Frequent Loss of NISCH Promotes Tumor Proliferation and Invasion In

Published OnlineFirst February 27, 2015; DOI: 10.1158/1535-7163.MCT-14-0911

Cancer Biology and Signal Transduction Molecular Cancer Therapeutics Frequent Loss of NISCH Promotes Tumor Proliferation and Invasion in Ovarian Cancer via Inhibiting the FAK Signal Pathway Jing Li1,2, Xiaoying He3, Ruofan Dong4, Yuan Wang4, Jinjin Yu4, and Haifeng Qiu2,5

Abstract

NISCH encodes the imidazoline receptor Nischarin and is a accelerated the growth of SKOV3 xenografts. In addition, NISCH known tumor suppressor in many human malignancies; how- significantly attenuated cell invasion by inhibiting the phos- ever, its roles in ovarian cancer are still largely unknown. Here, phorylation of FAK and ERK, which could be neutralized by PF- we aim to investigate the biologic functions of NISCH in ovarian 562271 (a FAK/Pyk2 inhibitor). Accordingly, NISCH knock- cancer. We found that NISCH was significantly downregulated, down xenografts exhibited increased peritoneal/pelvic metasta- which correlated considerably with advanced tumor stage, poor ses that were not present in counterparts treated with PF- differentiation, lymph node metastasis, and the serous/mucin- 562271. Furthermore, NISCH expression in primary ovarian ous subtypes in a panel of ovarian cancer tissues. Moreover, cancer cells predicted a cellular resistance to PF-562271. In NISCH gene silencing was mainly the product of promoter conclusion, we showed that NISCH was frequently silenced by hypermethylation, which could be reversed by treatment with promoter hypermethylation in human ovarian cancer. NISCH 5-aza-dC. In vitro, NISCH overexpression suppressed cell pro- manipulated cellular proliferation and invasion by arresting cell liferation and colony formation by hindering cell-cycle progres- cycle and inhibiting the FAK signal. Our findings revealed the sion, whereas the opposite was observed in NISCH knockdown biologic functions of NISCH in ovarian cancer, and might be counterparts. In vivo, abundant NISCH expression hindered the useful for treating patients with aberrant expression of NISCH. growth of HO8910 xenografts, whereas NISCH knockdown Mol Cancer Ther; 14(5); 1–11. 2015 AACR.

Introduction III and IV, respectively (5–7). Similar to other malignancies, ovarian cancer is characterized by rapid proliferation, invasion, Ovarian cancer is the fifth leading cause of cancer-related deaths and the formation of regional/distant metastasis (8). Thus, a among the global female population, resulting in the highest deeper understanding of the mechanisms underlying the initia- mortality among all the gynecologic malignancies (1). In 2014, tion and progression of ovarian cancer is urgently needed. there were estimated 21,980 new cases of and 14,270 deaths The NISCH gene localizes on human chromosome 3p21.1 and caused by ovarian cancer in the United States (2). Unfortunately, encodes the imidazoline receptor Nischarin, which binds the more than 70% of the ovarian cancer patients present with cytoplasmic tail of a5 integrin to regulate its translocation from advanced stage disease (stage III/IV) at the initial diagnosis, and the cell membrane to endosomes (9). Recently, several lines of are thus given a poor prognosis (3, 4). Despite many advances in evidence indicate that Nischarin directly interacts with PAK1 surgical techniques and chemotherapy during the past decades, (p21-activated kinase1), LIMK (LIM domain kinase), Rac1 the 5-year survival rates remain as low as 35% and 20% for stages (Ras-related C3 botulinum toxin substrate 1), and LKB1 (liver kinase B1), to inhibit cellular migration and invasion (10–13). NISCH 1Department of Oncology, the First Affiliated Hospital of Zhengzhou is frequently silenced by loss of heterozygosity (LOH) University, Zhengzhou, China. 2Key-Discipline Laboratory of Clinical in human breast cancer, whereas its overexpression is sufficient to 3 Medicine Henan, Zhengzhou, China. Department of Obstetrics and attenuate tumor growth and metastasis via a5 integrin signaling Gynecology, International Peace Maternity and Child Health Hospital NISCH Affiliated to Shanghai Jiaotong University School of Medicine, Shang- pathway inhibition (14). The roles of in ovarian cancer are hai, China. 4Department of Obstetrics and Gynecology, the Affiliated largely unknown; therefore, we investigated the role of NISCH in Hospital of Jiangnan University and The 4th People's Hospital of Wuxi, ovarian cancer initiation and progression. Jiangsu Province, China. 5Department of Obstetrics and Gynecology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China. Materials and Methods Note: Supplementary data for this article are available at Molecular Cancer Ovarian cancer tissue collection Therapeutics Online (http://mct.aacrjournals.org/). Two ovarian cancer tissue microarrays (OV952 and OV961) Corresponding Author: Haifeng Qiu, Department of Obstetrics and Gynecol- containing 12 normal ovaries, 38 serous, 28 endometrioid, 20 ogy, The First Affiliated Hospital of Zhengzhou University, No. 1, East Jianshe mucinous, and three clear cell ovarian cancers were purchased Road, Erqi district, Zhengzhou 450052, China. Phone: 0371-5675-5995; from Alenabio Biotechnology Company (Xi'an, Shanxi Province, Fax: 0371-5675-5995; E-mail: [email protected] China) for use in immunostaining assays. Another 30 ovarian doi: 10.1158/1535-7163.MCT-14-0911 cancer samples and adjacent normal ovary tissue collected at 2015 American Association for Cancer Research. the Affiliated Hospital of Jiangnan University (Wuxi, Jiangsu

