The authors have declared that no competing interests exist.
Conceived and designed the experiments: TL XY JT. Performed the experiments: TL Yueyang Liu XB XY. Analyzed the data: TL XY JT Yang Liu. Contributed reagents/materials/analysis tools: TL XY JT. Wrote the paper: TL XY XB JT.
Overwhelming evidence has demonstrated that the aberrant expression of the human trophoblast cell-surface antigen (TROP2) was associated with tumor aggressiveness and poor prognosis in a variety of human cancers, however the roles of TROP2 in cervical cancer have not been investigated. The purpose of our study was to elucidate the prognostic significance of TROP2 expression in patients with cervical cancer and determine its effect on tumor progression. Immunohistochemistry assay showed that 88.7% (94/106 cases) of cervical cancer specimens were positively stained with TROP2, and the overexpression of TROP2 was closely related with FIGO stage, histological grades, lymphatic metastasis, invasive interstitial depth and high expression of Ki-67. Patients with TROP2-positive staining exhibited a significantly decreased overall survival and progression free survival; it was also an independent predictor for prognosis according to multivariate analysis. Moreover, down-regulation of TROP2 mediated by siRNA in Siha and CaSki cells resulted in a strong inhibition of proliferation and invasion, TROP2 abrogation also elevated the apoptotic ratio and caused G1 arrest. Conversely, enforced expression of TROP2 in HeLa and C33A cells remarkably promoted cell growth, migration and invasion. In addition, the tumorigenic function of TROP2 was associated with the increased expressions of cyclin D1, cyclin E, CDK2 and CDK4 but reduced expression of p27 and E-cadherin via the activation of Erk1/2 signaling pathway. Furthermore, the inhibition of TROP2 expression in cervical cancer cell lines enhances sensitivity to cisplatin. The present study suggest that overexpression of TROP2 may play crucial roles in the development and pathogenesis of human cervical cancer, therefore, TROP2 may represent a prospective prognostic indicator and a potential therapeutic target of cervical cancer.
Cervical cancer is the third most prevalent malignancy among women worldwide
Early-stage patients (I–IIA) can get a satisfying outcome through radical surgery or radiotherapy, with an overall 5-year survival of >65%. Nevertheless, patients with advanced stage (IIB–IV) can only be treated with radiotherapy or plus chemotherapy, the 5-year survival rate for patients with stage III is 25 to 35%, but for stage IV is 15% or fewer
Trophoblast cell surface antigen 2 (TROP2) is a 36 kDa transmembrane glycoprotein belonging to tumor-associated calcium signal transducer (TACSTD) gene family. It was originally identified in human trophoblast cell lines, and elevated expression was found in various types of epithelial carcinomas while low or restricted expression was found in normal tissues
Recent work demonstrated that TROP2 behaved as a true oncogene leading to the tumorigenesis and invasiveness in colorectal cancer cell lines
A total of 160 samples obtained by punch biopsy, cone biopsy or hysterectomy were retrieved from the Department of Pathology, Qilu Hospital of Shandong University between April 2005 to October 2007 (
Characteristic | Total | TROP2 expression | p | |||||||
− | + | ++ | +++ | |||||||
20 | 11 (55.0%) | 8 (40%) | 1 (5%) | 0 (0%) | 0.000 | |||||
34 | 12 (35.3%) | 10 (29.4%) | 4 (11.8%) | 8 (23.5%) | ||||||
106 | 12 (11.3%) | 30 (28.3%) | 32 (30.2%) | 32 (30.2%) | ||||||
