Am J Cancer Res 2015;5(7):2320-2329 www.ajcr.us /ISSN:2156-6976/ajcr0010111 Original Article URI1 amplification in uterine carcinosarcoma associates with chemo-resistance and poor prognosis Yu Wang1,2, Michael J Garabedian2, Susan K Logan1,3 Departments of 1Urology, 2Microbiology, 3Biochemistry and Molecular Pharmacology, New York University School of Medicine, 550 First Avenue, MSB235, New York, NY 10016, USA Received May 11, 2015; Accepted June 11, 2015; Epub June 15, 2015; Published July 1, 2015 Abstract: Uterine carcinosarcoma (UCS) is a rare type of cancer and accounts for 5% of uterine malignancies. However, UCS patients suffer a high prevalence of chemo-resistance and a very poor prognosis compared to uterine cancer patients. URI is a chaperone with functions in transcription. We analyzed the somatic URI1 copy number variation in 57 post-menopausal non-metastatic UCS patients in comparison to 363 uterine corpus endometrial carcinomas. URI1 amplification was detected in 40% (23/57) of primary UCS and 5.5% (20/363) of uterine carci- nomas. UCS patients with URI1 amplification exhibited 13% (3/23) tumor-free survival compared to 41% (14/34) in the absence of URI amplification (P=0.023).URI1 amplification (OR=6.54, P=0.027), weight (OR=1.068, P=0.024), hypertension (OR=3.35, P=0.044), and tumor stage (OR=2.358, P=0.018) associated with poor survival. Patients treated with hormone replacement therapy (OR=15.87, P=0.011) displayed enhanced overall survival. Combined radiation and chemotherapy improved patient survival (median survival=2043 days) compared to single (median survival=597 days) or no treatment (median survival=317 days, P=0.0016). Importantly, patients with URI1 amplifi- cation had poor response to adjuvant treatment compared to control group (P=0.013). Tumors with URI1 amplifica- tion displayed decreased transcription of genes encoding tumor suppressor and apoptotic regulators and increased expression of genes regulating oncogenesis, survival and metastasis. Overexpression of URI1 in a cultured cell model induced ATM expression and resistance to cisplatin. Our findings suggest that high prevalence in UCS may associate with poor prognosis and worse response to adjuvant treatment. Keywords: URI1, uterine carcinosarcoma, prognosis, survival Introduction tives or postmenopausal hormone therapy reduces the risk of both types of cancers [10- Uterine carcinosarcoma (UCS), also known as 13]. The primary management of UCS is surgi- malignant mixed Mullerian tumor (MMMT), is cal, although adjuvant radiotherapy treatment an undifferentiated uterine malignancy with (RT) and/or chemotherapy are often used. characteristics of both carcinoma and sarco- While one study reported that adjuvant RT after ma. The endometrial carcinoma occurs within surgery decreased the risk of pelvic cancer the inner layer of tissue lining of the uterus, recurrence compared to surgery alone [14], it whereas the sarcoma arises from the outer did not provide any overall survival benefit [14]. layer of muscle of the uterus. USC makes up Additional studies have provided evidence to five percent of all uterine cancers [1]. In the support the benefit of aggressive adjuvant che- United States, approximately 2 per 100,000 motherapy that combines RT with DNA damage- women develop UCS annually [2]. Because of based chemotherapy [15-17]. Currently, it the aggressive nature of UCS, only 35% of remains challenging to predict outcomes for patients survive five years after diagnosis. UCS UCS patients and the mechanism underlying and endometrial carcinomas have similar risk patient relapse is unclear. Therefore, a reliable factors. Both malignancies are associated with preoperative biomarker that predicts primary obesity, diabetes, hypertension, smoking, nulli- surgical response and identifies patients who parity, and use of estrogen ortamoxifen [3-9]. would benefit from aggressive adjuvant therapy By contrast, progestin-containing contracep- is urgently needed. URI1 amplification associates with poor prognosis in UCS Table 1. Clinicopathologic characteristics of study reported URI1 amplification in 10% of the overall cohort ovarian cancers and increased URI protein level Characteristic Value correlates with tumor aggressiveness [24]. URI upregulation increases the expression of the Patient size 57 “p53 and DNA damage-regulated gene 1” Age (y) (PDRG1), suggesting a function for URI in DNA Median 68 damage repair [19]. Decreased URI1 expres- Range 51-90 sion inhibits cell proliferation and induces Race apoptosis in ovarian and liver cancer cells [25, White 44 26]. In cervical cancer, increased URI expres- Black 9 sion is also associated with a high tumor grade Asian 3 [27]. Unknown 1 Here, we hypothesize that URI1 amplification is Follow up (days) associated with poor clinical outcome in UCS Median 497 patients. We also reconstituted URI1 upregula- Range 8-3115 tion in a culture cell model and investigated URI Stage overexpression in the DNA damage response. I 22 II 5 Materials and methods III 20 Patient cohort IV 10 Positive pelvic lymph