Author Affiliations: Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, China (Wang XX, Liu ZR and Chen XJ); Department of Emergency (Li XZ) and Department of Pathology (Zhai LQ), Shanxi Provincial People's Hospital, Taiyuan 030012, China; and Department of Geriatric Oncology, Shanxi Academy of Medical Sciences, Shanxi Dayi Hospital, Taiyuan 030032, China (Wang XX and Pei Y)

Corresponding Author: Xiao-Xia Wang, MD, Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001, China (Tel/Fax: 86-351-4135277; Email: wxiaoxia99007@yahoo.com.cn)

 

© 2013, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(13)60085-5

 

 

Contributors: WXX proposed the study. WXX and LXZ performed research and wrote the first draft. CXJ analyzed the data. WXX and LXZ contributed equally to this work. All authors contributed to the design and interpretation of the study and to further drafts. WXX is the guarantor.

Funding: This study was supported by grants from the National Natural Science Foundation of China (81372676 and 30973401) and the Natural Science Foundation of Shanxi Province (2009011052-1).

Ethical approval: The study was approved by the Ethics Committee of Shanxi Tumor Hospital.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Pancreatic cancer is a highly aggressive malignant tumor with the lowest survival rate. A better understanding of the molecular mechanisms which contribute to pancreatic cancer occurrence and progression will aid in the development of new approaches to the early diagnosis, prevention, and treatment of this deadly disease. The scaffold protein IQGAP1 shows elevated levels in a variety of cancer types. Currently, we investigated whether or not IQGAP1 is also overexpressed in pancreatic cancer.

 

METHODS: IQGAP1 expression was examined in pancreatic cancer and normal tissues adjacent to cancerous tissues (adjacent tissues) by Western blotting and real-time RT-PCR as well as in paraffin sections of tissue microarray by immunohistochemistry. The correlations between IQGAP1 expression and various clinicopathological characteristics were analyzed.

 

RESULTS: Western blotting and real-time RT-PCR revealed that the levels of IQGAP1 protein and mRNA expression in pancreatic cancer tissues were significantly increased compared with adjacent tissues. Immunohistochemistry analysis on tissue microarray showed that IQGAP1 protein expression was significantly higher in pancreatic cancer (80.0%, 48/60) compared with adjacent tissues (18.3%, 11/60) (P<0.001). Moreover, overexpression of IQGAP1 was shown to be associated with the grades of tumor differentiation (P<0.05).

 

CONCLUSION: The overexpression of IQGAP1 may play an important role in pancreatic cancer occurrence and progression, and IQGAP1 may serve as a novel molecular target for the diagnosis and treatment of pancreatic cancer.

 

(Hepatobiliary Pancreat Dis Int 2013;12:540-545)

 

KEY WORDS: IQGAP1; pancreatic cancer; tissue microarray; immunohistochemistry

Pancreatic cancer is a highly aggressive malignant tumor with the lowest survival rate. Because the lack of specific symptoms in patients with pancreatic cancer makes early diagnosis difficult, the diagnosis is usually made in the advanced stage of the disease when local invasion and distant metastasis have occurred thereby precluding surgical resection.^[1-4] Chemotherapy has improved the prognosis in many cancers, but its effect on pancreatic cancer is very limited because pancreatic cancer has been found to be particularly resistant to chemotherapeutic agents, even radiation therapy and a combination of treatments.^[5] Therefore, a better understanding of the molecular mechanisms which contribute to pancreatic cancer occurrence and progression will aid in the development of new approaches to the early diagnosis, prevention, and treatment of this deadly disease. It has been shown that point mutations of the K-ras oncogene and the p53, p16 tumor suppressor genes are detected in pancreatic cancer.^[6-8] Gene silencing by DNA methylation (including CDKN1C, SPARC, RELN, TFPI2 and others)^[2] is often found in this malignant disease. In addition, the overexpression of pRB,^[5] Aurora A,^[9] COX-2,^[10] cyclin D1,^[11] EGF, EGFR and TGF-α^[12] contributes to the tumorigenesis and progression of pancreatic cancer. However, these are not sufficient to understand the common pathway of carcinogenesis and malignancy development of pancreatic cancer, and the exact molecular mechanisms of this neoplasm remain to be clarified.