0.037 | ||||||||||
CINI | 12 | 6 (50.0%) | 5 (41.7%) | 1 (8.3%) | 0 (0%) | |||||
CINII | 9 | 3 (33.3%) | 4 (44.4%) | 1 (11.1%) | 1 (11.1%) | |||||
CINIII | 13 | 3 (23.1%) | 1 (7.7%) | 2 (15.4%) | 7 (53.8%) | |||||
0.100 | ||||||||||
≤50 | 43 | 6 (14%) | 9 (20.9%) | 10 (23.3%) | 18 (41.9%) | |||||
>50 | 63 | 6 (9.5%) | 21 (33.3%) | 22 (34.9%) | 14 (22.2%) | |||||
0.023 | ||||||||||
Squamous | 77 | 6 (7.8%) | 18 (23.4%) | 28 (36.4%) | 25 (32.5%) | |||||
Adenocarcinoma | 29 | 6 (20.7%) | 12 (41.4%) | 4 (13.8%) | 7 (24.1%) | |||||
0.000 | ||||||||||
I | 41 | 10 (24.4%) | 12 (29.3%) | 16 (39.0%) | 3 (7.3%) | |||||
II | 59 | 2 (3.4%) | 18 (30.5%) | 14 (23.7%) | 25 (42.4%) | |||||
III and IV | 6 | 0 (0%) | 0 (0%) | 2 (33.3%) | 4 (66.7%) | |||||
0.000 | ||||||||||
Well | 35 | 7 (20.0%) | 17 (48.6%) | 5 (14.3%) | 6 (17.1%) | |||||
Moderate | 33 | 4 (12.1%) | 8 (24.2%) | 15 (45.5%) | 6 (18.2%) | |||||
Poorly | 38 | 1 (2.6%) | 5 (13.2%) | 12 (31.6%) | 20 (52.6%) | |||||
0.001 | ||||||||||
NO | 79 | 12 (15.2%) | 27 (34.2%) | 23 (29.1%) | 17 (21.5%) | |||||
YES | 27 | 0 (0%) | 3 (11.1%) | 9 (33.3%) | 15 (55.6%) | |||||
0.000 | ||||||||||
<1/2 | 53 | 12 (22.6%) | 21 (39.6%) | 18 (34.0%) | 2 (3.8%) | |||||
≥1/2 | 53 | 0 (0%) | 9 (17%) | 14 (26.4%) | 30 (56.6%) | |||||
0.255 | ||||||||||
d≤4 cm | 86 | 9 (10.5%) | 25 (29.1%) | 29 (33.7%) | 23 (26.7%) | |||||
d>4 cm | 20 | 3 (15.0%) | 5 (25.0%) | 3 (15.0%) | 9 (45.0%) |
Immunohistochemical staining was carried out on 4-µm thick sections of the paraffin-embedded tissues, and the staining process was strictly performed according to the streptavidin-biotin-peroxidase complex method. After deparaffinisation and rehydration, the sections were treated with 3% hydrogen peroxide to block endogenous peroxidase. Non-specific binding was blocked with normal goat serum for 30 min at 37°C, then the sections were incubated with anti-TROP2 (Santa Cruz, USA, 1∶500 dilution) or anti-Ki-67 monoclonal antibody (Dako, lostrup, Denmark, 1∶100 dilution) overnight at 4°C. After washing with PBS, the sections were incubated with a horseradish peroxidase-labeled polymer-conjugated anti-mouse secondary antibody (Beijing Zhong Shan Biotech Co. Ltd, BeiJing, China) at 37°C for 30 min. Then the sections were stained with 3,3-diaminobenzidine tetrahydrochloride for 5 min and nuclei were counterstained with hematoxylin for 3 min, and then mounted with neutral balsam. PBS was used to replace the antibody as a negative control.
The immunostaining results were evaluated by three pathologists who were blinded to the clinical details of patients. TROP2 expression was defined as the presence of yellow-brown membrane staining of tumor cells. Each sample should be estimated including the staining intensity and percentage of positive tumor cells with no less than 1000 cells and 5 high power fields. The intensity of staining was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong), while the percentage of positive cells was scored as 0 (0%), 1 (1–10%), 2(11–50%) and 3 (51–100%). The overall immunohistochemical staining results were based on the intensity score×percentage staining score as follows: − (score 0); +(score 1, 2, 3); ++(score 4, 6); +++(score 9)
Four human cervical cancer cell lines CaSki, Siha, HeLa and C33A cells were purchased from the American Type Culture Collection (Manassas, VA, USA), cultured in Dulbecco-modified Eagle medium (DMEM; Gibco Inc., Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Invitrogen) in a humidified incubator at 37°C and 5% CO2 atmosphere.