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DDC Province, China) were used for real-time PCR, quantitative meth- expression of NISCH was calculated using the 2 t method ylation-specific PCR (QMSP), and LOH analysis. All patients were with b-actin as an internal control. staged and graded following the criteria of International Feder- ation of Gynecology and Obstetrics (FIGO) for ovarian cancer. DNA bisulfite modification and QMSP Patient follow-ups were performed every 6 months for all avail- Genomic DNA was extracted from 30 pair-matched ovarian able cases until July 30, 2014. This study was approved by the cancer tissues using a DNA Kit (Tiangen Biotech) and treated with ethical committee of the Affiliated Hospital of Jiangnan Univer- bisulfite using the EZ DNA Methylation-Gold Kit (Zymo sity and written consents were obtained from all participants. Research) according to the manufacturer's instructions. The mod- For primary cultures, 12 freshly obtained ovarian cancer tissues ified genomic DNA then served as template DNA for QMSP were minced with scalpels before digestion in 0.1% collagenase IA reactions with the primers and conditions described in Table 1. dissolved in DMEM (Gibco) containing 10% FBS (Gibco) for 2 Samples were defined to be hypermethylated if the relative level of hours at 37 C. After a short centrifugation, cancer cells were methylated DNA in which exceeded a set threshold determined by 5 suspended with DMEM to a density of approximately 1 10 analyzing the overall methylation levels in the 30 pair-matched 4 cells/mL. For MTT assay, 1 10 cells per well were plated into the tissues and considering the best sensitivity and specificity). 96-well plates and incubated overnight before the 72-hour treatment with PF-562271. 5-aza-dC treatment Cells (1 106 cells/well) were seeded into 6-well plates and Immunohistochemical staining cultured overnight before treatment with 5 mmol/L 5-aza-dC All procedures were performed according to previously pub- (A3656; Sigma-Aldrich) or PBS vehicle for 48 hours. Real-time lished protocols (14). Anti-Nischarin mouse monoclonal anti- PCR and Western blotting were then used to assess alterations in body was purchased from BD Biosciences and used at a dilution of Nischarin at the mRNA and protein levels. 1:200. Slides were scored on the basis of average staining intensity by two pathologists individually. The criteria were as follows: Loss of heterozygosity analysis 0, no expression (negative staining); 1, weak (1%–25% positive); LOH analysis was performed using three microsatellite markers 2, moderate (26%–50% positive); 3, strong (>50% positive). (D3S3026, D3S3561, and D3S3688) with primers described in Table 1. The PCR products were loaded and separated on 7.5% polyacrylamide gels, and the allelic loss was determined Cell culture and reagents by ethidium bromide staining. Human ovarian cancer cell lines were obtained as follows: SKOV3 and IGROV were from the ATCC, A2780 from the Euro- Western blot analysis pean Collection of Cell Cultures, HO8910 from the Cell Bank of Total protein was extracted from cells using RIPA (Thermo the Chinese Academy of Sciences (CBCAS), and HEY was the kind Fisher Scientific). Equivalent amounts of protein were separated gift from Dr. Chunxiao Zhou (School of Medicine, University of on SDSPAGE gels (8% for Nischarin and 12% for other proteins) North Carolina at Chapel Hill, Chapel Hill, NC). In September of and transferred to polyvinylidene difluoride membranes. After 2013 and 2014, all working cell lines were authenticated using incubation with appropriate primary and secondary antibodies, short tandem repeat DNA profiling at CBCAS. Cell lines were the specific protein bands were visualized using a commercial ECL maintained at 37 C in a humidified atmosphere of 5% CO with 2 Chemiluminescent Kit (Beyotime). The following antibodies the following media: SKOV3 and HO8910 in DMEM with 10% were used: mouse monoclonal anti-Nischarin (1:1,000; BD Bios- FBS; IGROV in RPMI-1640 (Gibco) with 10% FBS; and HEY and ciences), rabbit monoclonal anti–phospho-FAKTyr397 (1:1,000; A2780 in RPMI-1640 with 5% FBS. Cell Signaling Technology), rabbit monoclonal anti-FAK (1:1,000; Cell Signaling Technology), rabbit monoclonal anti– RNA isolation and real-time PCR phospho-ERK1/2Thr202/Tyr204 (1:1,000; Cell Signaling Technolo- Total RNA was extracted using the TRizol reagent (Invitrogen) gy), rabbit monoclonal anti-ERK1/2 (1:1,000; Cell Signaling and quantified on a Nanodrop 2000 (Thermo Fisher Scientific). Technology), mouse monoclonal anti-cyclin D1 (1:1,000; Cell Real-time PCR was performed using the SYBR Premix ExTaq II Kit Signaling Technology), mouse monoclonal anti-CDK4 (1:1,000; (Takara) and the primers described in Table 1. The relative Cell Signaling Technology), and mouse monoclonal anti-Ki67

