Original Article Expression and Prognostic Significance of MCM-3 and MCM-7 in Salivary Adenoid Cystic Carcinoma

Int J Clin Exp Pathol 2018;11(11):5359-5369 www.ijcep.com /ISSN:1936-2625/IJCEP0085911

Original Article Expression and prognostic significance of MCM-3 and MCM-7 in salivary adenoid cystic carcinoma

Qing-Liang Wen1,2,3*, Sen-Miao Zhu5*, Lie-Hao Jiang2,3*, Fang-Yue Xiang6, Wen-Juan Yin4, Yang-Yang Qian1,2,3, Yu-Qing Huang3,7, Ke-Xin Yin7, Xin Zhu8, Ming-Hua Ge1,2,3

1The First Affiliated Hospital of Wenzhou Medical University, Ouhai District, Wenzhou, China; 2Department of Thyroid and Breast Surgery, Zhejiang Provincial People’s Hospital, Gongshu District, Hangzhou, China; Depart- ments of 3Head and Neck Surgery, 4Pathology, Zhejiang Cancer Hospital, Gongshu District, Hangzhou, China; 5The Second Affiliated Hospital of Wenzhou Medical University, Lucheng District, Wenzhou, China; 6Stomatology Col- lege, 7Second Clinical Medical College, Zhejiang Chinese Medical University, Binjiang District, Hangzhou, China; 8Zhejiang Cancer Research Institute, Zhejiang Province Cancer Hospital, Gongshu District, Hangzhou, China. *Equal contributors. Received September 24, 2018; Accepted October 25, 2018; Epub November 1, 2018; Published November 15, 2018

Abstract: This study sought to investigate minichromosome maintenance protein 3 (MCM3) and minichromosome maintenance protein 7 (MCM7) expression in salivary adenoid cystic carcinoma (SACC) samples, and to evaluate the relationship between clinicopathological characteristics and prognosis. The expressions of MCM3 and MCM7 were evaluated using immunohistochemistry of tissue sections from SACC patients, and statistical analyses were performed to evaluate the associations between MCM expression and clinicopathological variables and to analyze the disease-free survival (DFS) and prognostic factors. The positive expression rates of MCM3 and MCM7 in SACC were 98.8% and 96.6%, respectively. MCM3 expression correlated with T-stage and nerve invasion. MCM7 expression correlated with T-stage, adjacent tissue invasion, nerve invasion, and prognosis, and was negatively associated with DFS. However, there was no significant correlation between MCM3 expression and DFS. A kappa analysis demonstrated that MCM3 was closely associated with MCM7. MCM7 may be a favorable prognosis indicator in SACC.

Keywords: Salivary adenoid cystic carcinoma, MCM3, MCM7, immunochemistry, prognosis