Siha and CaSki cells were cultured on glass slides in a 6-well plate and incubated for 24 h. Cells were fixed with 4% paraformaldehyde and blocked with normal goat’s serum for 30 min at 37°C. After thorough washing with Tris-buffered saline (TBS), the cells were incubated with primary anti-TROP2 monoclonal antibody (Santa Cruz, USA, 1∶500 dilution) overnight at 4°C and stained with FITC conjugated anti-mouse IgG (Beijing Zhong Shan Biotech Co. Ltd, BeiJing, China, 1∶200 dilution) for 30 min. The fluorescence-labeled TROP2 was observed under a fluorescence microscope and photograph was taken.
To knock down endogenous TROP2 expression, two pairs of siRNA sequences targeting TROP2 were designed and synthesized by Genepharma Co., Ltd (Shanghai, China). These sequences were: siRNA-1100, 5′-GCACGCUCAUCUAUUACCUTT-3′, 5′-AGGUAAUAGAUGAGCGUGCTT-3; siRNA-550, 5′-CCAAGUGUCUGCUGCUCAATT-3′, 5′-UUGAGCAGCAGACACUUGGTT-3′. Negative scramble control sequences were: 5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′. To study the effects of TROP2 enforced expression, the TROP2 isoform expressing plasmid was employed in HeLa and C33A cells. The human TROP2 full length cDNA was amplified and inserted into the pcDNA 3.1 vector (Genepharma Co., Ltd Shanghai, China) to obtain pcDNA3.1-TROP2. For transfection, cells were seeded into 6-well plates and expected to be 50% confluency next day. After cell attachment, TROP2 siRNA or the recombinant pcDNA3.1-TROP2 were transfected into cells in Opti-MEM (Invitrogen) using the Lipofectamime 2000 transfection reagent (Invitrogen) according to the manufacturer’s instruction, culture medium was replaced after 6 hours of incubation. After 48 h transfection, cells were counted and subjected to cell viability assay and western blot analysis. Untransfected cells were thought to be blank control, cells transfected with scrambled siRNA or pcDNA3.1(empty vector) were considered as negative control.
Cell viability was determined using a cell counting kit-8 (CCK-8) in accordance with the manufacturer’s protocol (Jingmei biotech, Shanghai, China). Briefly, control and transfected cells were seeded at 5000 per well in 96-well plates, after treated with cisplatin (Sigma, St. Louis, MO, USA) or MEK inhibitor U0126 (Cell Signaling Technology, Beverly, MA, USA) at indicated time points, 10 ul of CCK-8 was added to each well, then incubated for an additional 2 h at 37°C. The optical density (OD) was measured using a microplate reader (Bio-Rad Model 680, Richmond, CA, USA) at 450 nm wavelength. The inhibitory concentrations of 50% proliferation (IC50) of cisplatin were calculated by GraphPad Prism software. The experiment was repeated three times.
At 48 h after transfection, the cells were collected and washed twice with cold PBS, resuspended in 400 uL Annexin V-FITC binding buffer at a density of 1×106 cells/ml. Cells were stained with 5 uL of Annexin V-FITC and 10 ul propidium iodide (PI) according to the Apoptosis Detection Kit (Jingmei biotech, Shanghai, China) instructions. Then subjected to flow cytometry (BD, San Jose, CA, USA) to detect cell apoptosis. This experiment was conducted three times.