Table 1. Primer sequences used in this study LOH analysis D3S3026 Forward 50-GCATCTTTGGTCCCAGCTAC-30 Reverse 50-TAATGGAACACCCTGTGGT-30 D3S3561 Forward 50-TCCTGGGGACTGTGATG-30 Reverse 50-GGTGACTGGAGGTTCAAG-30 D3S3688 Forward 50-CACCACTGCACTCCAG-30 Reverse 50-TGATTTGTTATTATCTCTTATGGG-30 Real-time PCR NISCH Forward 50-CCCCAGGGCTCCTTTGC-30 Reverse 50-CTCTGCTGGGACCTCCTG-30 b-Actin Forward 50-TCTGGCACCACACCTTCTAC-30 Reverse 50-GATAGCACAGCCTGGATAGC-30 QMSP NISCH Forward 50-TTTTTTTCGTATAGAGTTCGT-30 Reverse 50-CTAAACCTCTCTAAAATTCG-30 ACTB Forward 50-TGGTGATGGAGGAGGTTTAGTAAGT-30 Reverse 50-AACCAATAAAACCTACTCCTCCCTTAA-30

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(1:1,000; Cell Signaling Technology). Mouse monoclonal anti– Establishment of the tumor growth and metastasis mouse b-actin (1:1,000; Cell Signaling Technology) was used as an models endogenous control. All the animal experiments were permitted and supervised by the Animal Care and Use Committee of Jiangnan University. To 6 Establishment of stable cell lines investigate tumor growth, 1 10 SKOV3 or HO8910 cells were s. fl The NISCH overexpression and NISCH-shRNA vectors were c. injected into the right posterior ank of 6- to 8-week-old female constructed as previously described (14). The full-length clone of BALB/c nude mice. Starting at day 10, tumor diameters were human NISCH was amplified by PCR using the following primers: measured once a week to calculate tumor volume using the ¼ 2 fi forward, 50-CTAGGAATTCGCCACCATGGAGCAGAAACTGATC- formula: volume (length width )/2. All mice were sacri ced TC-30; reverse, 50-CTAGGGATCCCTAGCCGGGCCACCTGGCAC- on day 38 and tumor tissues were collected for IHC analysis. NISCH C-30. The 4545 bp PCR product was then cloned between BamHI To determine the effects of expression on tumor metas- 6 NISCH and EcoRI sites of pcDNA3.1 (Invitrogen). The NISCH-shRNA tasis, 5 10 SKOV3-luciferase cells with or without sequence was: 50-TGCTGTTGACAGTGAGCGCGCCTGTATTCT- knockdown were injected into the peritoneal cavity of 6- to 8- CTATTCCAATTAGTGAAGCCACAGATGTAATTGGAATAGAGAA- week-old female BALB/c nude mice. Fifteen mice were randomly NISCH TACAGGCATGCCTACTGCCTCGGA-30. HO8910 and SKOV3 arranged into three groups: negative control, -shRNA, NISCH þ clones with NISCH overexpression and knockdown, respectively, -shRNA PF-562271. PF-562271 was given to the mice were obtained and used in analysis with empty vector control from days 21 to 38 at a dose of 25 mg/kg/d (i.p. injection). counterparts. SKOV3 cells with stable luciferase expression Bioluminescent density was measured once a week and the mice fi (Genechem Technology) were transfected with shRNA-NISCH were sacri ced 4 weeks later to collect tumor tissues for IHC to generate Nischarin-deficient SKOV3-luciferase cells. Empty analysis. vector SKOV3-luciferase cells were used in experiments as negative controls. Statistical analysis All in vitro experiments were performed in triplicate and repeat- ed at least three times. Statistical analysis was performed using Cell-cycle analysis SPSS 17.0 (IBM Software). The c2 or t tests were used for cate- Cell-cycle distribution was determined on a FACS Caliber gorical or quantitative data, respectively. P values <0.05 were System (BD Biosciences). In brief, 1 106 cells were plated into defined as statistically significant. a 