Correlation of Twist upregulation and senescence bypass during the progression and metastasis of cervical cancer

Abstract

Cervical carcinoma is associated with high propensity for local invasion and lymph node metastasis. However, the molecular alterations that drive progression and metastasis of cervical cancer remain unclear. Cellular senescence has been proposed as the mechanism that protects an organism against cancer progression and metastasis. In addition, Twist, a basic helix-loop-helix transcription factor, has been suggested as an oncogene because it is overexpressed in many types of human cancer. This gene also exhibits a positive function in regulating invasion and metastasis. In this study, Twist was strongly and positively expressed in normal tissue, squamous cell carcinoma (SCC) IA--IIA, and SCC IIB--IIIB (4.3%, 44%, and 88.9%, respectively). The strong positive expressions of the senescence marker CBX3 were 39.1%, 32%, and 15.6%, respectively. The strong positive expressions of Twist in the SCC groups with or without lymph node metastasis were 80.8% and 50%. For CBX3, such expressions were 7.7% and 29.5%, respectively. Results also showed that the expression of Twist was inversely correlated with that of CBX3. Moreover, the knockdown of Twist with target siRNA in SiHa triggered the induction of the chromatin marker of the cellular senescence CBX3 and senescence-associated β-galactosidase activity. Our results suggested that the expression of Twist increased during the progression and metastasis of cervical cancer. Furthermore, Twist-induced senescence bypass is important in this process.

Keywords

cervical cancer / senescence / Twist / CBX3 / lymph node metastasis

Introduction

Cervical cancer is the second most common malignancy in women, and 86% of cervical cancer cases are reported in developing countries; human papillomaviruses are the common causative agents of cervical cancer [1]. Despite the generally good prognosis of patients diagnosed with an early-stage cervical cancer, approximately one-third of these patients die as a result of metastasis and recurrence [2]. As potential targets of cancer diagnosis and treatment, molecules involved in cancer development, metastasis, and outcome should be identified and functionally characterized. In this way, mortality associated with cervical cancer can be reduced.

Cellular senescence has been proposed as a mechanism that protects an organism against the initiation and progression of cancer [3]. Oncogene-induced senescence (OIS) is characterized by the accumulation of CBX3 (HP1γ), a marker of senescence-associated heterochromatin foci [4,5], and the upregulation of p53 and p16Ink4a, two effectors of cell cycle arrest [6,7]. Numerous reports have demonstrated the occurrence of OIS in patients and various experimental models and OIS bypass might be an important mechanism for a tumor to progress to malignancy and metastasis. [8]. Fewer studies have examined the effects of OIS in the metastasis of cervical cancer compared with other solid tumors, such as mammary and prostate tumors.

Twists are highly conserved basic helix-loop-helix transcription factors that function as important regulators during embryogenesis and tumor progression and metastasis [9]. In 2008, Ansieau et al. reported that the ablation of Twist expression in murine breast cancer cells or in melanoma cells can cause G₁ cell cycle arrest and cellular senescence [10]. In addition, the overexpression of Twist suppresses the senescence of immortalized human prostate epithelial cell lines [11]. Despite the increasing reports linking Twist to the progression and metastasis of human cancers, the clinical significance of Twist expression and its correlation with senescence in cervical cancer have not been adequately studied.

In this study, the expression and the association of the transcript factor Twist and the senescence marker CBX3 were analyzed in different stages of squamous cell carcinoma (SCC) with respect to tumor progression and metastasis by immunohistochemistry. The functions of Twist-induced senescence bypass were further identified in cervical cancer cell lines.

Materials and methods

Specimens

A total of 93 archival formalin-fixed, paraffin-embedded cervical specimens were obtained from the Department of Pathology at Tongji Hospital from 2011 to 2012. The following specimens were included: 23 cases of normal cervical tissue and 70 cases of SCC. The SCC specimens were futher divided into two histological groups: 25 cases of early stage SCC (IA-IIA) and 45 cases of late stage SCC (SCC IIB-IIIB). Patients who received a cytology test and cervical therapy or preoperative treatment, such as radio-therapy and chemotherapy, within sixβmonths were excluded. One pathologist independently reviewed the slides of these cases to confirm the diagnosis. All of the cases were also reviewed by two pathologists by using a multi-headed microscope to establish a consensus diagnosis. Informed consent was obtained from each patient before the samples were used.

Immunohistochemistry

Immunohistochemistry was performed as previously described [12]. The sections were incubated with monoclonal anti-Twist (1:200, Abcam, USA) or polyclonal anti-CBX3 (1:200, Santa Cruz, USA) in a humidified chamber overnight at 4°C. The sections were then incubated with secondary antibody and processed with universal SP histostain™-plus kit (ZYMED, Carlsbad, CA, USA). The slides were counterstained with hematoxylin. Normal mouse IgG was used as a negative control for Twist and normal rabbit IgG was used for CBX3. The specimens from mammary gland carcinoma were used as the positive control samples. Staining was scored as strong, moderate, weak, and negative. In particular, immunostaining intensity was scored based on the percentage positivity of the stained cells as negative (0% to 10%), weak (10% to 30%), moderate (30% to 50%), and strong (>51%). Each case was scored independently by two different investigators.