Cells transfected with TROP2 siRNA or pcDNA3.1-TROP2 were harvested at 48 h after transfection, collected by trypsinization and fixed in 75% cold ethanol for 1 h at −20°C. After being washed with PBS, cells were incubated with 100 µl RNase A (100 µg/mL) and 400 µl propidium iodide respectively for 30 min at 37°C. Finally, the cell cycle was measured by flow cytometry using a FACScan flow cytometer (BD, San Jose, CA, USA) at 488 nm, and the relative ratios of the G1, S, and G2 phases was analyzed by FlowJo 2.8 software. The experiment was performed in triplicate.
The monolayer wound healing assay was used to assess cell migration ability. Cells (5×105) were seeded in 6-well plates, incubated overnight, then transfected with TROP2 siRNA or pcDNA3.1-TROP2. After achieving 90% confluency, the cell monolayer was scratched with a sterile pipette tip, floating cells were removed with PBS and cultured again in RPMI 1640 medium containing 1% FBS. Photographic images were taken at 0, 24 and 48 h along the scrape line by microscope. Results were expressed as relative scratch width, based the distance migrated relative to the original scratched distance. The experiment was conducted in triplicate.
The invasive ability of cervical cancer cells was assessed using a 24-well transwell chamber (cell invasion assay kit), by calculating the cells passed through a polycarbonate membrane (8-µm pore size) (Corning Costar, New York, USA). The polycarbonate surface of each chamber was covered with 20 uL matrigel (BD Biosciences, USA; 1∶4 dilution) to create an artificial basement membrane. Cells (1×105 cells) transfected with TROP2 siRNA or pcDNA3.1-TROP2 were suspended in 200 ul serum-free 1640 medium and cultured in the upper transwell chamber for 24 h at 37°C, the lower chamber was filled with 600 uL of 1640 medium supplemented with 10% FBS. The non-invading cells attached to the upper surface of the membrane were removed with a sterile cotton swab, and the invasion cells penetrated through the membrane were stained with 0.1% crystal violet for 20 min at room temperature. The numbers of cells were calculated under a Leica microscope in eight random fields. The experiment was repeated three times for each group.
At 24 h after transfection, the transfected cells and control cells were exposed to different concentrations of cisplatin for 48 h. Then the cells were fixed with 4% paraformaldehyde for 15 minutes followed by staining with 100 µg/ml of Hoechst 33258 (Beyotime, Shanghai, China) at room temperature in the dark for 30 minutes, the apoptotic features were assessed by observing chromatin condensation or fragments under a fluorescence microscope with an excitation wavelength of 340 to 360 nm.
Equivalent amount of the cell protein samples were loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF membranes using the Bio-Rad electrotransfer system. After blocked by 5% w/v nonfat dried milk for 1 h, the membranes were incubated with primary antibodies specific to TROP2 (Santa Cruz Biotechnolog, CA, USA; 1∶1000 dilution), E-cadherin, cyclin D1, cyclin E, CDK2, CDK4, Erk1/2, p-Erk1/2, P27, bcl-2 and bax (Cell Signaling Technology, Beverly, MA, USA; 1∶1000 dilution) overnight at 4°C. Then incubated with horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody (Beijing Zhong Shan Biotech Co. Ltd, BeiJing, China; 1∶4000 dilution ) for 1 h, the protein bands were visualized by enhanced chemiluminescence (Millipore) and analyzed densitometrically using Quantity One Image software (Bio-Rad, USA). β-actin (Beijing Zhong Shan Biotech Co. Ltd, Beijing, China; 1∶1000 dilution) was used as internal control for protein loading and analysis.
SPSS (SPSS Inc, Chicago, IL, USA) 18.0 software was used to perform statistical analysis.
Quantitative data were expressed as the mean values ± standard deviation (SD). Differences between groups were evaluated by unpaired Student’s t test or one-way ANOVA. Pearson chi-square test or Fisher’s exact test was used for analysis between the degree of staining and clinical parameters, correlation analysis was performed with Spearman rank correlation coefficient. Kaplan–Meier curves were plotted to describe the survival information by the log-rank test. Multivariate analysis was performed using a Cox’s proportional hazards regression model. P<0.05 was considered statistically significant.