 

The human IQGAP protein family, as scaffold proteins, includes IQGAP1, IQGAP2 and IQGAP3. Named after their isoleucine-glutamine (IQ) and GTP-activating protein (GAP) domains, IQGAP family members contain multiple protein-interacting domains. Among these, IQGAP1 has been best characterized, which is expressed ubiquitously in human tissues. By interacting with its target proteins, human IQGAP1 participates in multiple cellular functions, such as cell-cell adhesion, cytoskeletal architecture, cell polarization and directional migration, cell growth and transformation.^[13-17] Several studies^[18-25] have also implicated that the overexpression of IQGAP1 is seen in certain cancers and more frequently associated with tumor progression and a lower survival rate, suggesting a role of this molecule in human carcinogenesis. However, IQGAP1 expression has not been characterized in pancreatic cancer.

 

In this study, we examined IQGAP1 expression in pancreatic ductal adenocarcinomas by Western blotting, real-time RT-PCR and immunohistochemistry.

 

 

Four fresh specimens of pancreatic carcinoma and matched adjacent tissue were collected from patients treated at Shanxi Tumor Hospital, Taiyuan, China. All the patients were diagnosed as pancreatic ductal adenocarcinomas. None had undergone chemotherapy and radiotherapy before operation. The specimens were snap-frozen after surgical excision and then stored at -80 �� before processing for protein and RNA extraction. All patients who were enrolled in this study gave written informed consent, and the study was approved by the Ethics Committee of the hospital.

 

Frozen tissue samples were sectioned into small pieces, homogenized and dissolved in lysis buffer containing 10 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 2 mmol/L ethylenediaminetetraacetic acid (EDTA), 100 µg/mL phenylmethanesulfonyl fluoride (PMSF), 2 µg/mL aprotinin, 2 µg/mL leupeptin and 1% Triton X-100 on ice for 30 minutes. After centrifugation at 12 000 g for 15 minutes, the supernatant was collected, and protein concentration was determined by Bradford method. Equal amounts of total protein (50 µg) from each sample were loaded and separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes. The membrane was blocked with 5% nonfat dry milk in phosphate buffered saline (PBS) with 0.1% Tween-20 and incubated with anti-IQGAP1 antibody (BD Biosciences) at 1:5000 dilution overnight at 4 ��. After washing, the membrane was incubated for 1 hour with horseradish peroxidase-conjugated anti-mouse antibody at 1:3000 dilution. The target protein was detected with enhanced chemiluminescence Western blotting detection reagents (Amersham Life Science). Parallel Western blotting was probed with an anti-β-actin monoclonal antibody (Santa Cruz Biotechnology) as loading control.

 

Total RNAs from the frozen tissue specimens were extracted using Trizol reagent (Invitrogen) in accordance with the manufacturer's protocol. Reverse transcription of total RNA into cDNA was conducted using Reverse Transcription Reagents (Takara) at 37 �� for 15 minutes, followed by 85 �� for 5 seconds. Real-time PCR was performed using SYBR Premix Ex Tap (Takara) in an ABI Prism 7500 sequence detector (Applied Biosystems) in accordance with the manufacturer's protocol. Primer sequences were as follows: IQGAP1, forward: 5'-GGA GCA CAA TGA TCC AAT CC-3'; reverse: 5'-ATG GTT CGA GCA TCC ATT TC-3', and GAPDH, forward: 5'-GGC CTC CAA GGA GTA AGA CC-3'; reverse: 5'-AGG GGT CTA CAT GGC AAC TG-3'. RT-PCR reactions were initiated at 95 �� for 30 seconds and cycled 40 times at 95 �� for 5 seconds, 55 �� for 30 seconds and 72 �� for 30 seconds. Experiments were performed in triplicate for each data point. Data analysis was performed using the SDS software (Applied Biosystems) and the quantity of IQGAP1 mRNA was normalized to GAPDH.