nodes 15 (26%) We analyzed UCS and uterine corpus endome- Positive aortic lymph nodes 9 (16%) trial cacinoma samples with corresponding nor- Tumor recurrence 29 (51%) mal tissue from clinically annotated patients in Surgical margin 12 (21%) The Cancer Genome Atlas (TCGA) Data Portal. Depth of myometrial invasion % All samples met freedom-to-publish criteria Median 40 without restriction or limitations. The uterine Range 0-100 corpus endometrial carcinomas cohort has Residual tumor been reported previously [28]. All UCS patients were postmenopausal (prior bilateral ovariec- R0 34 tomy or >12 months since last menstrual peri- R1 2 od with no prior hysterectomy). All UCS speci- R2 10 mens were surgically resected prior to systemic Rx 11 treatment and all patients received complete Adjuvant treatment surgery as the primary treatment. UCS Patients No 14 did not have metastasis at the time of surgery. Radiation treatment 7 The clinical stage, age at diagnosis, tumor inva- Chemotherapy 17 sion percent, local lymphatic status, and surgi- Radiation+chemo 19 cal margin, were recorded. Patient race, weight, menopausal hormone therapy, hormonal con- traceptives use, tamoxifen use, hypertension, The unconventional prefoldin RPB5 interactor 1 diabetes, full-term pregnancies, and history of (URI1) was originally identified as a scaffold other malignancies were also recorded. protein that binds RNA polymerase II [18]. URI Treatment response, time of disease relapse plays important roles in regulating gene expres- and date of death after initial diagnoses were sion. URI interacts with several transcription recorded after the initial treatment. Lymph factors including transcription factor IIF (TFII), node positivity was determined by H & E stain- and the androgen receptor (AR) in prostate can- ing and immunohistochemistry. We excluded cer cells [19-21]. It also interacts with a chro- patients who received neoadjuvant treatment. matin remodeling complex (PAF1) and transla- We also excluded patients with a history of tion initiation factors [22, 23]. Current clinical tamoxifen use or with colorectal cancer, as findings have shown the correlation between some colon cancers can metastasize to the URI1 and human cancers. For example, one uterus. Using these criteria, we identified 57 2321 Am J Cancer Res 2015;5(7):2320-2329 URI1 amplification associates with poor prognosis in UCS Table 2. URI1 amplification associates with patient pathol- CA), cBioPortal [30], and SPSS version ogy and prognosis 13.0 (SPSS, Inc., Chicago, IL). All in P- vitro experiments were performed URI1 amplification No Yes value three times independently and the Sample size n=34 n=23 error bars represent the standard deviation with significance calculated Age (yrs) 69.2±9.9 70.0±8.4 0.57 by nonparametric t-test. Stage 0.2 I 16 (47%) 6 (26%) Cell survival assay and reagents II 3 (9%) 2 (8%) III 9 (26%) 11 (48%) RL 95-2 cells (ATCC, Manasas, VA), a IV 6 (18%) 4 (17%) uterine/endometrial cancer line, were cultured in DMEM: F12 medium with Primary therapy outcome success1 21 (61%) 10 (43%) 0.18 0.005 mg/ml insulin and 10% FBS. Tumor free survival 14 (41%) 3 (13%) 0.023 Cells were plated in 96-well plates at a Tumor recurrence 18 (53%) 11 (48%) 0.7 concentration of 1,000 cells per well. Days of follow-up 743±206 800±208 0.79 After 48 hours of treatment, cell viabil- Median survival days 771±142 685±198 0.51 ity was measured using the CyQUANT Pelvic lymph nodes 7 (21%) 8 (35%) 0.23 assay (Life Technologies, Waltham, Aortic lymph nodes 5 (15%) 4 (17%) 0.31 MA), as per manufacturer’s instruc- Depth of myometrial invasion %2 33±10.2 55±15.2 0.19 tion. Cisplatin is from Sigma-Aldrich Surgical Margin 5 (15%) 7 (30%) 0.15 (St. Louis, MO). ATM inhibitor is from Selleckchem (Houston, TX) (Catalog 1Defined as complete or partial remission; 2Depth of myometrial invasion divided by depth of myometrial thickness. #S7136). Anti-URI1 antibody is from Santa Cruz Biotechnology (Santa Cruz, CA) (Catalog #376011). Anti-phospho- UCS patients and determined URI1 copy num- ATR (Catalog #2853), phospho-ATM (Catalog ber variation and mRNA expression. #5883), ATM (Catalog #2873), ATR (Catalog #2790), and phospho-γH2AX (Catalog #9718) Statistical analysis antibodies are from Cell Signaling (Berverly, MA). Anti-tubulin antibody (Catalog #489P) is Primary therapy outcome, tumor-free status, from Biolegend (San Diego, CA). new tumor event, and surgical margins between URI1 amplification and control groups were Quantitative reverse transcription-polymerase compared using the chi-square test. The differ- chain reaction (qRT-PCR) ences in age at diagnosis, and follow up time between two groups were evaluated with an qRT-PCR to quantitate ATM mRNA expression unpaired t-test. Tumor stage between control used SYBR Green (Applied Biosystem, Foster and URI1 amplifed groups was evaluated with City, CA). Expression of RPL-19 was used as the the Mann-Whitney test. Spearman’s rank cor- internal control. ATM primers are as following: relation coefficient was used to analyze pair- F: AGACCGCGTGATACTGGATG.