Introduction appears to be the effectiveness of radical surgical resection [4]. Patients who are insensitive Adenoid cystic carcinoma (ACC), while being an to radiotherapy and chemotherapy often have uncommon malignancy, is one of the most com- poor prognoses, developing local recurrence mon salivary gland cancers. ACC accounts for and distant metastases [5]. approximately 10-25% of all salivary gland malignancies [1]. Salivary adenoid cystic carci- In order to more effectively diagnose and treat noma (SACC) is characterized by aggressive this cancer, the molecular mechanism underly- growth, a high rate of recurrence, nerve and ing SACC malignant progression needs to be blood vessel invasion, and frequent lung metas- further explored. The discovery of novel bio- tasis, which lead to a poor outcome [2]. SACC markers associated with metastasis and pro- usually occurs in middle-aged and elderly peo- gression is urgently needed so that drugs for ple (50-60 years). There are currently no obvi- effective targeted molecular therapies against ous risk factors associated with the occurrence SACC may be developed. and development of SACC. In addition, it is unknown whether smoking affects the inci- Tumorigenesis results from abnormal cell prolif- dence of SACC [3]. Until now, treatment for eration caused by cell cycle dysregulation, SACC has involved radical surgical resection which is related to the initiation of DNA replica- with additional radiotherapy and chemothera- tion. Sustained DNA replication plays a crucial py. The most significant factor in prognosis role in the occurrence and development of can- MCM-3 and MCM-7’s role in cystic carcinoma cer [6]. Proliferative proteins related to the cell regulated by, Cyclin D1-dependent kinase cycle, such as the minichromosome mainte- (CDK4), to bind the tumor suppressor protein nance (MCM) nuclear proteins, proliferating cell RB [15]. nuclear antigen (PCNA), and Ki-67, are involved in carcinogenesis [7]. It has been found that Ki67, a commonly-used proliferative marker, is not expressed in cells Members of the MCM protein family have simi- entering G1 from the G0 phase but is only lar molecular structures and biological func- expressed in actively-dividing cells. In contrast tions and include six major isoforms (MCM2-7), to Ki67, MCM is significantly highly expressed which are involved in the initiation and exten- in the G1 and S phases of the cell cycle, so it sion of DNA replication, and which prevent re- can potentially be used to detect cells entering replication in the same cell cycle [8]. It has G1 from the G0 phase. PCNA, another widely been confirmed that the expression level of used marker, is involved not only in DNA repli- MCM proteins is related to the periodicity of the cation, but also in its repair, and may thus be cell cycle, and that they are significantly highly present in large quantities when cells are not expressed in the G1 and S phases. However, actively proliferating. In contrast, MCM proteins the expression level of MCM is gradually are related only to the replication of DNA [16]. decreased, and may even be undetectable, Thus, MCM proteins may have higher accuracy during the G0 phase, differentiation, and than the traditional markers Ki67 and PCNA in senescence [9]. In the G1 phase, the replica- assessing cellular proliferative activity. In addition licensing factor (RLF), which possesses tion, several studies have revealed that elevat- helicase activity and is composed mainly of ed expression of MCM is associated with poor stable heterohexamers formed by MCM2-7 pro- prognosis and malignant processes [17-19]. teins, combines with other related replication However, few studies have investigated the initiation factors to form a pre-replicative com- expression and prognostic utility of MCM in plex (pre-RC) that participates in DNA replica- SACC. In the present study, we aimed to investi- tion. The MCM proteins dissociate from the pre- gate the expression of MCM3 and MCM7 in RC and are in a free state during the S phase, SACC samples and evaluated the relationship and do not participate in the assembly of pre- between clinicopathological characteristics RC during replication. This prevents DNA repli- and prognosis. cation from re-initiating, ensuring that the DNA replicates only once per cell cycle [10]. Materials and methods

It has been found that, in order to perform their Patients and samples biological function, MCM proteins assembled in the cytoplasm form heterohexamers before A total of 89 patients diagnosed with primary entering the nucleus. This is because mono- SACC by postoperative histopathological exam- meric MCM protein can be easily excreted from ination in Zhejiang Cancer Hospital from June the nucleus. Nuclear localization sequences 2002 to May 2013 were enrolled in this study. present in MCM2 and MCM3 proteins suggest All 89 clinical tumor samples, acquired from that they are responsible for providing chromo- the patients who had not undergone preopera- some-binding targets for other MCM proteins. tive chemotherapy or radiotherapy, were recon- The removal of MCM proteins from the nucleus firmed by three expert SACC pathologists, and is regulated by cyclin-dependent kinases. their clinical information was retrospectively However, it remains to be determined whether investigated. Tumor-node-metastasis (TNM) cyclin-dependent kinases promote the degra- staging, carried out according to the recom- dation of MCM polymers [11-13]. Moreover, mendation of the International Union Against each particular subunit has its own specific Cancer, was used to assess the extent of SACC function. For instance, MCM3 proteins have [20]. All the patients provided informed con- been shown to interact directly with MCM5/ sent before surgical treatment, and the study CDC46, which interacts with MCM3AP before obtained approval from the Ethics Committee being acetylated by MCM3AP (a chromatin- of Zhejiang Cancer Hospital. associated acetyltransferase). The initiation of DNA replication and cell cycle progression is The prognoses of the 89 primary SACC patients inhibited by the acetylation of MCM3 proteins were evaluated by regular follow-ups, which [14]. MCM7 proteins interact with, and may be included oral ultrasound and enhanced compu-

