Non-small cell lung cancer (NSCLC) is the most commonly diagnosed cancer and the most frequent cause of cancer-associated mortality worldwide. Tesmin (MTL5) is a 60 kDa protein which has cysteine rich motifs, characteristic of metallothioneins. Tesmin expression was first observed in germ cells during spermatogenesis. Increased tesmin expression in NSCLC has been described previously. Minichromosome maintenance proteins (MCMs) serve a critical role in replication and cell cycle progression, i.e. in NSCLC. The aim of the present study was to evaluate the localization and intensity of tesmin, MCM5 and MCM7 protein expression in NSCLC and their association with the clinicopathological data of patients. Archival paraffin blocks of 243 cases of NSCLC and 104 non-cancerous tissue samples from the surgical margin (control) were obtained from patients treated at the Clinic of Thoracic Surgery of Wroclaw Medical University (Wroclaw, Poland) between 2010 and 2016, and were used for tissue microarrays and immunohistochemical (IHC) experiments. Laser capture microdissection was used for the isolation of cancer cells from 36 frozen samples of NSCLC and 8 control samples, and subsequently,
Lung cancer is currently one of the most common cancers and one of the most frequent causes of death worldwide (
Tesmin (Testis-specific metallothionein-like protein, also known as metallothionein-like 5 protein, MTL5) is a 60 kDa protein which has cysteine-rich motifs, characteristic of metallothioneins (MTs) (
The minichromosome maintenance protein complex (MCM) is a family of highly conservative, homogenous proteins (MCM2-MCM7) that play an active role in DNA synthesis (forming the prereplication-complex, which is a part of the replication forks) (
Many studies suggest that MCM proteins are expressed in cancer cells with a high proliferation activity. Consequently, they are considered as useful markers of proliferation in many cancers, including NSCLC (
The aim of this study was to examine the expression of tesmin (MTL5), MCM5 and MCM7 in NSCLC cases, as well as their associations with the clinicopathological data of patients.
The clinical material consisted of 243 paraffin blocks from patients operated on for non-small cell lung cancer (NSCLC), including 92 cases of squamous cell lung carcinoma and 151 cases of lung adenocarcinoma. Additionally, 104 paraffin blocks from the surgical margin were used, which constituted the control for the analysed cases. In doubtful cases, immunohistochemical (IHC) anti-p63 (squamous cell lung carcinoma) and anti-TTF-1 (lung adenocarcinoma) reactions were performed in order to establish the final histopathological diagnosis. Moreover, the presence of necrosis in the neoplastic tumour was assessed by determining the percentage of the area of the histological preparation (stained with the HE method) covered by necrosis. After surgery, the median time that patients were followed for a 27 months (range: 0–81 months, mean of 31.82±18.89). Three patients did not report for a check-up after surgery. During the follow-up period, 95 patients died.
The tumour fragments and the tissue fragments from the surgical margin were frozen in liquid nitrogen, then the tumor cells and the normal lung tissue cells were isolated by laser microdissection and used to analyse
All patients were operated on in the years 2010–2016 at the Department of Thoracic Surgery of the Medical University in Wroclaw.