In total, 160 samples were determined by immunohistochemistry, including 20 normal cervical tissues, 34 CIN tissues and 106 cervical cancer tissues. Demographic data and tumor characteristics are described in
A–B. Negative and weak TROP2 expression in normal cervical tissues. C. CIN III with strong TROP2 membrane staining (score +++). D–E. Strong and weak TROP2 expression in cervical sqamous cell carcinoma (score +++, +). F. Strong TROP2 expression in cervical adenocarcinoma (score +++). G–H. Ki-67 expression in CINIII and cervical cancer (score ++, +++). Magnifications: ×200 (A, B, C, D, E) and ×400 (F, G, H).
For the sake of verifying the association between TROP2 expression and cancer progression, we also analyzed the expression of Ki-67 in the same sets of cervix specimens by immunohistochemistry assay (
Ki-67 expression | ||||||||||
Total | − | + | ++ | +++ | p | |||||
20 | 15 (75.0%) | 3 (15.0%) | 2 (10.0%) | 0 (0%) | <0.001 | |||||
34 | 15 (44.1%) | 6 (17.6%) | 7 (20.6%) | 6 (17.6%) | ||||||
106 | 22 (20.7%) | 22 (20.7%) | 25 (23.6%) | 37 (34.9%) | ||||||
− | 12 | 7 (58.3%) | 4 (33.3%) | 1 (8.3%) | 0 (0%) | <0.001 | ||||
+ | 30 | 8 (26.7%) | 6 (20%) | 9 (30%) | 7 (23.3%) | |||||
++ | 32 | 6 (18.7%) | 4 (12.5%) | 7 (21.9%) | 15 (46.8%) | |||||
+++ | 32 | 1 (3.1%) | 8 (25%) | 8 (25%) | 15 (46.8%) |
The clinical significances of TROP2 overexpression in patients with cervical cancer were summarized in
To evaluate the impact of TROP2 expression and clinicopathological features on clinical outcomes of the patients, we used Kaplan -Meier analysis and the log-rank test for censored survival data. As displayed in
Parameter | Univariate analysis | Multivariate analysis | ||||||||||
p |
p |
p |
RR | 95% CI | p |
RR | 95% CI | |||||
0.000 | 0.000 | 0.022 | 1.891 | 1.097–3.261 | 0.016 | 1.952 | 1.136–3.357 | |||||
(Positive vs Negative) | ||||||||||||
0. 427 | 0.402 | |||||||||||
(>50 vs ≤50) | ||||||||||||
0.583 | 0.578 | |||||||||||
(Squamous vs Adenocarcinoma) | ||||||||||||
0.001 | 0.001 | 0.616 | 1.194 | 0.598–2.383 | 0.599 | 1.208 | 0.597–2.444 | |||||
(I vs II vs III–IV) | ||||||||||||
0.000 | 0.000 | 0.023 | 1.804 | 1.084–2.999 | 0.040 | 1.706 | 1.026–2.838 | |||||
(well vs moderately vs poorly) | ||||||||||||
0.000 | 0.000 | 0.002 | 0.343 | 0.173–0.678 | 0.005 | 0.375 | 0.190–0.743 | |||||
(yes vs no) | ||||||||||||
0.000 | 0.000 | 0.547 | 0.759 | 0.309–1.863 | 0.535 | 0.753 | 0.307–1.844 | |||||
(<1/2 vs ≥1/2) | ||||||||||||
0.211 | 0.201 | |||||||||||
(≤4 cm vs >4 cm) |
Overall survival(OS).
Progression-free survival (PFS).
RR: relative risk.