 

Sixty patients with pancreatic ductal adenocarcinomas diagnosed were selected from the formalin-fixed paraffin embedded archives of the Shanghai Biochip Company. All samples were analyzed in a TMA format. Histopathologically representative tumor regions were defined from hematoxylin and eosin-stained sections and marked on the donor blocks. TMA sections with 60 pairs of intratumoral and matched peritumoral samples were constructed by using punch cores that measured 1.0 mm in the greatest dimension from the non-necrotic area of tumor foci and tissue adjacent to tumor, and then transferred these tissue cores to a recipient block using a tissue microarrayer (Beecher Instruments, Silver Spring, MD). Five-micron sections were cut from TMAs and were stained with hematoxylin and eosin to confirm the presence of the expected tissue histology within each tissue core. The clinicopathologic characteristics are summarized in Table 1.

 

Tissue sections from the TMA were deparaffinized in xylene and dehydrated with a series of graded ethanol, and incubated in 3% hydrogen peroxide for 15 minutes to inactivate endogenous peroxidase activity after microwave treatment in an antigen-unmasking solution. The tissue sections were covered with normal goat serum (dilution 1:10) in PBS for 2 hours at 37 �� and incubated overnight with anti-IQGAP1 antibody in a dilution of 1:200 at 4 ��. The sections were then washed with PBS three times for 5 minutes, and incubated with biotinylated secondary antibody at 1:200 dilution for 30 minutes and then with horseradish peroxidase-labeled streptavidin for 30 minutes. After washing, the sections were incubated with diaminobenzidine (DAB) chromogen. Immediately after visualization, the sections were counterstained with hematoxylin, dehydrated through graded ethanol and xylene, and mounted. Negative controls were created by replacing the primary antibodies with PBS.

 

All sections were graded by two experienced investigators who had no knowledge of the clinical data. At least 10 randomly selected high-power fields were scored. The rate of positive cells was graded: 0 (<10%), 1 (10%-25%), 2 (26%-50%), 3 (51%-75%), and 4 (>75%). Staining intensity was graded: 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong). An overall staining score was obtained by the product of the two scores above. Scores of 0-2 were defined as "negative expression" (-); scores of 3-4 as "weakly positive expression" (±); scores of 5-6 as "moderately positive expression" (+); and scores of >6 as "strongly positive expression" (++). The scores of ≥5 were considered as positive expression.

 

The data were analyzed statistically using SPSS 12.0 software package (SPSS Inc., Chicago, IL, USA). Paired samples t test was used to analyze the IQGAP1 expression between the two groups. The Chi-square test was used to examine the association between IQGAP1 expression and clinicopathological parameters. A P value of <0.05 was considered statistically significant.

 

 

Western blotting revealed that there were only weak or absent expression of IQGAP1 in adjacent tissues. The high expression of IQGAP1 was found in pancreatic ductal adenocarcinoma tissues (Fig. 1A). Consistent with Western blotting results, real-time RT-PCR showed that the levels of IQGAP1 mRNA expression in pancreatic ductal adenocarcinoma tissues were increased more significantly (P<0.01) than in adjacent tissues (Fig. 1B).

 

Immunostaining of IQGAP1 was observed in the cytoplasm and at the cellular membrane. In adjacent tissues, we observed membrane staining and negative or weak cytoplasmic staining (Fig. 2A and B). In contrast, the tumors often showed strong cytoplasmic staining compared with the adjacent tissues. A representative tumor with intense staining due to IQGAP1 expression in the cytoplasm was shown in Fig. 2C-H. A small amount of expression at the cellular membrane was also observed in the tumors, but we classified them only according to the percentage of cytoplasmic staining. The immunohistochemistry data are shown in Table 2. In the adjacent tissues, the positive rate of IQGAP1 cytoplasmic staining was 18.3% (11/60). The positive expression of IQGAP1 was up-regulated more significantly in pancreatic cancer (80.0%, 48/60) than in the adjacent tissues (P<0.001).