The specific clinicopathological data of the patients are presented in
The TMA method is a accepted method of archiving material in paraffin blocks with many advantages including economic aspects, stability of IHC reaction conditions and the time of evaluation of IHC results with relatively small restrictions (
The paraffin blocks with NSCLC cases were cut into 4-µm sections. The IHC reactions were performed using anti-tesmin rabbit polyclonal antibody (Novus Biologicals; cat. no. NBP2-13624) in a 1:400 dilution, anti-MCM5 (Santa Cruz Biotechnology, Inc.; sc-165994) in a 1:100 dilution, anti-MCM7 (Santa Cruz Biotechnology, Inc.; sc-9966) in a 1:50 dilution and anti-Ki-67 mouse monoclonal antibody Clone MIB-1 (Dako) ready-to-use. The IHC reactions were performed using the DAKO Autostainer Link 48 (Dako). The visualization of the reactions was carried out using EnVision™ FLEX High pH (Link) reagents (Dako), according to the manufacturer's instructions. Positive IHC reaction for the tesmin antigen was assessed using the immunoreactive score (IRS) scale by Remmele and Stegner (
Additionally, the nuclear expression intensity for the Ki-67, MCM5 and MCM7 proteins was determined using a scale that analyses the percentage of the number of cancer cells with positive nuclear expression of the antigens studied, according to the following scale: 0%, 0 p.; 1–10%, 1 p.; 11–25%, 2 p.; 26–50%, 3 p.; 51–100%, 4 p. All specimens were assessed using an OLYMPUS BX-41 light microscope (Olympus) by two independent pathologists. In cases with divergent scores the evaluation was reassessed and discussed until consensus was reached. The final result was the mean of the IHC expression scores of 3 TMA cores. Moreover, p63 and TTF-1 antigen expressions were used to confirm the histological type of the tumor (TTF-1(+) adenocarcinoma; p63(+) squamous cell carcinoma).
Lung cancer cell line NCI-H1703 (lung squamous cell carcinoma) was obtained from the American Type Culture Collection. RPMI-1640 cell culture medium was used. The medium was additionally supplemented with L-glutamine to a final concentration of 2 mM, and with foetal bovine serum to a final concentration of 10%. All of the cell culture media and reagents were provided by Sigma-Aldrich; Merck KGaA. In addition, we performed
The count of cultured cells (NCI-H1703) in the phases of the cell cycle was evaluated using the FACSCanto II flow cytometer (BD Biosciences) and FxCycle PI/RNase Staining Solution (Life Technologies; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.
Frozen tissue samples of 36 NSCLC cases and 8 cases of non-malignant lung tissue (NMLT) (control) were used for RNA extraction. Tissue sections of 10-µm thickness were prepared with use of a Leica CM1950 cryostat (Leica Microsystems) and placed on a polyethylene terephthalate membrane slide (MMI). The slides were fixed in 70% isopropyl alcohol and stained with HE by using the H&E Staining Kit for LCM (MMI). LCM was performed using the MMI CellCut Plus System (MMI). The dissected neoplastic and normal cells were collected onto the adhesive lid of 500-µl tubes (MMI). Total RNA was isolated from the tissue samples by using the RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. The protocol included on-column DNase digestion to eliminate genomic DNA. First-strand cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen), according to the manufacturer's instructions.
The mRNA expression of
The Western blot technique (WB) was used to determine tesmin, MCM5 and MCM7 expression levels on the cell cultures of NCI-H1703. Whole protein lysates from the cell culture samples were obtained using the T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Inc.) with the addition of the Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, Inc.) and 0.2 mM PMSF (Sigma-Aldrich; Merck KGaA). Protein concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) and the NanoDrop™ 1000 (Thermo Fisher Scientific) spectrophotometer. Equal amounts of total protein (30 µg) were mixed with Laemmli sample buffer and resolved on 10% acrylamide gel by SDS-PAGE. After electrophoresis, the samples were transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Merck KGaA) in the XCell SureLock™ Mini-Cell Electrophoresis System (Thermo Fisher Scientific, Inc.). Next, the membranes were blocked in 4% bovine serum albumin solution (Merck KGaA) in TBST buffer (0.2 M Tris; 1.5 M NaCl; 0.1% Tween-20). After blocking, the membranes were incubated overnight at 4°C with the primary rabbit anti-human tesmin polyclonal antibody (NBP2-13624; Novus Biologicals), diluted at 1:200, anti-MCM5 antibody (Santa Cruz Biotechnology, Inc.; sc-165994) diluted at 1:200 and anti-MCM7 (Santa Cruz Biotechnology, Inc.; sc-9966) antibody diluted at 1:200. Further, the membranes were incubated with secondary HRP-conjugated donkey anti-rabbit antibody (715-035-152; Jackson ImmunoResearch), diluted at 1:3,000 for 1 h at room temperature. Finally, the membranes were rinsed and treated with the Luminata Classico (Merck KGaA) chemiluminescent substrate. The reactions were visualized using the ChemiDoc Imaging System (Bio-Rad Laboratories). β-actin detected with primary rabbit anti-human β-actin antibody (4970; Cell Signaling Technology) diluted at 1:1,000 and secondary HRP-conjugated donkey anti-rabbit antibody (711-035-152; Jackson ImmunoResearch) diluted at 1:3,000 were used as an internal control to normalize the amount of tesmin. A densitometric analysis of the results obtained was performed with the use of the Image Lab software (Bio-Rad Laboratories).
