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And Cyclin D1 in Endometrial Cancer

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And Cyclin D1 in Endometrial Cancer Int J Clin Exp Pathol 2017;10(3):3335-3343 www.ijcep.com /ISSN:1936-2625/IJCEP0045526 Original Article Overexpression of peptidyl-prolyl isomerase 1 (Pin1) and cyclin D1 in endometrial cancer Rongrong Yan1, Xingguo Wu1, Yongmei Wang1, Chunli Yu1, Hua Li1, Lan Zhang2 1Department of Gynecology, Taian City Central Hospital, Tai’an, Shandong, China; 2Second Department of Gyne- cology, Taian City Central Hospital, Tai’an, Shandong, China Received December 1, 2016; Accepted January 18, 2017; Epub March 1, 2017; Published March 15, 2017 Abstract: The aim of this study is to investigate the expression and correlation of peptidyl-prolyl isomerase 1 (Pin1) and cyclin D1 in endometrial cancer. This retrospective study included 52 newly diagnosed endometrial cancer pa- tients in Tai’an Central Hospital from January 2011 to June 2015. Endometrial cancer samples were obtained from all 52 patients. Adjacent non-cancerous endometrium was also collected from 15 patients and used as control. The endometrium from 29 patients with uterine leiomyomata in the same period was also obtained as control. The ex- pression of Pin1 and cyclin D1 in endometrium was detected by immunohistochemical staining (SABC method). The expression of Pin1 and cyclin D1 in different groups was compared by chi-square tests. The association between protein expression and clinical characteristics was also analyzed by chi-square tests. The relationship between Pin1 and cyclin D1 expression was analyzed by Pearson’s correlation test. The expression of Pin1 and cyclin D1 in endometrial cancer was significantly higher compared with both normal endometrium and adjacent non-cancerous endometrium (P < 0.05). High expression of Pin1 and cyclin D1 was significantly associated with high clinical stage, pathological grade, lymphatic metastasis and the depth of myometrial invasion (P < 0.05). The expression of Pin1 was positively correlated with cyclin D1 (P = 0.001). In conclusion, Pin1 and cyclin D1 are highly expressed in en- dometrial cancer, and is associated with clinical stage, pathological grade, lymphatic metastasis and the depth of myometrial invasion, suggesting their regulatory role in the occurrence and development of endometrial cancer. Keywords: Endometrial cancer, Pin1, Cyclin D1, immunohistochemical analyses Introduction physiological and pathological processes, in- cluding immune response, apoptosis and vari- Endometrial cancer, the epithelial malignancy ous types of cancers [9-11]. Cyclin D1, a mem- that arises from the endometrium, is the third ber of the cyclin family, is a key regulator of cell most common cancer of the female reproduc- cycle in eukaryotic cells. It binds to cyclin- tive system, behind ovarian cancer and cervical dependent kinase CDK4 or CDK6, forming the cancer [1]. Recently, the incidence rate of endo- cyclin D-CDK4/6 complex, which is required for metrial cancer has been gradually increased cell cycle progression in G1 phase. Moreover, probably due to the increasing number of elder- cyclin D1 can regulate cell functions through ly people and obesity patients [2]. The occur- promoting the phosphorylation of tumor sup- rence and development of endometrial cancer pressor Rb and its family members [12, 13]. is a complex, multi-step process involving a Numerous studies have reported cyclin D1 series of oncogenes and signal pathways [3]. overexpression in a variety of cancers, includ- ing lung cancer, breast cancer, esophageal can- Pin1, a prolyl cis/trans isomerase (PPIase) from cer, colorectal cancer, and pancreatic cancer the parvulin family, can specifically recognize [14-18]. It is believed that cyclin D1-induced cell the phosphorylated serine/threonine-proline cycle disorder is associated with the occur- sequences (pSer/Thr-Pro), leading to conforma- rence and progression of cancers [12, 19]. tional and functional changes of proteins [4, 5]. Overexpression of Pin1 can stimulate the Pin1 is also known as an important regulator of expression of downstream genes including cell cycle [6-8]. It has been known that abnor- cyclin D1 through Ras, Wnt/β-catenin, and mity in Pin1 expression is involved in several C-Jun/AP-1 pathways, leading to abnormal cell Pin1 and cyclin D1 in endometrial cancer metabolism and cell cycle, excessive prolifera- Neutral gum was purchased from Specimen tion, and even tumorigenesis [20, 21]. and Model Factory (Shanghai, China). All other reagents were purchased from (ZSGB Biotech, Although Pin1 inhibitors and Pin1-targeted Beijing, China) unless specified otherwise. gene therapy has received considerable atten- tion, the expression of Pin1 and its association Immunohistochemical staining with cyclin D1 in endometrial cancer has not yet been clarified. In this study, we examined the The expression of Pin1 and cyclin D1 in endo- expression of Pin1 and cyclin D1 in 52 cases of metrium was detected by immunohistochemi- endometrial cancer, and further analyzed their cal staining (SABC). Briefly, paraffin sections association with clinical characteristics of the were