Cancer Letters 353 (2014) 104–114

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The telomere/telomerase binding factor PinX1 regulates paclitaxel sensitivity depending on spindle assembly checkpoint in human cervical squamous cell carcinomas Xiao-Peng Tian a,1, Dong Qian a,1, Li-Ru He b,1, He Huang d, Shi-Juan Mai a, Chang-Peng Li a, Xiao-Xia Huang a, Mu-Yan Cai a,c, Yi-Ji Liao a, Hsiang-fu Kung a, Yi-Xin Zeng a, Dan Xie a,⇑ a

State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, China Department of Radiation Oncology, Sun Yat-Sen University Cancer Center, Guangzhou, China c Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, China d Department of Gynecology, Sun Yat-Sen University Cancer Center, Guangzhou, China b

a r t i c l e

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Article history: Received 13 May 2014 Received in revised form 3 July 2014 Accepted 9 July 2014

Keywords: Cervical squamous cell carcinomas PinX1 Paclitaxel Telomere dysfunction Spindle assembly checkpoint

a b s t r a c t Paclitaxel is a main ingredient in the combination chemotherapy treatment of advanced human cervical squamous cell carcinomas. We investigated the roles and underlying molecular mechanisms of PinX1 in cervical squamous cell carcinomas (CSCC) cells response to paclitaxel and its clinical significances. The expression dynamics of PinX1 was first examined by immunohistochemistry in 122 advanced CSCC patients treated with cisplatin/paclitaxel chemotherapy. The expression of PinX1 was significantly associated with the effects of cisplatin/paclitaxel chemotherapy in advanced CSCCs (P < 0.05). High expression of PinX1 correlated with CSCC’s response to cisplatin/paclitaxel chemotherapy, and was an independent predictor of shortened survival (P < 0.05). A series of in vivo and in vitro assays were performed to elucidate the function of PinX1 on CSCC cells chemosensitivity to paclitaxel and underlying mechanisms. In CSCC cells, the levels of PinX1 were only associated with the cytotoxicity and sensitivity of paclitaxel, in which knockdown of PinX1 dramatically enhanced paclitaxel cytotoxicity, whereas the reestablishment of PinX1 levels substantially reduced the paclitaxel-induced killing effect. In addition, we identified that the ability of PinX1 to stabilize the tension between sister kinetochores and maintain the spindle assembly checkpoint was the main reason CSCC cells undergo apoptosis when treated with paclitaxel, and further studies demonstrated that shortened distance between sisters kinetochores by nocodazole confers upon PinX1-replenished cells a sensitivity to the death inducing paclitaxel effects. Furthermore, our study of CSCC cells xenografts in nude mice confirmed the role of PinX1 in paclitaxel sensitivity in vivo. Our data reveal that PinX1 could be used as a novel predictor for CSCC patient response to paclitaxel, and the role of PinX1-mediated paclitaxel sensitivity might represent a new direction for the development of a new generation of microtubule drugs. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction Across the world cervical carcinoma is the third most common malignancy in women. Unfortunately, in developing countries, it is still the second most frequent cause of cancer-related death in women and its incidence rate is still increasing [1]. The clinical prognosis for most patients with cervical squamous cell carcinomas ⇑ Corresponding author at: State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, No. 651, Dongfeng Road East, 510060 Guangzhou, China. Tel.: +86 20 87343193; fax: +86 20 87343170. E-mail address: [email protected] (D. Xie). 1 These authors contributed equally to this work. 0304-3835/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

(CSCC) is poor unless the lesions are detected early [2,3]. Chemotherapy is an essential part of the treatment plan for women with CSCC. In some cases, chemotherapy is used concurrently with localized radiotherapy for those in advanced stage cancers or chemotherapy the primary treatment administrated in recurrent or metastatic cases [4–6]. The typical treatment plan for CSCC patients is a combination chemotherapy regimen, especially the cisplatin/ paclitaxel regimen. The dual chemotherapy treatment is considered to be superior to a single chemotherapy drug because many followup and pilot studies have demonstrated benefits in terms of overall survival (OS) and disease-free survival (DFS) [7–9]. On the other hand, this dual treatment option also has a potential risk in increased toxicity [10]. Thus, it is critical to identify reliable