The Kolmogorov-Smirnov test was used to evaluate the normality assumption of the examined groups. The Student's t-test, unpaired t-tests, Mann-Whitney, Kruskal-Wallis test with Dunn's multiple comparison test and MANOVA with Bonferroni post hoc tests were used to compare the differences in the expression of the examined markers in
The analysis of the cell cycle by means of the assessment of the DNA content in the NCI-H1703 cells stained with propidium diodide and then analysed in the flow cytometer showed that in the NCI-H1703 cell line with a silenced
Moreover, the statistical analysis of the results obtained by RT-qPCR showed a significantly lower expression of
The analysis of the expression of the mRNA isolated by laser microdissection showed the expression of
In the analysed NSCLC cases, positive cytoplasmic (95.6%) and nuclear (83.4%) IHC expression of tesmin were demonstrated (
Additionally, no correlation was found between the expression of tesmin (both cytoplasmic and nuclear) and other clinicopathological data such as patient's age, sex, tumour size, pN, pM, clinical stage or tobacco smoking status (
The analysis of the correlation of the intensity of the expression of tesmin (IHC) and the proliferation markers showed that the nuclear (but not the cytoplasmic) expression of tesmin correlated moderately positively with the nuclear expression of MCM5, MCM7 and Ki-67 (
The analysis of the survival of the NSCLC cases showed that patients without confirmed (by the IHC method) cytoplasmic expression of tesmin in the tumour cells lived longer that patients with confirmed tesmin expression (P=0.052, Gehan-Breslow-Wilcoxon test;
In the presented study, we have demonstrated for the first time a relationship between an increased nuclear expression of tesmin and a higher expression of MCM5 and MCM7 at the protein and mRNA levels in NSCLC. A positive correlation between the expression of tesmin and Ki-67 in NSCLC, which we have as well demonstrated in this study, was also previously observed by us (
The role of tesmin in the process of proliferation of NSCLC cells is additionally confirmed by an
Despite this, the role of tesmin in cancer is not fully understood. An increased expression of this protein in NSCLC cells compared to the control tissue has already been described by us (
The positive correlations between the expression of tesmin (
Due to the fact that tesmin is indicated as a co-activator that differentiates the activity of the ligands aldosterone and deoxycorticosterone for the mineralocorticoid receptor (this receptor is a ligand-dependent transcription factor), it can be assumed that tesmin, as a co-activator, may regulate the expression of other genes, e.g. those related to the proliferation process (
Moreover, as in our previous study, we were also able to demonstrate that an increased expression of tesmin is a negative prognostic factor in NSCLC, both at the mRNA and the IHC levels (
One of the limitation of the study was lack of clinical data in the form of presence of the EGFR gene mutation. This is due the fact that the mutation testing of NSCLC in Poland is not routinely performed in cases of I and II stage of non-small cell lung cancer. The cases in these stages that are mostly treated surgically and constitute the dominant group of cases in the presented study. Unfortunately, we also do not have such clinical data for a much smaller group of patients in stage III and IV. Because the main cause of the EGFR gene mutation is smoking we compared the expression of tesmin in smokers and non-smokers but we did not reveal any significant differences in the expression of tesmin in these groups.
The present study was carried out using the NCI-H1703 squamous cell carcinoma because the analysis of
In conclusion, the role of tesmin in NSCLC seems to be related to the cell proliferation process of this tumour, and a worse prognosis of NSCLC patients with a higher expression of it may indicate the role of this protein in the progression of NSCLC.