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Figure 1. Clinical significance of MCM3 and MCM7 in SACC. Oncomine data mining analysis of MCM3 and MCM7 mRNA levels in Frierson HF salivary-gland statistics between normal salivary gland versus SACC. MCM3 and MCM7 expression in SACC by IHC. tational tomography of the oral cavity, at three- Evaluation of immunohistochemical staining month intervals after surgical treatment. We calculate the survival of the time interval Immunohistochemical assessment of the between the diagnosis day and the latest fol- expression of the MCM3 and MCM7 was per- low-up (or relapse or death). The median dura- formed by scanning each slide under low mag- tion of the follow-ups was 48 months (range nification (×100) to identify regions containing 3-145 months). Disease-free survival (DFS) positive immunoreactivity. At high magnifica- data were available for all the 89 (100%) tion (×400), immunostaining was further evalu- patients, and 37 (41.5%) out of the 89 patients ated by two experienced pathologists using a relapsed or died after surgery. method similar to the one described by Deraco et al. [21]. Briefly, the results were indepen- Immunohistochemistry (IHC) dently evaluated based on staining intensity and the proportion of positively-stained cells by A standard immunohistochemical analysis was two experienced pathologists. Doubtful cases performed on 4 μm sections of paraffin-embed- were discussed by the two pathologists until a ded tissue samples. All paraffin sections were consensus was achieved. The positive expres- deparaffinized with xylene and dehydrated with sion of MCM3 and MCM7 in SACC is localized in alcohol. Following dehydration, a citrate buffer (0.01 M) was used to retrieve the antigen. After the nucleus. Staining intensity score was cate- neutralizing endogenous peroxidase with a 3% gorized as negative (score 0), weak (score 1), hydrogen peroxide solution, all sections were moderate (score 2), or strong (score 3). The per- preincubated with blocking serum for 30 min- centage of tumor cells positive for MCM3 and utes at 25°C. Then, the sections were incubat- MCM7 was independently scored by patholo- ed with the primary anti-MCM7 (Proteintech, gists based on the interquartile range. The per- 11225-1-AP, Wuhan, China) or the anti-MCM3 centage of MCM3 positive tumor cells was (Proteintech, 15597-1-AP, Wuhan, China) anti- scored as follows: 0 if < 15%; 1 if ≥ 15% and < body at a dilution in 1:100 at 4°C overnight. 30%; 2 if ≥ 30% and < 50%; 3 if ≥ 50% and < After washing three times in PBS, the sections 70%; 4 if ≥ 70%. The percentage of MCM7 posi- were incubated with horseradish peroxidase- tive tumor cells was scored as follows: 0 if < labelled goat anti-mouse/rabbit secondary 5%; 1 if ≥ 5% and < 10%; 2 if ≥ 10% and < 20%; antibody (Dako, Glostrup, Denmark) for 50 min- 3 if ≥ 20% and < 30%; 4 if ≥ 30%. A final score utes at room temperature, followed by a further based on adding two score values was classi- three washes in PBS. Immunoreactivity was fied as negative (score 0), weakly positive (score visualized with 3,3’-diaminobenzidine (DAB, 1-2), positive (score 3-4), strongly positive Dako, Glostrup, Denmark). The sections were (score 5-6). Those with a final score of < 3 were counterstained with hematoxylin. Finally, the defined as ‘low expression’ and those with a sections were dehydrated and mounted in neu- final score of ≥ 3 were considered ‘high tral resin. expression’.