Given the evidence that TROP2 elevated expression was closely associated with tumor aggressiveness and poor clinical prognosis, we further investigated the functional consequences of TROP2 expression in cervical cancer cell lines. First of all, we examined the TROP2 expression in four cervical cancer cell lines Siha, HeLa and CaSki and C33A by Western blot (
A. The expression of TROP2 was found in four cervical cancer cell lines by western blot, Siha and CaSki cells showed higher expression compared with HeLa and C33A cells, β-actin was used as internal control. B. Immunofluorescence assay demonstrated there was a layer of green fluorescent staining of the cytomembrane in Siha and CaSki cells. C. Western blot analysis to confirm the siRNA mediated knockdown and pcDNA3.1-TROP2 mediated overexpression of TROP2 in different cervical cancer cell lines. D. Cells were transfected with TROP2 siRNA or pcDNA3.1-TROP2, the viability was assessed using a CCK-8 assay at five time points (0, 1, 2, 3, 4 days, respectively). At 2 days after transfection, knockdown of TROP2 elicited a significant inhibit effect on cell proliferation in Siha and CaSki cells, while enforced expression of TROP2 increased cell viability of HeLa and C33A cells. Data are expressed as mean ± SD of three independent experiments, *p<0.05, **p<0.01.
CCK-8 assay was used to determine the effect of TROP2 expression on cell proliferation. Our results revealed that knockdown of TROP2 evoked a markedly inhibition effect on the proliferation after transfection in Siha and CaSki cells (p<0.05), whereas the cell viability in the pcDNA3.1-TROP2 transfection group was improved significantly (p<0.05;
It has been demonstrated that cell apoptosis plays a considerable role in the progression and development of tumors, we further explored whether the cell proliferation inhibition due to the apoptosis-induced function. Flow cytometry was used to determine cell apoptosis at 48 h after transfection. The results showed that the total cell apoptosis rates of the TROP2 siRNA transfection group (siRNA-1100 and siRNA-550) was significantly higher compared with negative control group in both Siha and CaSki cells (p<0.05;
A. Down-regulation of TROP2 induced cell apoptosis in Siha and CaSki cells. Cells were harvested at 48 h after transfection, followed by apoptosis assay using the annexin V-FITC apoptosis detection kit. Cells in the right lower and upper quadrants are consider as early and late apoptosis respectively, in the left upper quadrants are considered as dead cells. The results were analyzed by FlowJo software. B. Enforced expression of TROP2 significantly inhibited cell apoptosis C. Western blot analysis showed that down-regulation of TROP2 reduced the expression of bcl-2 and increased the expression of bax in Siha and CaSki cells. D. Enforced expression of TROP2 in HeLa and C33A cells increased the expression of bcl-2 and reduced the expression bax. These data are expressed as mean ± SD of three independent experiments, *p<0.05, **P<0.01.
To obtain a more comprehensive understanding of TROP2 gene function, we further investigated the effects of TROP2 expression on the cell cycle by flow cytometry analysis. As shown in
A. Knockdown of TROP2 induced G1 arrest in Siha and CaSki cells. Siha and CaSki cells were transfected with TROP2 siRNA or scrambled siRNA. 48-regulation of TROP2 induced cell cycle arrest in G1 phase. B. Overexpression of TROP2 promoted G1-S and G2-M transitions in HeLa and C33A cells. C, D. TROP2 regulated cell cycle factors via ERK1/2 signaling pathway in cervical cancer cells. Siha and CaSki cells (C) were transfected with TROP2 siRNA or scrambled siRNA. 48 h later, Western blot analysis showed that the changes in cell cycle distribution was accompanied by reduced expression of cyclin D1, cyclin E, CDK4, CDK2 and increased expression p27. Knockdown of TROP2 also inhibited the phosphorylation of ERK1/2. HeLa and C33A cells (D) were transfected with pcDNA3.1-TROP2, and 48 h post transfection, protein were extracted and subjected to Western blot analysis of related proteins. Data are expressed as mean ± SD of three independent experiments, *p<0.05, **P<0.01.