deparaffinized in xylene, rehydrated with cancer. The current study should elucidation serial ethanol (100, 90, and 80% ethanol), and the role of both proteins in the pathogenesis of rinsed with running water. The sections were endometrial cancer, and might provide new boiled in antigen retrieval solution for 5 min, insight into gene therapy of the malignant and incubated in 3% hydrogen peroxide for 10 disease. min to block the endogenous peroxidases. The sections were then blocked with 5% Bovine Materials and methods serum albumin (BSA) for 20 min, and incubated with appropriate primary antibody overnight at Subjects and sample collection 4°C. The sections were rinsed and incubated with biotinylated mouse anti-rabbit secondary This study selected 52 newly diagnosed endo- antibody at 37°C for 20 min. The sections were metrial cancer patients in Tai’an Central rinsed and incubated with SABC at 37°C for Hospital from January 2011 to June 2015. The 20 min, followed by DAB color development for inclusion criteria were as follows: patients with- 5 min. The sections were further counter- out any prior cancer treatment including radio- stained with hematoxylin, dehydrated through therapy, chemotherapy, surgery, etc.; the diag- serial alcohols, cleared in xylene and covered nosis was confirmed by post-operative path- with neutral gum. The endometrium from 29 ological examination; without other concurrent uterine leiomyomata patients and adjacent primary malignancies; without any hormone- non-cancerous endometrium from 15 endome- related diseases; and paraffin specimens were trial cancer patients were used as control. PBS well preserved. Endometrial cancer samples was used as negative control, and breast can- were obtained from all 52 patients. Adjacent cer tissue was used as positive control. non-cancerous endometrium was also collect- ed from 15 patients and used as control. In Determination of expression level addition, the endometrium from 29 patients with uterine leiomyomata in the same period The sections were independently examined for was also obtained as control. The tissues were the extent and intensity of staining using a fixed with 4% paraformaldehyde for 72 h, Zeiss axiovert 40 c inverted microscope by two embedded in paraffin and cut into 4-μm sec- experienced pathologists in a blinded manner. tions. This study was approved by the Research Yellow or brown-stained granules in cytoplasm Ethics Committee at the Tai’an Central Hospital. and nuclei were identified as positive expres- All patients were required to sign the informed sion. The staining intensity and positive stain- consent for the research use of tissues. ing rate of five randomly selected visual fields was quantified using the Image-Pro Plus 5.0 Main reagents software. Pin1 was primarily localized in cell cytoplasm and/or nuclei (Figure 1). (-) was Rabbit anti-human Pin1 polyclonal antibody defined as negative staining or light brown and rabbit anti-human cyclin D1 monoclonal staining, (+) as moderate-brown, (++) as brown, antibody was purchased from Abcam (Cam- and (+++) as extensive dark-brown particles in bridge, MA, USA). SABC immunohistochemistry both cytoplasm and nuclei. Positive staining kit and DAB chromogenic reagent kit were pur- rates of 0%-9%, 10%-33%, 34%-66%, and 67% chased from Boster Biotech. (Wuhan, China). or higher were scored as 0, 1, 2 and 3, respec- 3336 Int J Clin Exp Pathol 2017;10(3):3335-3343 Pin1 and cyclin D1 in endometrial cancer Figure 1. Representative immunohistochemical images (SABC method) comparing the expression of Pin1 in dif- ferent groups. A. High Pin1 expression in normal endometrium (200×); B. Low Pin1 expression in normal endome- trium (200×); C. High Pin1 expression in endometrial cancer (400×); D. Low Pin1 expression in endometrial cancer (400×). tively. If the sum of the average intensity score relationship between Pin1 and cyclin D1 expres- and average stinging rate score was above 3, sion was analyzed by Pearson’s correlation the sample was considered high Pin1 expres- test. P values smaller than 0.05 were consid- sion. Otherwise, the sample was considered ered statistically significant. low expression. Cyclin D1 was also mainly dis- tributed in cell nuclei (Figure 2). Colorless, light Results brown, brown and dark brown were scored as Clinical characteristics (-), (+), (++) and (+++), respectively. Positive staining rates of 0-4%, 5-25%, 26-50%, 51-75% Of the 52 patients in this study, 21 patients and 76-100% were scored as 0, 1, 2, 3 and 4, were aged below 50 and 31 were aged 50 or respectively. If the sum of the two scores was older. There were 38 cases of adenocarcinoma, above 1, the sample was considered cyclin 10 adenocarcinoma with squamous differenti- D1-positive. A sample with a score of 1 or lower ation and 4 serous adenocarcinoma. Based on was considered negative expression. the International Federation of Gynecologists and Obstetricians (FIGO) classification 2009, Statistical analyses 23 patients were in stage I, 18 in stage II, and 11 in stage III/IV patients. There were 20 cases Data was analyzed using SPSS19.0 statistical of in G1 grade, 22 in G2 and 10 in G3.
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