60-mm dish and cultured for 24 hours before harvest. Cells were stained in 500 mL staining buffer containing PI (50 mg/mL) and RNase A (20 mg/mL) for 15 minutes. Data were then analyzed in Results FCS4 Express software (Denovo Software). NISCH was significantly downregulated in human ovarian cancer We first investigated Nischarin protein expression in two ovar- Colony formation assay ian cancer panels by immunohistochemistry. As expected, Ovarian cancer cells with forced overexpression or knockdown Nischarin was significantly downregulated in ovarian cancer of NISCH were seeded into the 6-well plates (100 cells/well) and tissues when compared with normal controls (P ¼ 0.025, Fig. cultured for 12 days. Resulting colonies were then fixed with 10% 1A and B) and was associated with advanced tumor stage (Fig. 1B, cold methanol for 30 minutes and stained in 0.1% crystal violet P ¼ 0.047), high histologic grade (Fig. 1C, P ¼ 0.029), and lymph for 15 minutes. Colonies containing more than 50 cells were then node metastasis (Fig. 1D, P ¼ 0.031). A notable loss of Nischarin counted using an inverse microscope. was also observed in serous and mucinous tumor subtypes (Fig. 1E, P ¼ 0.006 and 0.022, respectively), but not in endome- Invasion assay trioid or clear cell ovarian cancer (Fig. 1E, P ¼ 0.198 and 0.351, Cell invasion assay was performed using the Transwell system respectively). Survival data were available for 76 patients at the (Applied Biosystems). In brief, the upper chamber was precoated last follow-up, including 34 alive and 42 deaths. Follow-up with 40 mL Matrigel (BD Biosciences) and loaded with 300 mL periods ranged from 19 to 73 months with a median of 42.0 6 (1 10 cells/mL) cell suspension. Medium containing 10% FBS months. Significantly, Kaplan–Meier survival analysis revealed (500 mL) was added to the lower chamber. After incubated for 24 that high Nischarin expression associated with a much better hours, noninvasive cells were removed by scraping. Cells that overall survival (OS; Fig. 1F, P ¼ 0.032). remained on the membrane were fixed in 4% paraformaldehyde Consistently, the mRNA level of NISCH in fresh ovarian cancer solution and stained with 0.1% crystal violet. Ten fields (100 tumor samples was much lower than that observed in matched magnification) were randomly selected to count colony numbers normal ovary tissues (Fig. 1G, P < 0.001). As shown in Fig. 1H, the on an optical microscope. Nischarin expression was notably higher at the mRNA and protein levels in mildly invasive cell lines (SKOV3 and A2780) than in the MTT assay moderately invasive IGROV cell line, and was lowest in highly Cells were seeded at 3 103 cells per well into 96-well plates invasive cell lines (HEY and HO8910). and cultured overnight prior treatment with the FAK inhibitor PF-562271 (M2302; Abmole) for 72 hours. Then, 5 mL MTT NISCH silencing was mediated by promoter hypermethylation solution (5 mg/mL; Sigma-Aldrich) was added into each well. in ovarian cancer After a 1-hour incubation, media were removed and 100 mL The diminished NISCH expression observed in human breast DMSO per well was added to dissolve the formazan crystals. cancer is reportedly due to LOH (14). Through our analysis, we Absorbance at 570 nm was then measured using a plate reader. found that NISCH promoter hypermethylation and LOH