ERK1/2 (extracellular signal regulated kinase) is a key member of MAPK (mitogen-activated protein kinases) signaling pathway, can be activated by a variety of stimuli, the posphorylation of ERK1/2 is involved in a lot of biological behaviors, such as cell proliferation, cell cycle, adhesion and invasion, as well as survival and metastasis. Recent studies have demonstrated that an involvement of TROP2 in MAPK signaling pathway, so we hypothesized that the posphorylation of ERK1/2 may also contribute to the changes of cell cycle mediated by TROP2 in cervical cancer. As shown in
Our immunohistochemical analysis demonstrated that high expression of TROP2 was positively correlated with lymphatic metastasis, therefore we further tested whether TROP2 deletion would inhibit invasion and migration of cervical cancer cells. The serum-stimulated matrigel invasion assay showed that the number of Siha and CaSki cells that passed through the polycarbonat emembrane in the TROP2 siRNA transfection group was significantly less than that in the negative control group (p<0.05;
A. Down-regulation of TROP2 inhibited cell invasion of Siha and CaSki cells. At 24 h after transfection, cells (1×105) were reseeded in the upper Transwell chamber for 24 h and then stained with crystal violet. TROP2 siRNA group contained significantly less invasive cells than the control groups. B. Up-regulation of TROP2 significantly increased the invasive ability of HeLa and C33A cells at 48 h after transfection. Data are mean ± SD, *p<0.05, **P<0.01.
A. Knockdown of TROP2 exhibited a slower wound recovery compared to the control groups at 24-cadherin expression of Siha and CaSki cells. D. Enforced expression of TROP2 reduced the E-cadherin expression of HeLa and C33A cells.
Previous results showed that knockdown of TROP2 inhibited the proliferation and invasion of Siha and CaSki cells, we further investigated whether TROP2 plays a role in the chemotherapy of cervical cancer. Cisplatin is a widely used anticancer agent, has also been the primary agent used in the combination chemotherapy for cervical cancer patients. To determine the sensitivity of cells to cisplatin, at 24 h after transfection with siRNA-1100, CaSki cells were exposed to different concentration of cisplatin (CDDP) ranging from 0 to 50 µM for 48 h, as Siha cells turned out to be more resistant, they were treated with concentrations from 0 to 100 µM, the cell viability was examined by CCK-8 assay. As shown in
A. Effect of cisplatin on Siha and CaSki cells growth. Cells were transfected with TROP2 siRNA-1100 to deplete endogenous TROP2 expression, 24 h later, they were exposed to CDDP for another 48 h. Cell viability was determined by CCK-8 assay. B. Graphic representation of the mean IC50 values of cisplatin in Siha and CaSki cells. Cells transfected with TROP2 siRNA-1100 were more sensitive to cisplatin than control cells. C. Morphological transformation of Siha and CaSki cells exposed to cisplatin was observed under a fluorescence microscope, Hoechst 33258 staining indicated that there were more apoptotic cells in TROP2 siRNA group, compared with control groups. Red arrows indicate apoptotic cells. D. Western blot analysis showed TROP2 siRNA-1100 decreased the expression of pERK1/2 after cisplatin treatment. Siha cells were tansfected with TROP2 siRNA-1100 or scrambled siRNA, 24 h later, the cells were exposed to 30 µM cisplatin for another 48 h, and the levels of pERK1/2 was analyzed by western blot. E. ERK inhibitor U0126 enhanced TROP2 siRNA-1100 mediated cisplatin sensitivity of Siha cells. U0126 at the concentration of 10 µmol/l was used 2 h before cisplatin (30 µM) treatment. Data are presented as mean ± SD of three independent experiments, *p<0.05, **P<0.01.