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or Fisher’s exact test. Disease- free survival was analyzed using the Kaplan-Meier method, log-rank test. Parameters with a P-value < 0.1 in the univariate analyses were further tested using the multivariate Cox proportional hazards mo- del to evaluate prognostic factors for survival. P-values < 0.05 were considered to be statistically significant.

Results

Patient characteristics

Among the 89 SACC patients, 53 (59.5%) were newly diagnosed, 38 (42.6%) were male, and 45 (50.5%) were older than 57 years. 41 (46.1%) patients had advanced stage disease (stage IV or recurrence) and the other individu- als had lower stage disease (stages I, II, III). Three (3.3%) had distant metastases, 54 (60.7%) had nerve invasion, and 10 (11.2%) had lymphatic metastases. Adjacent tissue invasion data were available for 44 (49.4%) patients, and 36 (81.8%) out of the 44 showed adjacent tissue invasion. 27 (30.3%) patients showed tumors at major sites. Histologically, 55 (61.7%) patients showed cribriform type tumors.

MCM3 and MCM7 are abnor- mally overexpressed in SACC tissues

Previous reports have indicat- Figure 2. Representative images for MCM3 and MCM7 negative and positive ed that MCM3 and/or MCM7 staining. are involved in multiple types of tumors and promote cancer Statistical analysis progression [17-19]. First, to determine the clinical significances of both MCM3 and MCM7 in Statistical analyses were performed using patients with SACC, we performed data mining SPSS 20.0 (SPSS Inc, Chicago, Illinois, USA). and analyzed the expressions of both MCM3 The associations between MCM3 and MCM7 and MCM7, using the publicly available expression and clinicopathological variables Oncomine database. In Frierson HF salivary- were analyzed using Pearson’s chi-square test gland statistics, MCM3 and MCM7 gene

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Table 1. Association of MCM expression with clinicopathological Both MCM3 and MCM7 were categories in SACC patients observed in the nuclei of can- MCM3 MCM7 cer cells. Faint cytoplasmic Category All case P-valuea P-valuea staining was seen, which was High Low High Low disregarded as nonspecific Initial diagnosis staining. Representative im- Yes 53 40 13 0.364 42 11 0.293 ages for MCM3 and MCM7 No 36 24 12 25 11 negative and positive stain- Gender ing are presented in Figure 2. Male 38 29 9 0.425 28 10 0.763 According to the IHC scoring Female 51 35 16 39 12 system, among patients ex- Age pressing MCM3, 1 patient ≤ 57 44 34 10 0.266 32 12 0.581 showed score 0, 13 patients > 57 45 30 15 35 10 showed score 1, 11 patients Tumor site showed score 2, 28 patients showed score 3, 34 patients Major 27 22 5 0.185 20 7 0.862 showed score 4, and 2 pa- Minor 62 42 20 47 15 tients showed score 5. Am- T-stage ong those expressing MC- T1-3 49 31 18 0.045 32 17 0.016 M7, 3 patients showed score T4 and recurrence 40 33 7 35 5 0, 12 patients showed score Histologic types 1, 7 patients showed score 2, Cribriform 55 39 16 0.789 39 16 0.224 17 patients showed score 3, Tubular and solid 34 25 9 28 6 20 patients showed score 4, Adjacent tissue invasion 25 patients showed score 5, Yes 36 28 8 0.652 30 6 0.024 and 5 patients showed score 6. In total, of the 89 SACC No 8 5 4 3 5 patients, 88 (98.8%) stained Distant metastasis positive for MCM3 expres- Yes 3 2 1 0.838 2 1 0.725 sion, and 86 (96.6%) were No 86 62 24 65 21 positive for MCM7. Moreover, Nerve invasion 64 and 67 of the 89 SACC Yes 54 43 11 0.044 45 9 0.029 patients (71.9% and 75.2%) No 35 21 14 22 13 displayed high levels of MC- lymphatic metastasis M3 and MCM7 expression, Yes 10 7 3 1.000 8 2 1.000 respectively. No 79 57 22 59 20 Clinicopathological associa- Prognosis tions of MCM3 and MCM7 Relapse or death 37 29 8 0.252 32 5 0.039 expression Living 52 35 17 35 17 Para-carcinoma tissue The relationship between the (+) 20 6 0.393 1 0 1.000 expression of MCM3 and (-) 9 5 30 11 MCM7, and the clinicopatho- aBold-italic values are statistically significant P( < 0.05). logical characteristics of SA- CC are summarized in Table 1. There was no significant expression exhibited significant P-values (P < correlation between MCM3 expression and ini- 0.001) and gene ranks in the top 3% among all tial diagnosis (P = 0.364), gender (P = 0.425), differentially-expressed genes. Both MCM3 age (P = 0.266), tumor site (P = 0.185), histo- and MCM7 were up-regulated in SACC com- logic types (P = 0.789), adjacent tissue invasion pared with normal salivary gland (Figure 1). (P = 0.652), distant metastasis (P = 0.838), Next, we examined the expression levels of lymphatic metastasis (P = 1.000), or prognosis MCM3 and MCM7 in SACC tissues using IHC. (P = 0.252). However, MCM3 expression was