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Figure 1. NISCH expression in ovarian cancer and its associations with clinicopathologic characteristics. A, representative staining of Nischarin in normal ovary (A1), serous (A2), mucinous (A3), and clear cell (A4) ovarian cancer tissues. B, Nischarin is signiﬁcantly downregulated in ovarian cancer tissues (P ¼ 0.025), and correlates with advanced tumor stage (P ¼ 0.047). Loss of Nischarin associates with high histologic tumor grade (P ¼ 0.020; C) and lymph node metastasis (P ¼ 0.031; D). E, Nischarin downregulation is more prevalent in serous and mucinous ovarian cancer (P ¼ 0.004 and 0.022, respectively). F, increased Nischarin expression is predicative of better OS in ovarian cancer patients (P ¼ 0.032). G, NISCH mRNA is signiﬁcantly downregulated in ovarian cancer tissues as compared with matched normal tissue (P < 0.001). H, NISCH mRNA and protein levels are lower in highly invasive HEY and HO8910 cells, but elevated in minimally invasive SKOV3 and A2780 cells.

occurred in 36.7% (11/30; Supplementary Fig. S1A and S1B) and Moreover, we found that promoter hypermethylation, but not 6.7% (2/30; Supplementary Fig. S1C) of ovarian cancer tissues, LOH, associated with NISCH silencing in ovarian cancer (Fig. 2B and were not present in any matched normal tissue (Fig. 2A). and C; P < 0.001 and P ¼ 0.2115, respectively). After the

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Figure 2. NISCH promoter hypermethylation and LOH in ovarian cancer. A, NISCH exhibited promoter hypermethylation and LOH in 36.7% (11/30) and 6.7% (2/30) of paired ovarian cancer tissues, respectively. B, NISCH mRNA expression negatively correlates with promoter hypermethylation (P < 0.001), but did not associate with LOH (P ¼ 0.2115; C). D and E, NISCH mRNA and protein expression were rescued in HEY cells after treatment with 5-aza-dC (P ¼ 0.032).

demethylation treatment with 5-aza-dC, the mRNA and protein progression (P ¼ 0.021) and upregulated cyclin D1 and CDK4 levels of Nischarin were notably restored in HEY cells, but not in (Fig. 4B and Supplementary Fig. S2B). Moreover, the cell prolif- A2780 cells (NISCH was hypermethylated in HEY cells, Fig. 2D eration and colony formation were significantly enhanced by and E, P ¼ 0.032). NISCH silencing (Fig. 4C and D, P ¼ 0.042 and 0.029, respectively). In vivo, NISCH-knockdown SKOV3 xenografts progressed much faster than controls (Fig. 4E, P ¼ 0.012) and exhibited a Nischarin regulated cellular proliferation by restraining cell- significantly higher expression of cyclin D1 (Fig. 4F, P ¼ 0.03). cycle progression As shown in Fig. 3A, HO8910 cells transfected with pcDNA3.1- NISCH exhibited a significant overexpression of Nischarin (>20- Nischarin attenuated cell invasion by inhibiting FAK signal fold, P < 0.001). Notably, this overexpression induced a significant transduction G1 phase arrest (P ¼ 0.039) and lower cyclin D1 and CDK4 protein In HO8910 NISCH-overexpressing cells, we detected a sharp levels (Fig. 3B and Supplementary Fig. S2A) when compared with decline of phospho-FAK and phospho-ERK (Fig. 5A and B, P ¼ empty vector–negative controls, as well as abrogated the prolifer- 0.01 and 0.032, respectively). This finding persuaded us to assess ative and colony-forming abilities of HO8910 cells (Fig. 3C and D, the effects of NISCH expression on cell invasion. As shown in Fig. P ¼ 0.018 and P ¼ 0.025, respectively). In vivo, the HO8910 5C and D, NISCH overexpression significantly inhibited the NISCH-overexpressing xenografts grew much slower than controls invasiveness of HO8910 cells (Fig. 5C and D, P ¼ 0.029). In (Fig. 3E, P < 0.001) and exhibited downregulated cyclin D1 (Fig. contrast, NISCH-knockdown SKOV3 cells displayed an increased 3F, P ¼ 0.031). expression of phospho-FAK and phospho-ERK (Fig. 5E and F, P ¼ In SKOV3 cells, NISCH-shRNA decreased Nischarin protein 0.016 and 0.015, respectively) that can be reversed by the treat- expression by as much as 85% (P ¼ 0.005, Fig. 4A). Consistent ment with the FAK inhibitor PF-562271 (Fig. 5E and F, P ¼ 0.009 with our previous data, NISCH silencing accelerated cell-cycle and 0.033, respectively). Moreover, cell invasion was also