 

As summarized in Table 2, tumors with poor differentiation exhibited higher expression levels of IQGAP1 protein compared with those with good to moderate differentiation (P<0.05). In addition, there were no statistically significant correlations between IQGAP1 expression and tumor size and lymph node metastasis.

 

 

IQGAP1 is a 189-kDa scaffolding protein that plays a pivotal role in the control of many different cellular processes by interacting with a diverse number of cellular proteins. The changing intracellular IQGAP1 expression can alter these activities.^[14] The accumulated evidences have demonstrated that IQGAP1 is overexpressed in vivo, in a number of different cancer types, including colorectal cancer,^{[18, 19]} hepatocellular cancer,^{[16, 23]} gastric cancer,^{[20, 21]} ovarian cancer,^[22] breast cancer,^[24] thyroid cancer,^[25] etc. Furthermore, IQGAP1 overexpression is more frequently associated with tumor progression and a lower survival rate in a variety of human cancers.^[18-25] In the present study, we demonstrated the overexpression of IQGAP1 in pancreatic carcinoma by Western blotting, real-time RT-PCR and immunohistochemistry. The approximately 80.0% of the carcinoma samples exhibited a positive expression of the IQGAP1 protein, whereas adjacent tissue showed little or no IQGAP1 expression. Interestingly, the overexpression of IQGAP1 was shown to be associated with the grades of tumor differentiation. These findings suggested that IQGAP1 may play a role in the initiation and progression of pancreatic cancer although its function remains to be clarified.

 

To explore the precise mechanism by which IQGAP1 plays a role in cancer occurrence and progression, a better understanding of interaction of IQGAP1 and signal pathways is needed. Many binding partners of IQGAP1 are implicated in the development of cancer. Therefore, IQGAP1 is thought to contribute to the transformed cancer cell phenotype by interacting with many important cellular proteins involved in cell adhesion, proliferation, transformation and migration. IQGAP1 localizes to the sites of cell-cell contact and binds directly to E-cadherin which mediates intercellular adhesion. The overexpression of IQGAP1 reduces E-cadherin-mediated adhesion by interacting with β-catenin, which causes the dissociation of α-catenin from the cadherin-catenin complex in cells, inhibiting epithelial cell-cell adhesion.^{[26, 27]} Moreover, the overexpression of IQGAP1 increases β-catenin nuclear localization and β-catenin mediated transcriptional activation, leading to cell transformation.^{[28, 29]} In addition, IQGAP1 is a MAPK scaffold which modulates the activation of B-Raf, extracellular signal-regulated kinase (ERK) and MAPK/ERK kinase (MEK), promoting proliferation and inhibiting differentiation.^[30-32] IQGAP1 is also an important modulator of polarization and directional migration by regulating Rac1/Cdc42 signaling and actin dynamics at the leading edge migrating cells. Therefore, IQGAP1 overexpression increases the activity of Cdc42 and enhances cell migration.^{[33, 34]} Taken together, it is possible that IQGAP1 overexpression contributes to carcinogenesis and malignancy development of pancreatic cancer by altering properties of cell adhesion, proliferation and/or migration, and further studies are necessary to verify these possibilities both in vitro and in vivo. Moreover, these studies also suggest that the modulation of the signaling pathways linking IQGAP1 and other cellular proteins may provide a way for cancer therapies.

 

In conclusion, our data showed that IQGAP1 is overexpressed in human pancreatic ductal adenocarcinoma compared with that in adjacent tissue. Overexpression of IQGAP1 is associated with the grades of tumor differentiation. The current study offered significant information on the role of IQGAP1 in pancreatic cancer, and IQGAP1 may serve as a novel molecular target for the diagnosis and treatment of pancreatic cancer. Future studies will clarify the precise mechanisms that IQGAP1 contributes to neoplastic transformation and tumor progression.