Not applicable.
The present study was supported by the National Science Centre, Poland, under the programme ‘Preludium 12’ project no. 2016/23/N/NZ5/02570.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JG designed the study, evaluated the IHC reaction, prepared the database, performed the statistical analysis, analysed the results and created a draft of the manuscript. PSG evaluated the IHC reaction and prepared the database. AG performed microdissection, isolation of mRNA and quantitative PCR analysis. MO, KR and NGP prepared all
The experiment was performed in accordance with the ethical standards and following approval of the Ethics Committee of Wroclaw Medical University (decision no. KB 455/2009 and KB 40/2017) and all patients provided a written statement of informed consent for the use of the material samples for scientific research.
Not applicable.
The authors declare that they have no competing interests.
Analysis of the expression levels of
Immunohistochemical analysis of tesmin expression in controls and non-small cell lung cancer. (A) Low expression levels of tesmin in non-malignant lung tissue (mainly lung macrophages). (B) Nuclear-cytoplasmic expression of tesmin in non-small cell lung cancer cells. (C) Low and (D) high cytoplasmic expression of tesmin. (E) Low and (F) high MCM5 nuclear expression. (G) Low and (H) high MCM7 nuclear expression. Magnification, ×200. MCM, minichromosome maintenance protein.
Kaplan-Meier survival curves and survival analysis of patients with high and low MTL5 and tesmin expression. (A) Elevated cytoplasmic expression levels of tesmin in NSCLC cells are an unfavourable prognostic factor. (B) Similarly, patients with an elevated tesmin mRNA expression (cut-off, median) in NSCLC cells had significantly shorter survival (Gehan-Breslow-Wilcoxon test). NSCLC, non-small cell lung cancer; RQ, relative quantification.
Patients and tumour characteristics.
Parameter | IHC (n=243) | RT-qPCR (n=36) |
---|---|---|
Mean age, years (range) | 66.40±7.4 (50–84) | 65.29±7.52 (52–77) |
Sex, n (%) | ||
Male | 145 (59.67) | 21 (58.33) |
Female | 98 (40.33) | 15 (41.67) |
Tumour size, n (%) | ||
T1 | 76 (31.28) | 8 (22.22) |
T2 | 124 (51.03) | 20 (55.56) |
T3 | 24 (9.88) | 5 (13.89) |
T4 | 5 (2.06) | 1 (2.78) |
No data | 14 (5.76) | 2 (5.56) |
Lymph nodes, n (%) | ||
N0 | 147 (60.49) | 22 (61.11) |
N1,N2,N3 | 82 (33.74) | 12 (33.33) |
No data | 14 (5.76) | 2 (5.56) |
Grade, n (%) | ||
G1 | 3 (1.23) | 0 (0.00) |
G2 | 150 (61.73) | 25 (69.44) |
G3 | 90 (37.04) | 10 (27.78) |
No data | 0 (0.00) | 1 (2.78) |
pTNM, n (%) | ||
I | 104 (42.80) | 15 (41.67) |
II | 76 (31.28) | 15 (41.67) |
III | 46 (18.93) | 4 (11.11) |
IV | 2 (0.82) | 0 (0.00) |
No data | 15 (6.17) | 2 (5.56) |
Stage, n (%) | ||
Early | 180 (74.07) | 30 (83.33) |
Advanced | 48 (19.75) | 4 (11.11) |
No data | 15 (6.17) | 2 (5.56) |
Histology, n (%) | ||
Adeno | 151 (62.14) | 18 (50.00) |
SCC | 92 (37.86) | 18 (50.00) |
p63, n (%) | ||
Positive | 111 (45.68) | 20 (55.56) |
Negative | 89 (36.63) | 11 (30.56) |
No data | 43 (17.70) | 5 (13.89) |
TTF-1, n (%) | ||
Positive | 156 (64.20) | 20 (55.56) |
Negative | 44 (18.11) | 11 (30.56) |
No data | 43 (17.70) | 5 (13.89) |
Tesmin IHC, n (%) | ||
IRS | ||
0 | 12 (4.94) | – |
1-12 | 231 (95.06) | – |
Nuclear | ||
0 | 40 (16.46) | – |
1-4 | 203 (83.54) | – |
IHC, immunohistochemistry; RT-qPCR, reverse transcription-quantitative PCR; TTF-1, thyroid transcription factor 1; IRS, immunoreactive score; SCC, squamous cell carcinoma of the lung.