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Figure 3. The effect of NISCH overexpression on cellular phenotype in HO8910 cells. A, NISCH mRNA was upregulated more than 20 fold after the transfection with pcDNA3.1-NISCH (P < 0.001). B, NISCH overexpression results in G1 phase arrest (P ¼ 0.039) and cyclin D1 and CDK4 downregulation. NISCH overexpression decelerates cell proliferation (P ¼ 0.018; C), and inhibits colony formation in HO8910 cells (P ¼ 0.025; D). E, NISCH overexpression attenuates the growth of HO8910 xenografts (P < 0.001). F, cyclin D1 protein level is downregulated in HO8910 xenografts (P ¼ 0.031).

enhanced in NISCH-knockdown cells (Fig. 5G and H, P ¼ 0.029), signiﬁcantly lowered by treatment with PF-562271 (P ¼ 0.027). which was neutralized by treatment with PF-562271 (Fig. 5G and Consistently, the expression of phospho-FAK and Ki-67 in met- H, P ¼ 0.05). astatic tissues was notably increased following the knockdown of NISCH (Fig. 6C and D, P ¼ 0.023 and 0.02, respectively), as well as with PF-562271 treatment (Fig. 6C and D, P ¼ 0.037 and NISCH suppressed tumor metastasis via inhibiting FAK signal 0.041, respectively). in vivo To investigate the effect of NISCH expression on tumor metastasis, we established a mouse model of ovarian cancer with Silence of NISCH predicted cellular response to PF-562271 peritoneal/pelvic cavity metastasis suitable for bioluminescent To determine the correlation between NISCH expression and imaging. As shown in Fig. 6A and B, mice harboring NISCH- cellular sensitivity to PF-562271, we performed MTT assay with a shRNA xenografts exhibited a signiﬁcantly higher bioluminescent panel of 12 primary ovarian cancer cell cultures (Table 2). We signal than negative control counterparts (P ¼ 0.006), which was found that cells with lower mRNA expression of NISCH were