 

 

1 Li J, Wientjes MG, Au JL. Pancreatic cancer: pathobiology, treatment options, and drug delivery. AAPS J 2010;12:223-232. PMID: 20198462

2 Vincent A, Herman J, Schulick R, Hruban RH, Goggins M. Pancreatic cancer. Lancet 2011;378:607-620. PMID: 21620466

3 Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer statistics, 2003. CA Cancer J Clin 2003;53:5-26. PMID: 12568441

4 Ballehaninna UK, Chamberlain RS. The clinical utility of serum CA 19-9 in the diagnosis, prognosis and management of pancreatic adenocarcinoma: An evidence based appraisal. J Gastrointest Oncol 2012;3:105-119. PMID: 22811878

5 Plath T, Peters M, Detjen K, Welzel M, von Marschall Z, Radke C, et al. Overexpression of pRB in human pancreatic carcinoma cells: function in chemotherapy-induced apoptosis. J Natl Cancer Inst 2002;94:129-142. PMID: 11792751

6 Watanabe H, Ha A, Hu YX, Ohtsubo K, Yamaguchi Y, Motoo Y, et al. K-ras mutations in duodenal aspirate without secretin stimulation for screening of pancreatic and biliary tract carcinoma. Cancer 1999;86:1441-1448. PMID: 10526271

7 Yamaguchi Y, Watanabe H, Yrdiran S, Ohtsubo K, Motoo Y, Okai T, et al. Detection of mutations of p53 tumor suppressor gene in pancreatic juice and its application to diagnosis of patients with pancreatic cancer: comparison with K-ras mutation. Clin Cancer Res 1999;5:1147-1153. PMID: 10353750

8 Hu YX, Watanabe H, Ohtsubo K, Yamaguchi Y, Ha A, Okai T, et al. Frequent loss of p16 expression and its correlation with clinicopathological parameters in pancreatic carcinoma. Clin Cancer Res 1997;3:1473-1477. PMID: 9815833

9 Li D, Zhu J, Firozi PF, Abbruzzese JL, Evans DB, Cleary K, et al. Overexpression of oncogenic STK15/BTAK/Aurora A kinase in human pancreatic cancer. Clin Cancer Res 2003;9:991-997. PMID: 12631597

10 Okami J, Yamamoto H, Fujiwara Y, Tsujie M, Kondo M, Noura S, et al. Overexpression of cyclooxygenase-2 in carcinoma of the pancreas. Clin Cancer Res 1999;5:2018-2024. PMID: 10473081

11 Gansauge S, Gansauge F, Ramadani M, Stobbe H, Rau B, Harada N, et al. Overexpression of cyclin D1 in human pancreatic carcinoma is associated with poor prognosis. Cancer Res 1997;57:1634-1637. PMID: 9134998

12 Barton CM, Hall PA, Hughes CM, Gullick WJ, Lemoine NR. Transforming growth factor alpha and epidermal growth factor in human pancreatic cancer. J Pathol 1991;163:111-116. PMID: 1707959

13 Brown MD, Sacks DB. IQGAP1 in cellular signaling: bridging the GAP. Trends Cell Biol 2006;16:242-249. PMID: 16595175

14 White CD, Brown MD, Sacks DB. IQGAPs in cancer: a family of scaffold proteins underlying tumorigenesis. FEBS Lett 2009;583:1817-1824. PMID: 19433088

15 Briggs MW, Sacks DB. IQGAP proteins are integral components of cytoskeletal regulation. EMBO Rep 2003;4: 571-574. PMID: 12776176

16 Chen F, Zhu HH, Zhou LF, Wu SS, Wang J, Chen Z. IQGAP1 is overexpressed in hepatocellular carcinoma and promotes cell proliferation by Akt activation. Exp Mol Med 2010;42:477-483. PMID: 20530982

17 Osman M. An emerging role for IQGAP1 in regulating protein traffic. ScientificWorldJournal 2010;10:944-953. PMID: 20495773

18 Nabeshima K, Shimao Y, Inoue T, Koono M. Immunohisto-chemical analysis of IQGAP1 expression in human colorectal carcinomas: its overexpression in carcinomas and association with invasion fronts. Cancer Lett 2002;176:101-109. PMID: 11790459