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Table 2. Univariate and multivariate cox regression analysis of SACC Univariate analysis Multivariate analysis Variablea HR (95% CI) P-valueb HR (95% CI) P-value Initial diagnosis 0.841 (0.441-1.607) 0.601 Gender 0.922 (0.482-1.765) 0.807 Age 2.501 (1.273-4.913) 0.008 2.345 (1.182-4.654) 0.015 Tumor site 0.864 (0.417-1.789) 0.694 T-stage (T1-3 vs. T4 and recurrence) 2.855 (1.447-5.634) 0.002 2.398 (1.199-4.797) 0.013 Histologic types (cribriform vs. tubular, solid) 1.473 (0.420-5.163) 0.545 Distant metastasis 2.994 (0.710-12.616) 0.135 Nerve invasion 1.328 (0.671-2.628) 0.415 lymphatic metastasis 1.508 (0.628-3.620) 0.358 MCM3 1.143 (0.521-2.508) 0.739 MCM7 2.957 (1.144-7.642) 0.025 2.393 (0.905-6.326) 0.079 aAssociations determined by Cox proportional hazards regression and adjusted for initial diagnosis, gender, age, tumor site, T-stage, histologic types, distant metastasis, nerve invasion, lymphatic metastasis, MCM3, and MCM7. bBold-italic values are statistically significant (P < 0.05). statistically related to T-stage (P = 0.045) and 3D). However, there were no significant correla- nerve invasion (P = 0.044). Regarding MCM7 tions between DFS and other clinicopathologi- expression, statistical analysis indicated that cal parameters, including initial diagnosis, gen- T-stage (P = 0.016), adjacent tissue invasion (P der, tumor site, histologic types, distant me- = 0.024), nerve invasion (P = 0.029), and prog- tastasis, nerve invasion, lymphatic metastasis, nosis (P = 0.039) were correlated with the and MCM3 expression. expression of MCM7 (P < 0.05). Association between MCM3 and MCM7 Survival analysis and prognosis The MCM proteins assembled in the cytoplasm As shown in Table 2, univariate analysis re- form heterohexamers, which enter the nucleus vealed SACC patients with high MCM7 expres- to perform their biological functions, with the sion levels had higher relapse risk than those of MCM2 and MCM3 proteins providing chromo- low-level expression (P = 0.025). Additional sig- some-binding targets for these MCM proteins nificant associations of relapse with ageP ( = [11-13]. A chi-square analysis was used to 0.008), and T-stage (P = 0.002) were also found investigate the association between MCM3 by univariate analysis. Subsequently, these and MCM7 (Table 3), which demonstrated that parameters were investigated using multivari- MCM3 is closely associated with MCM7 (P = ate analysis, which indicated that both age (P = 0.001). 0.015) and T-stage (P = 0.013) were predictors Discussion of survival for SACC patients (Table 2). Un- fortunately, the multivariate analysis failed to In recent decades, proliferation markers have suggest MCM3 or MCM7 as independent prog- played an important role in tumor assessment nostic factors. Likewise, the Kaplan-Meier anal- [16-19, 21, 22]. MCM3 and MCM7 are both ysis failed to indicate that MCM3 expression (P critical proliferation markers in various malig- = 0.737) was a significant indicator of survival nancies, including esophageal squamous cell (Figure 3A). However, the Kaplan-Meier analy- carcinoma (ESCC), hepatocellular carcinoma, sis indicated that MCM7 expression (P = 0.019) ovarian cancer, T-cell lymphomas, and non- was a significant indicator of survival Figure ( small cell lung cancer [18, 19, 23-25]. However, 3B). Furthermore, significant associations be- the utility of MCM3 and MCM7 as markers for tween DFS and additional parameters, which SACC has not yet been reported. included age (P = 0.006) and T-stage (P = 0.002), were demonstrated, similar to the In this study, using IHC, we found that 88 of 89 results of the univariate analysis (Figure 3C, SACC patients (98.8%) were positive for MCM3