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Figure 4. The effect of NISCH knockdown on cellular phenotype in SKOV3 cells. A, NISCH mRNA was reduced as much as 85% (P ¼ 0.005) by the transduction with NISCH-shRNA. NISCH knockdown promotes cell-cycle progression (P ¼ 0.021) and cyclin D1 and CDK4 upregulation (B), and significantly accelerates cell proliferation (P ¼ 0.042; C). D, NISCH-deficient SKOV3 cells form more clones than those in blank and negative control groups (P ¼ 0.029). E, NISCH knockdown notably accelerated the growth of SKOV3 xenografts (P ¼ 0.012). F, cyclin D1 is upregulated in NISCH-knockdown SKOV3 xenografts (P ¼ 0.03). much more sensitive to PF-562271 than those containing higher NISCH is a known tumor suppressor; thus, we first sought to NISCH (Fig. 6E, P < 0.001, R2 ¼ 0.7073). investigate whether its expression was deregulated in ovarian cancer. As expected, we detected a frequent downregulation of NISCH in a panel of ovarian cancer tissues, which was significantly Discussion associated with advanced tumor stage, high histologic grade, During recent decades, several genes have been identified as lymph node metastasis, and specific tumor subtypes (serous and tumor suppressors that could inhibit the initiation and progres- mucinous). Our findings were supported by Lu and colleagues sion of human cancers (15–17). These genes often manipulate (18), study, which revealed mild loss of NISCH expression in all cellular proliferation, cell-cycle progression, migration, invasion, the four subtypes of ovarian cancer (Supplementary Fig. S3). apoptosis, autophagy, and senescence. Unfortunately, epigenetic Together, these results support an important role for NISCH in or genetic alterations (such as promoter hypermethylation, LOH, ovarian cancer. and mutation) can lead to the inactivation or deletion of these Furthermore, we demonstrated that the silencing of NISCH genes, subsequently allowing some normal cells to undergo observed in ovarian cancer is the result of promoter hyper- malignant transformation. methylation, rather than LOH. Consistently, NISCH expression

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Figure 5. The involvement of FAK signaling in NISCH-mediated cellular invasion. A and B, NISCH overexpression hinders the phosphorylation of FAK and ERK in HO8910 cells (P ¼ 0.01 and 0.032, respectively), and signiﬁcantly attenuates the invasiveness of HO8910 cells (P ¼ 0.029; C and D). E and F, NISCH-knockdown promotes FAK and ERK phosphorylation in SKOV3 cells (P ¼ 0.016 and 0.015, respectively), which can be reversed by the FAK inhibitor PF-562271 (P ¼ 0.009 and 0.033, respectively). GandH,NISCH knockdown enhances the invasiveness of SKOV3 cells (P ¼ 0.029), whereas PF-562271 abrogates this effect (P ¼ 0.05).

was notably restored after the treatment with 5-aza-dC in HEY Baranwal and colleagues (14) previously reported that NISCH cells that harbor a heavily methylated NISCH promoter. This LOH occurs in 60% (12/20) breast cancer patients and is result provides evidence that NISCH is mainly silenced by signiﬁcantly associated with its silencing. On the basis of these promoter hypermethylation in ovarian cancer. Interestingly, ﬁndings, we hypothesized that NISCH silencing might be

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Figure 6. The effects of NISCH expression on metastasis and sensitivity to PF-562271 in ovarian cancer. A, representative bioluminescent images of peritoneal metastasis in mice bearing SKOV3 cells with empty vector, NISCH-shRNA, or a combination of NISCH-shRNA and PF-562271. B, quantiﬁcation of bioluminescence data in the three groups: NISCH knockdown facilitates peritoneal/pelvic metastasis (P ¼ 0.006), which is impaired by the addition of PF-562271 (P ¼ 0.027). C and D, NISCH knockdown enhances the expression of p-FAK and Ki-67 (P ¼ 0.023 and 0.02, respectively), which is reversed by PF-562271 treatment (P ¼ 0.037 and 0.041, respectively). E, NISCH predicts the cellular response to PF-562271 in primary ovarian cancer cells (P < 0.0001, R2 ¼ 0.8112). induced by different mechanisms in various cancers, which Most ovarian cancers present with rapid proliferation and needs further exploration in the future. widespread metastasis that preferentially invades the adjacent Next, we investigated the functional importance of NISCH peritoneal and pelvic tissues. The 5-year survival rate for patients silencing in ovarian cancer. In vitro, the forced overexpression of with localized ovarian cancer is more than 90%; however, this NISCH HO8910 cells suppressed cell proliferation and led to their drops to 70% and 30% for those with regional and distant arrest in G1 phase, whereas the knockdown of NISCH enhanced metastasis, respectively (2, 3). Therefore, preventing the invasion cell proliferation and facilitated progression into S phase. In vivo, and metastasis of ovarian cancer seems to be a potent therapeutic NISCH expression resulted in delayed tumor growth and a nota- strategy. In the present study, we demonstrated that NISCH could ble downregulation of cyclin D1. Consistent with these results, suppress ovarian cancer invasion by inhibiting the phosphoryla- similar suppressive effects of NISCH on colony formation and tion of FAK. In contrast, NISCH knockdown cells exhibited xenograft progression have also been reported in breast cancer increased FAK phosphorylation and invasiveness, which could (14). be reversed by the FAK inhibitor PF-562271. In vivo, NISCH