As a calcium signal transducer, TROP2 has become a recent focus in tumor research. There are increasing evidences show that TROP2 overexpression is significantly associated with an aggressive malignant phenotype and poor prognosis in various cancer tissues
In present study, we characterized the expression pattern of TROP2 via immunohistochemistry in 106 cervical cancers, 34 CIN and 20 normal cervical tissues. The results showed that TROP2 was highly expressed in cervical cancer tissues (88.7%), but relatively low expression was found in normal (45%) and CIN tissues (64.7%) (P<0.001), and the percentage of TROP2 - positive cells increased progressively from CINI to CINIII, suggesting that TROP2 protein expression was significantly associated with the cervical oncogenesis and the development of cervical cancer. Moreover, the data of present study showed that the expression of TROP2 was tightly related with lymphatic metastasis and histological grade,patients with TROP2-positive staining exhibited a significantly decreased overall survival and progression free survival, it was also considered to be an independent prognostic predictor for cervical cancer patients according to multivariate analysis. The presented data herein is consistent with previous reports suggest that TROP2 may play a substantial role in tumor biological aggressiveness and metastasis
To better understand the potential connection between TROP2 expression and the biological features of cervical cancer cells, we studied the effect of gain-or-loss of TROP2 expression through ectopic over-expression or RNAi mediated knockdown. In accord with the results obtained in immunohistochemical analysis, vitro study also showed that TROP2 was able to promote cell proliferation by inhibiting cell apoptosis and accelerating cell cycle progression. Evasion of apoptosis is considered to be one of the major hallmarks of tumor development. Bcl-2 and Bax are two critical regulators of cell apoptosis and play pivotal roles in anti-apoptotic and pro-apoptotic, respectively
Using PI staining, down-regulation of TROP2 in Siha and CaSki cells induced the cells to accumulate in G1 phase but reducing the cells in the S phase, while enhanced TROP2 expression was required for the growth of HeLa and C33A cells through promoting G1-S transition. A tentative reason might be attributed to certain TROP2 -dependent signaling events that promote up-regulation of growth factors or the of down-regulation growth inhibitory factors, so we further analyzed several cell cycle regulators expression in the four cervical cancer cell lines. CyclinE/CDK2 complex are regulators at the late G1 stage and play an important role in the initiation of DNA replication
Our immunohistochemical assay showed that TROP2 expression was significantly related with lymphatic metastasis, suggested that TROP2 may be involved in invasion and metastasis of cervical cancer. Tumor metastasis is a complex and multistep process including cancer cell migration, adhesion and invasion and so on. By wound healing and transwell invasion assay, we found that TROP2 has some putative activities in promoting the invasive capability of tumor cells. E-cadherin is a calcium- dependent cell adhesion molecule, plays a considerable role in the formation of adhesion connections and the maintenance of epithelial cell function
Many researchers speculated that TROP2 might be a cell-surface signal transducer based on the finding that the cytoplasmic tail domain of TROP2 contains several potential phosphoinositide-binding sites. Recently, the regulatory network of TROP2 was seriously studied by S Alberti et al
Furthermore, we determined the association between TROP2 expression and CDDP sensitivity in cervical cancer cells. The results showed that down-regulation of TROP2 sensitizes Siha and CaSki cells to cisplatin treatment by decreased activity of pERK1/2. Using specific ERK inhibitor U0126 effectively strengthened TROP2- siRNA mediated cisplatin sensitivity. Theses finding indicated the role of ERK1/2 pathway in TROP2-siRNA mediated cisplatin sensitivity.
Major progress in immunotherapy targeted TROP2 has been achieved in lots of cancers.
Recent studies demonstrated that a humanized version of TROP2-specific monoclonal antibody (hRS7) labeled with a radioiodinated, diethylenetri-aminepentaacetic acid-appended peptide, 131I-IMP-R4, exhibited a desirable therapeutic effect in a breast cancer model in vivo
In conclusion, we have demonstrated that the elevated expression of TROP2 in cervical cancer contributes to aggressive phenotypes and poor survival. Meanwhile, silencing TROP2 expression in cervical cancer cells suppresses the proliferation and invasion, also sensitizes cells to cisplatin and induces cell cycle arrest by suppression of ERK1/2 signaling pathway. Therefore, TROP2 has potential as an attractive prognostic marker and a new target for cervical cancer treatment.