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Figure 3. Kaplan-Meier curve analysis. A. Association between MCM3 protein expression level and DFS of patients with SACC based on IHC. MCM3 failed to indicate the difference of DFS in SACC patients (P > 0.05). B. SACC patients with positive MCM7 expression had shorter DFS than those with negative MCM7 expression according to IHC (P < 0.05). C. Association between age and DFS of patients with SACC (P < 0.05). D. Association between T-stage and DFS of patients with SACC (P < 0.05). expression, and that 64 (71.9%) patients dis- ings may possibly be attributed to the small played high levels of MCM3 expression. In addi- sample size (17 cases) used in their study. tion, 86 of the SACC patients (96.6%) tested positive for MCM7 expression, with 67 (75.2%) There was no significant difference in the expressing it at high levels. These results dem- expression of the MCM3 protein between the onstrated that the MCM3 and MCM7 proteins para-carcinoma tissue and the corresponding are highly expressed in SACC. However, a study SACC tissue (P = 0.393). Twenty-six of forty conducted by Manuela et al. [26] reported the (65%) para-carcinoma tissues were positive for opposite, finding that MCM3 expression was MCM3 expression, suggesting that MCM3 pro- low in SACC. When considered alongside the tein does not distinguish well between SACC MCM3 expression data from the Oncomine and the corresponding para-carcinoma tissues. database and our own results, Manuela’s find- There was also no significant difference in the