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Table 2. Clinicopathologic characters of patients in primary culture. thus, evaluated NISCH expression as a biomarker for potential Case Age Stage Grade Histology sensitivity to FAK inhibition. According to our data, NISCH 1 68 IIIA III Serous mRNA levels notably predicted the sensitivity of primary ovarian 2 56 IIIC III Serous cancer cells to PF-562271, supporting the role of NISCH as a 3 60 IVB III Serous fi 4 63 IIIC III Serous biomarker for patient strati cation in clinical trials using FAK 5 77 IIIC III Serous inhibitors. However, due to the limited group size, further studies 6 69 IIIB II Endometrioid are necessary to verify these results. 7 51 IIIA III Endometrioid In conclusion, we demonstrate that NISCH is frequently 8 72 IIA III Mucinous silenced by promoter hypermethylation in human ovarian cancer, 9 75 IIIA III Mucinous thus enhancing cell proliferation and invasion by inhibiting 10 67 IIB II Serous 11 62 IVB III Serous FAK-dependent signal transduction. Our results reveal the bio- NISCH 12 65 IIIC III Serous logic functions of in ovarian cancer, which might be useful for treating patients with aberrant NISCH expression. knockdown enhanced the formation of peritoneal/pelvic metastasis, whereas the treatment with PF-562271 was able to neutral- Disclosure of Potential Conflicts of Interest fi NISCH ize this phenomenon. These ndings suggest that might No potential conflicts of interest were disclosed. suppress tumor metastasis by inhibiting FAK-mediated signal transduction in ovarian cancer. Tumor cell invasion is a hallmark of metastasis and plays an Authors' Contributions important role during cancer progression (19). In normal epi- Conception and design: J. Li, H. Qiu thelial and breast cancer cells, Nischarin (encoded by NISCH) Development of methodology: J. Li, X. He, R. Dong, Y. Wang, H. Qiu Acquisition of data (provided animals, acquired and managed patients, inhibits cellular invasion via selective binding with PAK1 to provided facilities, etc.): J. Li, X. He, Y. Wang inhibit its kinase activity (10, 11). Nischarin also could regulate Analysis and interpretation of data (e.g., statistical analysis, biostatistics, cell invasion by directly binding to the PDZ and kinase domains computational analysis): R. Dong, Y. Wang, J. Yu, H. Qiu of LIMK, and negatively modulating the LIMK–cofilin pathway Writing, review, and/or revision of the manuscript: J. Li, X. He, J. Yu, H. Qiu (12). Consistently, we found that NISCH silencing significantly Administrative, technical, or material support (i.e., reporting or organizing promoted tumor invasion by activating the FAK pathway, as this data, constructing databases): R. Dong, J. Yu Study supervision: H. Qiu effect was reversed by treatment with PF-562271. Collectively, these data support that NISCH inhibits tumor invasion by regu- lating various signal pathways. Grant Support Currently, most ovarian cancer patients receive similar systemic Our study was supported by grants to Y. Wang from the Scientific Bureau treatments that mainly include cytoreductive surgery and plati- (CSE31N1418) and the Health Bureau of Wuxi City (MS201417 and num-based chemotherapy. However, despite an initial objective FYKY201401). The costs of publication of this article were defrayed in part by the payment of response rate of approximately 60%, many patients succumb to page charges. This article must therefore be hereby marked advertisement in their disease due to chemoresistance and tumor relapse (7, 20). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Recently, several FAK inhibitors were shown to suppress ovarian cancer progression by resensitizing cells to routine chemothera- Received October 23, 2014; revised February 16, 2015; accepted February 17, pies (21–23). Because NISCH is an upstream regulator of FAK, we 2015; published OnlineFirst February 27, 2015.

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www.aacrjournals.org Mol Cancer Ther; 14(5) May 2015 OF11

Frequent Loss of NISCH Promotes Tumor Proliferation and Invasion in Ovarian Cancer via Inhibiting the FAK Signal Pathway

Jing Li, Xiaoying He, Ruofan Dong, et al.

Mol Cancer Ther Published OnlineFirst February 27, 2015.

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