19 Hayashi H, Nabeshima K, Aoki M, Hamasaki M, Enatsu S, Yamauchi Y, et al. Overexpression of IQGAP1 in advanced colorectal cancer correlates with poor prognosis-critical role in tumor invasion. Int J Cancer 2010;126:2563-2574. PMID: 19856315

20 Takemoto H, Doki Y, Shiozaki H, Imamura H, Utsunomiya T, Miyata H, et al. Localization of IQGAP1 is inversely correlated with intercellular adhesion mediated by e-cadherin in gastric cancers. Int J Cancer 2001;91:783-788. PMID: 11275980

21 Walch A, Seidl S, Hermannstädter C, Rauser S, Deplazes J, Langer R, et al. Combined analysis of Rac1, IQGAP1, Tiam1 and E-cadherin expression in gastric cancer. Mod Pathol 2008;21:544-552. PMID: 18246045

22 Dong P, Nabeshima K, Nishimura N, Kawakami T, Hachisuga T, Kawarabayashi T, et al. Overexpression and diffuse expression pattern of IQGAP1 at invasion fronts are independent prognostic parameters in ovarian carcinomas. Cancer Lett 2006;243:120-127. PMID: 16387427

23 White CD, Khurana H, Gnatenko DV, Li Z, Odze RD, Sacks DB, et al. IQGAP1 and IQGAP2 are reciprocally altered in hepatocellular carcinoma. BMC Gastroenterol 2010;10:125. PMID: 20977743

24 Jadeski L, Mataraza JM, Jeong HW, Li Z, Sacks DB. IQGAP1 stimulates proliferation and enhances tumorigenesis of human breast epithelial cells. J Biol Chem 2008;283:1008-1017. PMID: 17981797

25 Liu Z, Liu D, Bojdani E, El-Naggar AK, Vasko V, Xing M. IQGAP1 plays an important role in the invasiveness of thyroid cancer. Clin Cancer Res 2010;16:6009-6018. PMID: 20959410

26 Li Z, Kim SH, Higgins JM, Brenner MB, Sacks DB. IQGAP1 and calmodulin modulate E-cadherin function. J Biol Chem 1999;274:37885-37892. PMID: 10608854

27 Kuroda S, Fukata M, Nakagawa M, Fujii K, Nakamura T, Ookubo T, et al. Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin- mediated cell-cell adhesion. Science 1998;281:832-835. PMID: 9694656

28 Fukata M, Kuroda S, Nakagawa M, Kawajiri A, Itoh N, Shoji I, et al. Cdc42 and Rac1 regulate the interaction of IQGAP1 with beta-catenin. J Biol Chem 1999;274:26044-26050. PMID: 10473551

29 Briggs MW, Li Z, Sacks DB. IQGAP1-mediated stimulation of transcriptional co-activation by beta-catenin is modulated by calmodulin. J Biol Chem 2002;277:7453-7465. PMID: 11734550

30 Ren JG, Li Z, Sacks DB. IQGAP1 modulates activation of B-Raf. Proc Natl Acad Sci U S A 2007;104:10465-10469. PMID: 17563371

31 Roy M, Li Z, Sacks DB. IQGAP1 is a scaffold for mitogen-activated protein kinase signaling. Mol Cell Biol 2005;25:7940-7952. PMID: 16135787

32 Roy M, Li Z, Sacks DB. IQGAP1 binds ERK2 and modulates its activity. J Biol Chem 2004;279:17329-17337. PMID: 14970219

33 Mataraza JM, Briggs MW, Li Z, Entwistle A, Ridley AJ, Sacks DB. IQGAP1 promotes cell motility and invasion. J Biol Chem 2003;278:41237-41245. PMID: 12900413

34 Watanabe T, Wang S, Noritake J, Sato K, Fukata M, Takefuji M, et al. Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration. Dev Cell 2004;7:871-883. PMID: 15572129