated with an increased risk of post-operative recurrence [24] and patients with non-small cell lung cancer for whom a low level of AChE protein is correlated with a decreased survival rate [27]. These results strengthen the idea of a link between low ACh-hydrolysing activity and/or AChE protein and tumour growth in some cancers. Because of the heterogeneity in AChE levels between different tumours, the role of AChE should be specific for each type of cancer. In addition, the results shown in Table 1 suggest a probable causal relationship between cancer-promoting lifestyles (smoking or alcohol intake) and decreases in AChE activity. Our results support the notion that a lower AChE activity in LSCC is associated with poorer prognosis, likely due to cholinergic overactivation arising from an increased level of ACh in the neighbourhood of cancerous cells. The function of BChE remains a puzzle. It has been suggested that BChE acts as a scavenging enzyme in the detoxification of natural compounds [35]. BuChE is also able to hydrolyse ACh but much less efficiently than AChE. Therefore, BChE could partially compensate for the drop of AChE in tumour tissues as occurs in AChE knockout mice [36]. The presence in both unaffected and malignant respiratory tract epithelia of principal amphiphilic AChE dimers (G2A), most likely consisting of GPI-linked AChE (Fig. 1), agrees with the molecular profiles observed in studies of unaffected and cancerous epithelial tissues [8,25,31]. The predominance in epithelial tissues and blood cells of GPI-linked G2A AChE, which arises from AChE–H mRNA, reinforces the hypothesis that the AChE–H mRNA variant is the principal source (if not the unique) of AChE activity in the airway epithelium and other non-nervous tissues [37]. Interestingly, the AChE gene expression was found to vary in larynx cancer according to the anatomical location of the tumours. Thus, whilst the supraglottic tumours exhibited either unchanged or even increased
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levels of AChE mRNA, the three alternatively spliced mRNAs of AChE tended to be decreased in glottic tumours (Fig. 2). In addition, the decreased AChE–T mRNA level in glottis-located tumours and its increased level in supraglottis tumours indicated that AChE–T mRNA is not likely the leading transcript for AChE activity in unaffected and cancerous pieces. Instead, the decreased AChE–H mRNA levels in glottis and supraglottis tumours strongly support the proposal of AChE–H mRNA as the principal source of enzyme activity in non neural peripheral tissues [37]. Moreover, the opposite changes in the AChE–T mRNA levels in glottis and supraglottis tumours suggest that the particular environment surrounding tumour cells may determine the transcriptional and post-transcriptional events in the biology of AChE. Post-translational events, including conversion of catalytically incompetent into competent subunits, oligomerization, and rapid secretion of AChE–T made oligomers may explain the lack of correlation between the discrepant levels of AChE–T mRNA and of AChE hydrolyzing activity found in supraglottis tumours (Table 1). If AChE–T mRNA is the source of a catalytically incompetent AChE protein needed for apoptosis, the lack of correlation between the discrepant levels of AChE–T mRNA and AChE hydrolysing activity may be explained. Several studies hint at a causal relationship of cholinergic activation with tumour growth in different cancers (review in [4]). Interestingly, the differences between smokers and non-smokers in the gene expression of nicotinic receptor subunits in the airway epithelium [10] firmly support the possibility that the tobacco components drive phenotypic changes favouring proliferation and tumour progression. Thus, it is tempting to speculate that daily and persistent nicotine/nitrosamine exposure may lead to changes in the expression of ACh-related proteins in favour of the formation of a pro-tumorigenic non-neuronal cholinergic system in airway epithelia. The feeding of the system by endogenous ACh would explain the high risk of tumourigenesis that former smokers show long after smoking cessation. The combination of increased nicotine-induced ACh synthesis and decreased degradation due to down-expressed ChEs can result in an increase in available ACh in the tumour and in an increase in proliferative stimuli to both ‘tumoral’ mAChR and nAChR. If this hypothesis is proven, it may be especially relevant for the carcinogenesis of smoking-related cancer. To summarize, the fact that larynx carcinomas expressing low AChE activity exhibit a poor prognosis raises the possibility of using the AChE activity measurement as a reliable prognosis predictor. Any new biomarker for the assessments of smoking-related cancer risk as well as for targeted therapy must be a priority in cancer research. Abbreviations LSCC larynx squamous cell carcinoma GSCC glottic squamous cell carcinoma SGSCC supraglottic squamous cell carcinoma ANCT adjacent non-cancerous tissue ACh acetylcholine AChE acetylcholinesterase BChE butyrylcholinesterase nAChR nicotinic acetylcholine receptor mAChR muscarinic acetylcholine receptor Competing interests The authors declare that they have no competing interests. Acknowledgements This work was supported in part by grants from FIS (Project 01/3025), MINECO (Projects SAF2006-070040-C02-01 and SAF2006-070040-C0202) and Fundación Séneca de la Región de Murcia (Project 10/15265). A.C.C-G and S.N-C were granted by FFIS (Murcia). J.N.R-L. was supported by a grant from Ministerio de Economia y Competitividad (MINECO) (SAF2013-48375-C2-1-R).
Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
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Please cite this article as: A.C. Castillo-González, et al., Unbalanced acetylcholinesterase activity in larynx squamous cell carcinoma, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.05.011