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Table 3. Association of MCM3 expression In our analysis, SACC patients with advanced with MCM7 expression in SACC patients T-stage, adjacent tissue invasion, nerve inva- MCM7 sion, and poor prognosis were more likely to Category P-valuea have high MCM7 expression. A survival analy- High Low sis demonstrated that patients with higher MCM3 MCM7 levels had a shorter DFS and poorer High 54 10 0.001 prognosis after surgery. This suggested that Low 13 12 MCM7 may drive malignant aggression by regu- aBold-italic values are statistically significant P( < 0.05). lating abnormal cell proliferation, leading to poor prognoses, which is consistent with the study results for several tumors. For instance, expression of the MCM7 protein between para- Feng et al. [29] found that high LIs of MCM7 are carcinoma tissue and corresponding SACC tis- related to the development and metastasis of sue (P = 1.000). However, this does not mean oral squamous cell carcinoma (OSCC), which that there is no difference between them. Of may enable early OSCC diagnosis, and predict 42 para-carcinoma tissues, only one (2.38%) its prognosis. In another study with similarities was positive for MCM7 expression when com- to our own, Tamura et al. [28] reported that LIs pared with the adjacent SACC tissue, suggest- of MCM7 in OSCCs were associated with histo- ing that the protein is highly specific to SACC logical grade, and that MCM7 is a reliable prog- tissues. This suggests that MCM7 may be a nostic marker for this cancer. The nuclear new indicator for judging benign and malignant expression of MCM7 in HCC was linked with lesions of the salivary gland. One study similar hepatitis B viral infection, intrahepatic metas- to our own, Alina et al. [19], demonstrated that tasis, vascular invasion, and shorter overall the expression level of MCM7 is lower in benign survival (OS). Moreover, MCM7 was shown to inflammatory dermatoses than in malignant be an independent prognostic factor for overall lymphoproliferative disorders. Kimura et al. survival by multivariate analysis [30]. Mean- [27] confirmed that MCM7 labelling indices while, Qu et al. [25] also demonstrated that (LIs) were significantly higher in malignant high MCM7 expression was related to poorer mesothelioma cells than in reactive mesotheli- OS in patients with HCC. Ota et al. [31] suggest- al cells, indicating that they can be a reliable ed that MCM7 could serve as a potential prog- biomarker for distinguishing between malig- nostic factor for DFS in patients with high-grade nant mesothelioma cells and reactive meso- ovarian serous carcinomas. thelial cells. Zhou et al. [28] found that the MCM7 protein is expressed exclusively in can- MCM7 is known to promote cancer progression cer tissues of patients with hepatocellular car- by activating the MCM7-MAPK-cyclin D1-de- cinoma (HCC). Choy et al. [24] reported that pendent signaling pathway in HCC [25]. In addi- MCM7 protein expression increased in a step- tion, the overexpression of MCM7 promotes wise manner with increasing lesion degree in ESCC progression through the AKT1/mTOR sig- squamous epithelium, columnar cell metapla- naling pathway [32]. Further studies are needed to investigate the molecular mechanisms sia, Barrett’s esophagus, low-dysplasia, high- underlying these findings. dysplasia, and squamous cell carcinoma, indicating that MCM7 expression positively co- A previous study investigated the expression of rrelates with disease progression, and thus MCM3 among 15 pleomorphic adenomas (PA), may play a critical role as a sensitive prolifera- 17 mucoepidermoid carcinomas (MEC), and 18 tive marker in evaluating esophageal lesions. It ACC. Their results suggested that the LIs of is worth noting that the P-value of the associa- MCM3 was significantly lower in PA compared tions between MCM3 and MCM7 and lymph to MEC and ADCC [33]. Unfortunately, this study node metastasis is 1.000, indicating that the did not refer to the relationship between MCM3 expression levels of these two proteins are expression and clinicopathological variables almost the same in the high-expression and and prognosis. In our research, MCM3 expres- low-expression groups. In other words, the sion was found to have a lower association with expression levels of MCM3 and MCM7 are not clinicopathological variables than MCM7, and indicative of lymph node metastasis. only correlated with T-stage and nerve invasion.