X.-P. Tian et al. / Cancer Letters 353 (2014) 104–114

molecular characteristics in CSCC patients to determine who will benefit from the combination chemotherapy regimen. Paclitaxel (aka Taxol) is a pharmaceutical agent that targets microtubules and is a major component in the chemotherapy treatment for patients with advanced cervical cancer [9]. Paclitaxel interacts with b-tubulin to destroy the normal dynamics and reorganization of the network of microtubules. This unbalance of the network leads to tubulin depolymerization and microtubules stabilization, which results in mitotic arrest at metaphase and anaphase [11]. Furthermore, paclitaxel not only has been demonstrated as microtubule-binding drugs, but also has the function of spindle assembly checkpoint activation. [12]. An increasing body of evidence indicates that paclitaxel stabilization of the kinetochore microtubules in cells in metaphase results in a loss of tension at kinetochores [13]. This lack of tension induces a continuous activation of the spindle assembly checkpoint resulting in the arrest of cells at mitosis [14]. Therefore, the effects of paclitaxel cytotoxicity and sensitivity could likely be reversed by other molecules, which interfere with the spindle assembly checkpoint [12]. PinX1 was first identified in a yeast two-hybrid screen as a TRF1bind protein [15]. This conserved nuclear protein is an intrinsic telomerase inhibitor and therefore, also a putative tumor suppressor gene due to its role in telomerase activity [16]. In support of the tumor suppressor role, PinX1-null mice are embryonic lethal, while a majority of PinX1± mice spontaneously develop malignant tumors, which was demonstrated to be due to chromosome instability [17]. Recently, our group made the important discovery that not only does PinX1 contribute to telomere maintenance but also affects the sensitivity of cancer cells to DNA damage, which induces telomere dysfunction and apoptosis. Additionally, we observed that anthracyclines have the ability to disrupt telomere maintenance by increasing the ubiquitination and degradation of PinX1 and, thus, changing telomerase activity [18,19]. Furthermore, we revealed a correlation between PinX1 and esophageal squamous cell carcinoma (ESCC) resistance to chemo-radiotherapy. The mechanism of this resistance is likely associated with altered PinX1 activity in maintaining telomere stability and therefore, reducing ESCC cell death by RT-induced mitosis catastrophe (MC) [20]. Yonekawa et al. recently discovered that localization of PinX1 extends beyond the tail of telomere, suggesting a novel function(s) outside the established roles [21,22]. PinX1 has also been shown during mitosis to interact with the outer plate of kinetochores and the periphery of chromosomes. Knockdown of PinX1 causes a delay mitotic entry during mitosis stage. Also, shown by small interference RNA, loss of PinX1 abrogates faithful chromosome segregation, which causes anaphase chromatid bridges in mitosis and micronuclei in interphase [23]. Therefore, given the important role of PinX1, we investigated the role of PinX1 in advanced CSCC and the effects of the chemotherapy agent paclitaxel. We discovered both in vitro and in vivo that the efficacy of paclitaxel in CSCC is associated with the expressions levels of PinX1. This is the first report establishing a novel predictor for selecting patients with CSCC who would benefit from paclitaxel therapy and provides a promising new direction for the discovery of novel microtubule drugs.

Materials and methods Patients and tissue specimens In this study, 122 advanced CSCC patients were assigned from the Department of Gynecology, Cancer Center, Sun Yat-sen University between 2002 and 2010. Patients eligibility was determined by either International Federation of Gynecology and Obstetrics stage IVB or recurrent/persistent disease not suited to curative treatment with surgery or radiation therapy. The combination chemotherapy (D + P) group received paclitaxel at 135 mg/m2 and cisplatin at 50 mg/m2. This treatment was administered for six cycles for advanced CSCC patients, including


those with stable disease. Patients with a partial response and an acceptable toxicity level were continued treatment. The clinico-pathological characteristics of the patients are summarized in Table 1. After completion of the D + P chemotherapy, patients were evaluated as follows: physical examination, chest X-ray, abdominal ultrasonography, pelvic magnetic resonance imaging (MRI) and bone scan. The clinical response was defined according to the criteria adopted by the Response Evaluation Criteria in Solid Tumors (RECIST) (complete response: disappearance of all target and non-target lesions for at least 4 weeks; partial response: sum of the maximum diameter of all lesions decreased by >30%, no new lesions and no progression of disease; stable disease: sum of the maximum diameter of lesions decreased by 46b