Correlations of IHC tesmin expression and
Markers | r | P-value |
---|---|---|
Tesmin IRS vs. tesmin nuclear | −0.007 | 0.9086 |
Tesmin IRS vs. Ki-67 | 0.015 | 0.8060 |
Tesmin IRS vs. MCM5 | 0.049 | 0.4436 |
Tesmin IRS vs. MCM7 | 0.034 | 0.5954 |
Tesmin nuclear vs. Ki-67 | 0.239 | <0.0010 |
Tesmin nuclear vs. MCM5 | 0.336 | <0.0001 |
Tesmin nuclear vs. MCM7 | 0.315 | <0.0001 |
Ki-67 vs. MCM5 | 0.634 | <0.0001 |
Ki-67 vs. MCM7 | 0.603 | <0.0001 |
MCM5 vs. MCM7 | 0.722 | <0.0001 |
0.421 | <0.0500 | |
0.553 | <0.0100 | |
0.861 | <0.0001 |
IHC, immunohistochemistry; MTL5, tesmin; MCM, minichromosome maintenance protein; IRS, immunoreactive score.
Univariate and multivariate Cox analyses of overall survival of patients with non-small cell lung cancer.
Univariate Cox analysis of survival | Multivariate Cox analysis of survival | |||||||
---|---|---|---|---|---|---|---|---|
Characteristics | P-value | Hazard ratio | 95% CI lower | 95% CI upper | P-value | Hazard ratio | 95% CI lower | 95% CI upper |
p63 (negative vs. positive) | 0.7645 | 1.0680 | 0.6945 | 1.6424 | ||||
TTF-1 (negative vs. positive) | 0.8613 | 1.0484 | 0.6168 | 1.7822 | ||||
Histological type (adeno vs. SCC) | 0.1486 | 0.7301 | 0.4765 | 1.1187 | ||||
Clinical stage (I–II vs. III–IV) | 0.0003 | 1.5542 | 1.2215 | 1.9775 | 0.6312 | 1.1109 | 0.7231 | 1.7064 |
pT (pT1-2 vs. pT3-4) | 0.0024 | 1.5170 | 1.1593 | 1.9852 | 0.0061 | 1.7928 | 1.1809 | 2.7216 |
pN (pN0 vs. pN1-3) | 0.0023 | 1.9072 | 1.2604 | 2.8861 | 0.0068 | 1.4608 | 1.1105 | 1.9216 |
Necrosis (%) (continuous) | 0.3824 | 0.9458 | 0.8347 | 1.0718 | ||||
Histological grade (G1 vs. G2-3) | 0.1886 | 1.3095 | 0.8761 | 1.9572 | ||||
Tesmin IRS (0 vs. 1–12) | 0.1184 | 3.0555 | 0.7521 | 12.4124 | ||||
Tesmin nuclear (0 vs. 1–4) | 0.3831 | 1.2978 | 0.7225 | 2.3310 | ||||
MCM-5 (0–2 vs. 3–4) | 0.4159 | 0.8169 | 0.5019 | 1.3297 | ||||
MCM-7 (0–2 vs. 3–4) | 0.5114 | 1.3192 | 0.5770 | 3.0160 | ||||
Ki-67 (0–2 vs. 3–4) | 0.8906 | 0.9641 | 0.5730 | 1.6224 |
IRS, immunoreactive score; MCM, minichromosome maintenance protein; TTF-1, thyroid transcription factor 1; SCC, squamous cell carcinoma of the lung.