Cells, plasmids, and transfection procedures

The cervical cancer cell line SiHa was obtained from the American Type Culture Collection. SiHa was maintained and propagated in vitro by serial passage in DMEM (GIBCO) supplemented with 10% fetal bovine serum.

Small interfering RNA against Twist (siTwist) was transfected into SiHa cells by using lipofectamine transfection reagent according to the manufacturer’s instructions (Life). The target sequence (5′-CCTCTGCATTCTGATAGAA-3′) and the negative control siRNA (siNC) were purchased from Ribobio (Guangzhou, China).

Real-time PCR

Total RNA was extracted from cultured cells by using Trizol reagent (Takara, Japan). For each sample, 2 µg of RNA was reverse-transcribed using the ReverTra Ace qPCR RT kit (Toyobo, Japan) according to the manufacturer’s protocols. Real-time PCR was performed using the SYBR Green qPCR Master Mix (DBI, USA) on a CFX Connect real-time system (Biorad, USA). The following conditions were used: 40 cycles of three-step PCR (95°C for 40 s, 60°C for 50 s, and 72°C for 30 s) after initial denaturation (95°C for 5 min). All of the primers were supplied by Invitrogen.

The following primers were used: for Twist expression, F 5′-GAGCGACGAGATGGACAATAAGA-3′ and R 5′-ATGCGCCACACGGAGAA-3′ (product size, 84 bp); and for GAPDH (housekeeping gene) expression, F 5′-TGCACCACCAACTGCTTAGC-3′ and R 5′-GGCATGGACTGTGGTCATGAG-3′ (product size, 87 bp).

Western blot

After transfection, the cells were lysed in ice-cold RIPA lysis buffer containing a protease inhibitor cocktail. Approximately 60 µg of protein was separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in Tris-buffered saline containing 5% skim milk at room temperature for 1 h and then incubated with primary antibody at 4°C for 12 h. Afterward, these membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse/rabbit antibody at a dilution of 1:3000 at room temperature for 1 h. Signals were detected on an X-ray film by using the ECL detection system (Pierce, Rockford, IL, USA). Loading differences were normalized using a monoclonal GAPDH antibody.

Migration and invasion assays

Cells that migrated through a filter or invaded through a biological barrier were determined by transwell insert chambers (Becton Dickinson, Franklin Lakes, NJ) with 8 µm pore filters. Cells at a density of 2 × 10⁵ cells/ml were seeded in the upper chambers with 200 µl of serum-free DMEM, and the lower wells were filled with 750 µl of DMEM containing 10% FBS as an inducer of cell migration. For the invasion assay, a matrix barrier was formed by coating Matrigel at 5 mg/ml in serum-free DMEM. After an incubation period of 48 h at 37°C, the cells that migrated to the lower surface of the filter were fixed with 5% glutaraldehyde in PBS for 10 min, stained with 0.5% toluidine blue (Sigma-Aldrich, St. Louis, MO), and counted. Each experiment was carried out in triplicate to ensure consistency.

Immunocytochemistry

For immunocytochemistry staining, the cells were seeded onto a 12-well chamber slide, treated under the indicated conditions, fixed with 4% paraformaldehyde, and permeabilized by PBS. Afterward, the permeabilized cells were blocked with goat blocking serum, incubated with primary antibody at 4°C overnight and washed. The cells were also incubated with secondary antibody and processed using the universal SP histostain™-plus kit. The slides were then counterstained with hematoxylin.

SA-β-galactosidase (SA-β-gal) staining

The cells were fixed in 3% formaldehyde and washed thrice with PBS. These cells were then incubated at 37°C for 12 h in a buffer containing 40 mM sodium phosphate (pH= 6), 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆, 150 mM NaCl, 2 mM MgCl₂, and 1 mg/ml X-Gal according to the manufacturer’s instructions (Beyotime).

Statistical analysis

Statistical analysis was performed using SPSS 13.0 (Chicago, IL, USA). The differences in Twist and CBX3 expressions from the three histological groups of specimens were analyzed using Mann-Whitney test. Pearson’s chi-square test and Fisher’s exact test (wherever applicable) were used to compare the clinical characteristics of patients with or without lymph node metastasis and the differences between Twist or CBX3 expression. For all tests, P<0.05 was considered significant.

Results

Twist upregulation is correlated with cervical cancer progression

Fig. 1 and Table 1 reveal the expression of Twist in all SCC specimens, with 44% (11/25) of SCC IA-IIA and 88.9% (40/45) of SCC IIB-IIIB showing a high level of staining (moderate and strong). By contrast, only 4.3% (1/23) of normal cervical tissue showed similar intensity levels of staining. Statistical analysis showed that Twist expression levels in SCC were significantly higher than that in normal cervical tissue (P<0.05).