63 59

28(47.8) 25(38.2)

35(52.2) 34(61.8)


Performance status 0 (fully active) 1 (restricted physically)

64 58

31(48.4) 22(37.9w)

33(51.6) 36(62.1)


Grade G1 G2 G3

7 70 45

4(57.1) 32(47.1) 17(37.8)

3(42.9) 38(52.9) 28(62.2)


Site of disease Pelvic Distant Both

51 59 12

21(41.2) 27(45.8) 5(41.7)

30(58.8) 32(54.2) 7(58.3)


Chemotherapy response Response (CR + PR) No response (SD + PD)

50 72

29(58.0) 27(37.5)

21(42.0) 45(62.5)


Abbreviations: CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease. a Chi-square test. b Median age. Immunofluorescence (IF)

Statistical analysis

To examine the telomere dysfunction-induced foci (TIF), cells were stained with the DNA damage factor cH2AX and the constitutive telomere protein component TRF1 in co-localizations studies [25]. Immunostaining with mouse anti-TRF1 antibody (1:100, GeneTex, San Antonio, TX, USA) and rabbit anti-cH2AX antibody (1:100, Cell Signaling Technology, Beverly, MA, USA) was performed as previously described [19].

All statistical analysis was performed using the SPSS software (SPSS Standard version 17.0). The associations between PinX1 expression and clinico-pathological features were assessed with the Chi-square test. Data derived from cell line and xenografts experiments are presented as mean ± SE and assessed by the student’s t test. P values of 0.05, student’s t-test). After paclitaxel treatment (5 mg/kg), the mean tumor volume in the C33A-PinX1shRNA group was 368 ± 85 mm3 and 864 ± 143 mm3 in the C33A-mock group (n = 8, P < 0.05, student’s t-test) (right). The values represent mean tumor volume ± SE. Mock, no-transfected cells; PinX1, PinX1-replenished cells. *, compare with the Mock group, **, compare with the Mock + paclitaxel group.


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after the paclitaxel treatment, P < 0.05). These results correlate with our in vitro data to illustrate that the reduced PinX1 protein levels enhance paclitaxel induced apoptosis of CSCC tumors in vivo. Discussion The telomerase-inhibitory domain of PinX1-C (amino acids 254-328) has been shown to block telomerase activity, shorten telomere, induce cellular disfunction and suppresses tumor growth [34]. However, PinX1 gene regulation and protein expression are vastly different in various tissues and tumors and thus, suggests that abnormal gene regulation and/or protein functions of PinX1 in tumourigenesis are complicated and may likely be tumortype-specific [35,36]. Previously, we described PinX1 as a novel predictor for CRT response in ESCC patients. In this study, we expanded our investigation of the role of PinX1 in a different tumor type, cervical squamous carcinoma. We discovered that PinX1 protein levels influence the cytotoxicity and sensitivity to paclitaxel treatment in CSCC cells and we also uncovered the underlying mechanism of PinX1 in paclitaxel treatment. We started our study by examining the PinX1 protein expression levels in 122 CSCC patients treated primarily with combination of cisplatin/paclitaxel chemotherapy. The expression of PinX1 in these patients was significantly associated with the response to chemotherapy, with higher levels of PinX1 reducing the efficacy of the chemotherapy. To further understand this PinX1/cisplatin/paclitaxel relationship, we moved our investigation to in vitro experiments. Our previous study demonstrated that in ESCC, PinX1 knockdown had no impact on ESCC cell sensitivity to cisplatin and moreover, cisplatin could not influence TIF incidences in PinX1 knocking-down ESCC cells [20]. In the present study, utilizing CSCC cell lines engineered to have PinX1 knocked down or overexpressed, we also determined that the levels of