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In addition, we did not find any significant rela- References tionship between MCM3 and prognosis. Interestingly, our analysis did reveal a signifi- [1] Senft E, Lemound J, Stucki-Koch A, Gellrich cant relationship between age and T-stage in NC, Kreipe H, Hussein K. Expression of cyclin- dependent kinase inhibitor 2A 16, tumour pro- relation to DFS. In a multivariate analysis, how- tein 53 and epidermal growth factor receptor ever, only the presence of age and T-stage were in salivary gland carcinomas is not associated independent prognostic factors of DFS in SACC with oncogenic virus infection. Int J Oral Sci patients. A previous study reported similar 2015; 7: 18-22. results to our present study, suggesting that [2] Jiang LH, Ge MH, Hou XX, Cao J, Hu SS, Lu XX, age and T-stage have important roles in the Han J, Wu YC, Liu X, Zhu X, Hong LL, Li P, Ling aggressive behavior of ACC [34]. Our results ZQ. miR-21 regulates tumor progression th- suggested that SACC patients with T4 stage rough the miR-21-PDCD4-Stat3 pathway in hu- cancer, recurrence, or with age > 57 years had man salivary adenoid cystic carcinoma. Lab Invest 2015; 95: 1398-1408. a shorter DFS. In addition, the MCM3 was [3] Coca-Pelaz A, Rodrigo JP, Bradley PJ, Vander closely associated with MCM7 (P = 0.001), Poorten V, Triantafyllou A, Hunt JL, Strojan P, which was consistent with previous studies [18, Rinaldo A, Haigentz M Jr, Takes RP, Mondin V, 19]. Teymoortash A, Thompson LD, Ferlito A. Ade- noid cystic carcinoma of the head and neck-- In conclusion, MCM3 expression has no rela- an update. Oral Oncol 2015; 51: 652-661. tion to prognosis. Our results suggest that [4] Cordesmeyer R, Kauffmann P, Laskawi R, Rau MCM7 might be a more reliable and useful pro- A, Bremmer F. The incidence of occult metas- liferation marker than MCM3 in SACC. Also, it tasis and the status of elective neck dissection can be combined with age and T-stage to pre- in salivary adenoid cystic carcinoma: a single dict prognosis in post-operative SACC patients. center study. Oral Surg Oral Med Oral Pathol Oral Radiol 2018; 125: 516-519. Further studies should be undertaken to deter- [5] Xu J, Zhu X, Li Q, Chen C, Guo Z, Tan Z, Zheng mine the usefulness of this marker in the pre- C, Ge M. Loss of PIM1 correlates with progres- diction of clinical behavior, such as adjacent sion and prognosis of salivary adenoid cystic tissue invasion or nerve invasion. MCM7 may carcinoma (SACC). Cancer Cell Int 2018; 18: be a novel molecular biomarker for SACC 22. patients, and downregulation of its expression [6] Hanahan D and Weinberg RA. Hallmarks of may provide a therapeutic method for improv- cancer: the next generation. Cell 2011; 144: ing their DFS. 646-674. [7] Tachibana KE, Gonzalez MA, Coleman N. Cell- Acknowledgements cycle-dependent regulation of DNA replication and its relevance to cancer pathology. J Pathol This study was supported by grants from 2005; 205: 123-129. [8] Nowińska K, Dzięgiel P. Białka MCM i Ich rola w Medical Innovation Discipline of Zhejiang proliferacji komórek i procesie nowotworowym Province of China (Head and Neck Oncology) the role of MCM proteins in cell proliferation and the High-level Talents Special Support Plan and tumorigenesis. Postepy Hig Med Dosw On- of Zhejiang Province of China. line 2010; 64: 627-635. [9] Ritzi M, Knippers R. Initiation of genome repli- Disclosure of conflict of interest cation: assembly and disassembly of replication-competent chromatin. Gene 2000; 245: None. 13-20. [10] Chang F, Riera A, Evrin C, Sun J, Li H, Speck C, Address correspondence to: Dr. Ming-Hua Ge, De- Weinreich M. Cdc6 ATPase activity disengages partment of Head and Neck Surgery, Zhejiang Cdc6 from the pre-replicative complex to pro- Cancer Hospital, No. 1 East Banshan Road, Gongshu mote DNA replication. Elife 2015; 4: e05795. District, Hangzhou, China. Tel: +86-13605813782; [11] Nguyen VQ, Co C, Irie K, Li JJ. Clb/Cdc28 kinases promote nuclear export of the replica- Fax: 0571-81708102; E-mail: [email protected]; tion initiator proteins Mcm2-7. Curr Biol 2000; Dr. Xin Zhu, Zhejiang Cancer Research Institute, 10: 195-205. Zhejiang Province Cancer Hospital, Gongshu Dis- [12] Gómez EB, Catlett MG, Forsburg SL. Different trict, Hangzhou, China. Tel: +86-13588453286; phenotypes in vivo are associated with ATP- Fax: 0571-81708102; E-mail: [email protected] ase motif mutations in Schizosaccharomyces

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