Univariate and multivariate Cox analyses of overall survival of patients with lung adenocarcinoma.
Univariate Cox analysis of survival | Multivariate Cox analysis of survival | |||||||
---|---|---|---|---|---|---|---|---|
Characteristics | P-value | Hazard ratio | 95% CI lower | 95% CI upper | P-value | Hazard ratio | 95% CI lower | 95% CI upper |
p63 (negative vs. positive) | 0.0508 | 1.7264 | 0.9982 | 2.9859 | 0.4915 | 1.1647 | 0.7543 | 1.7984 |
TTF-1 (negative vs. positive) | 0.3777 | 0.5910 | 0.1837 | 1.9012 | ||||
Clinical stage (I–II vs. III–IV) | 0.0056 | 1.4877 | 1.1231 | 1.9707 | 0.5683 | 1.1346 | 0.7352 | 1.7510 |
pT (pT1-2 vs. pT3-4) | 0.0277 | 1.4084 | 1.0383 | 1.9104 | 0.0068 | 1.4608 | 1.1105 | 1.9216 |
pN (pN0 vs. pN1-3) | 0.0036 | 2.1450 | 1.2833 | 3.5852 | 0.0061 | 1.7928 | 1.1809 | 2.7216 |
Necrosis, % (continuous) | 0.9781 | 0.9973 | 0.8222 | 1.2097 | ||||
Histological grade (G1 vs. G2-3) | 0.1548 | 1.4197 | 0.8761 | 2.3007 | ||||
Tesmin IRS (0 vs. 1–12) | 0.5856 | 0.7525 | 0.2707 | 2.0914 | ||||
Tesmin nuclear (0 vs. 1–4) | 0.2839 | 1.4119 | 0.7514 | 2.6530 | ||||
MCM-5 (0–2 vs. 3–4) | 0.6605 | 0.8876 | 0.5213 | 1.5112 | ||||
MCM-7 (0–2 vs. 3–4) | 0.3379 | 1.5103 | 0.6499 | 3.5102 | ||||
Ki-67 (0–2 vs. 3–4) | 0.9732 | 1.0101 | 0.5603 | 1.8212 |
IRS, immunoreactive score; MCM, minichromosome maintenance protein; TTF-1, thyroid transcription factor 1.
Univariate Cox analysis of overall survival of patients with squamous cell carcinoma of the lung.
Univariate Cox analysis of survival | ||||
---|---|---|---|---|
Characteristics | P-value | Hazard ratio | 95% CI lower | 95% CI upper |
TTF-1 (negative vs. positive) | 0.7255 | 0.8774 | 0.4227 | 1.8213 |
Clinical stage (I–II vs. III–IV) | 0.0614 | 1.6079 | 0.9775 | 2.6447 |
pT (pT1-2 vs. pT3-4) | 0.0206 | 1.9206 | 1.1052 | 3.3377 |
pN (pN0 vs. pN1-3) | 0.4125 | 1.3751 | 0.6420 | 2.9452 |
Necrosis, % (continuous) | 0.6559 | 0.9575 | 0.7908 | 1.1592 |
Histological grade (G1 vs. G2-3) | 0.3497 | 0.5671 | 0.1727 | 1.8619 |
Tesmin IRS (0 vs. 1–12) | 0.4507 | 1.7353 | 0.4143 | 7.2686 |
Tesmin nuclear (0 vs. 1–4) | 0.5495 | 1.8384 | 0.2503 | 13.5045 |
MCM-5 (0–2 vs. 3–4) | 0.9938 | 0.9920 | 0.1321 | 7.4486 |
MCM-7 (0–3 vs. 4) | 0.3132 | 0.8703 | 0.5433 | 1.4610 |
Ki-67 (0–2 vs. 3–4) | 0.4511 | 0.7507 | 0.3562 | 1.5824 |
IRS, immunoreactive score; MCM, minichromosome maintenance protein; TTF-1, thyroid transcription factor 1.