PinX1 do not correlate with the cytotoxicity of cisplatin, but the cell viability was dependent on paclitaxel treatment. These results clearly indicate that with the patients’ response to the combination chemotherapy is likely due to the impact of PinX1 on cytotoxicity of paclitaxel. Furthermore, the rest of our in vitro data, annexin staining and flow cytometry assay, western blotting analysis and colony forming assay, also confirm the specific associated of PinX1 to the cytotoxicity and sensitivity in CSCC cells treated with paclitaxel. We previously demonstrated that PinX1 acts in conjunction with telomerase to regulate telomere maintenance. This action of PinX1 and telomerase prohibited the chemotherapy-induced apoptosis in telomerase-positive cancer cells. Other studies have indicated that paclitaxel promotes chromosomal instability via two mechanisms, stimulating chromosomal end-to-end fusion and delaying multinucleation in cells with telomere dysfunction [28,37]. Therefore, we hypothesized that the function of PinX1 in CSCC would impact the cytotoxicity and sensitivity of paclitaxel by mediating telomere dysfunction, TERT degradation and telomerase inactivity. Surprisingly our study demonstrated that PinX1/ paclitaxel associated cytotoxicity and sensitivity was actually independent of the telomere function, TERT expression and telomerase activity. Since the PinX1/paclitaxel effect in CSCC is independent of telomere function, we examined the role of PinX1 on kinetochore because the central region of PinX1-M (amino acids 92-254) mediates interactions with the kinetochore and spindle poles during mitosis. Furthermore, PinX1 binds to microtubules in a dosedependent manner [38]. Paclitaxel stabilizes microtubules through the promotion of microtubule assembly and prevention depolymerization, which ultimately leads to cell cycle arrest in the G2/ M phase and induces apoptosis. To assess if PinX1 expression and paclitaxel treatment in CSCC results in mitotic arrest, we

Fig. 6. A model for the role of PinX1 associated with the cytotoxicity and sensitivity of paclitaxel in cancer cells. Cells treated with paclitaxel induce the tension-sensing CENP-E cascade, generate an active form of Mad2, and trigger spindle assembly checkpoint activation, which inhibits the degradation of cyclinB and securin, ultimately resulting in mitotic arrest and cellular apoptosis. PinX1 is located on the spindle microtubules and the outer plate of kinetochores, which stabilizes the microtubulekinetochore association. The replenishment of PinX1 maintains the tension between kinetochore, while the depletion of PinX1 relaxes the distance between kinetochore thus silencing the checkpoint.

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examined the knockdown of PinX1 via flow cytometry and discovered an increased number of G2 phase cells while the reintroduction of PinX1 decreased the number of G2 phase cells. We also checked the expression of CyclinB1, the regulatory subunit of the Cyclin-dependent kinase1 (CDK1), which is a key factor in maintaining the G2-M transition [39]. In our PinX1/paclitaxel system, the expression of CyclinB1 is increased in PinX1 KD cells and reduced in PinX1-replenished cells. Taken together, these results demonstrate that PinX1 plays a role in the paclitaxel-induced mitotic arrest in CSCC. PinX1 also has a function in faithful chromosome segregation as well as functioning to promote the M phase transition during the cell cycle. To investigate if PinX1 is influencing the paclitaxelinduced spindle assembly checkpoint during mitotic arrest, we measured the distance between sister kinetochores and counted aberrant chromosome attachments in CSCC cells with different PinX1 expression. We discovered that PinX1 functions to stabilize sister kinetochore. The integrity of spindle assembly checkpoint components, especially CENP-E, is necessary for response to paclitaxel treatment [40] since knockdown of CENP-E by siRNA results in moderate increase in paclitaxel sensitivity [41,42]. Therefore, we analyzed the expression of CENP-E in response to paclitaxel treatment in PinX1 shRNA and PinX1-replenished cells and found no difference in CENP-E levels. These results clearly demonstrate that increased PinX1 levels are associated with paclitaxel resistance and sensitivity, which influences the spindle assembly checkpoint. Nocodazole is a microtubule drug that impacts the distance between kinetochores but has a small effect on microtubule depolymerization at low concentrations (

telomerase binding factor PinX1 regulates paclitaxel sensitivity depending on spindle assembly checkpoint in human cervical squamous cell carcinomas.

Paclitaxel is a main ingredient in the combination chemotherapy treatment of advanced human cervical squamous cell carcinomas. We investigated the rol...
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