Twist upregulation is correlated with the possibility of metastasis

Carcinoma cells invade the lymphatic vessels and this invasion is a critical step in the formation of distant metastasis of cervical cancer cells. Thus, the presence of cervical cancer cells in lymph nodes is considered as an indicator of metastatic potential.

We analyzed 70 specimens of cervical cancer. In the SCC groups with or without lymph node metastasis, the strong positive expressions of Twist were 88.5% and 63.6%, respectively, and the differences were statistically significant (Fig. 1 and Table 2; P<0.05).

Expression of CBX3 in cervical specimens

The expression of CBX3 in the three histological stages was studied. Fig. 1 and Table 3 reveal that a decrease in the number of cases showed the strong positive expressions of CBX3 in the progression of cervical cancer: 39.1% (9/23) of normal cervical epithelium; 32% (8/25) of SCC IA-IIA; and 15.6% (7/45) of SCC IIB-IIIB. Statistical analysis showed that CBX3 expression levels in SCC were significantly lower than that in normal cervical tissue (P<0.05).

We analyzed the expression of CBX3 in SCC subgroups with or without lymph node metastasis. Fig.1 and Table 4 show that the expressions of CBX3 were significantly different between the subgroups with or without lymph node metastasis (P<0.05).

Twist regulates motility and inhibits cell senescence in human squamous cervical cancer cells

To further validate the association between Twist and senescence, we transfected squamous cervical cancer cell SiHa with siRNA targeting Twist and found that Twist expression was effectively silenced at both mRNA and protein levels (Fig. 2A and 2B). In addition, the inhibition of Twist significantly reduced the migration and invasion of SiHa (Fig. 2C and 2D). Moreover, the knockdown of Twist invariably triggered the cellular senescence characterized by the induction of the chromatin marker of cellular senescence CBX3 and SA-β-gal activity (Fig. 2E).

Discussion

Cervical cancer is the second most frequently diagnosed malignancy in women worldwide; this type of cancer is also considered as a major gynecologic malignancy staged clinically according to the International Federation of Obstetrics and Gynecology (FIGO) recommendations [13]. In patients with cervical cancer, invasion and metastasis are the primary causes of treatment failure and subsequent death. Metastasis to lymphatic vessels is an indicator of an unfavorable prognosis, even in an early FIGO stage [14]. However, the molecular alterations that drive the invasion and metastasis of cervical cancer and predict prognosis have not been well established yet. Such alterations should be determined to reveal the targets of diagnosis and therapy and provide insights into the molecular mechanisms of the invasion and metastasis of cervical cancer.

Cellular senescence is an intrinsic protective mechanism that removes uncontrolled proliferating cells and functions as a safeguard against immortalization, malignant transformation, and progression [15]. Numerous reports have demonstrated the occurrence of senescence in patients and various experimental models [16]. Moreover, Ansieau and coworkers [10] challenged the view that metastasis corresponds to the final step of the tumorigenic cascade. They particularly proposed that the dissemination of primary tumor cells residing at the invasive edge couples the metastatic spread to the bypass of senescence. Thus, mechanisms that allow a cancer cell to bypass senescence should exist so that a tumor could progress to malignancy. Several EMT-inducing transcription factors, such as Twist, have been shown to mitigate the effects of OIS.

Twist is a key factor that promotes epithelial-mesenchymal transition during the metastasis of breast cancer [17]. Since then, numerous studies have reported the positive function of Twist in the development and progression of cancer by providing protection against apoptosis [18] and promoting cancer cell invasion by many signaling pathways [19]. However, few studies have presented the relationship of Twist and senescence with cervical cancer progression and metastasis.

We initially analyzed the association between Twist/senescence marker CBX3 and cervical cancer progression and metastasis. We also investigated the relationship between Twist and senescence in cervical cancer specimens. Furthermore, we demonstrated a negative correlation between Twist and CBX3 in the same patients. In addition, we provided the first evidence showing that the silencing of Twist upregulated the CBX3 expression and induced the SA-β-gal activity.

Twist is important in solid tumors. For example, high Twist expression induces EMT, mediates senescence, and correlates with poor prognosis of solid tumors [11,20]. Studies have shown that Twist also induces EMT and functions as a prognostic predictor of the clinical outcome of patients with cervical cancer [21]. Therefore, our findings strengthen the idea that proteins, such as Twist, Snail, and Zeb1, can elicit a double effect on tumor progression and metastasis by controlling the safeguard mechanism against cancer (cellular senescence) in addition to EMT [22]. Specifically, the dissemination of primary tumor cells undergoing EMT at the invasive edge may commence early in response to environmental cues; as a result, metastasis spreads to the bypass of senescence [23]. Our study provided evidence supporting that of Ansieau et al. [10] and elucidated the possible mechanism of cervical cancer progression and metastasis.

In summary, Twist performs a novel function in suppressing cellular senescence. This study also revealed the association of Twist with cervical cancer progression and metastasis. Therefore, Twist may enhance the progression and metastasis of cervical cancer by inducing senescence bypass.

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