www.elsevier.com/locate/humpath
Human Pathology (2014) xx, xxx–xxx
Original contribution
Aberrant activation of Sonic hedgehog signaling in chroniccholecystitis and gallbladder carcinoma☆,☆☆
Fang Xie PhDa,1, Xiaoping Xu PhDa,1, Angao Xu PhDb, Cuiping Liu PhDa,Fenfen Liang MSa, Minmin Xue MSa, Lan Bai PhDa,c,⁎
aGuangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Nanfang Hospital, SouthernMedical University, Guangzhou 510515, ChinabHuizhou Medical Institute, Huizhou 516003, ChinacDepartment of Huiqiao Building, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
Received 22 July 2013; revised 15 October 2013; accepted 16 October 2013
T
0h
Keywords:Sonic hedgehog pathway;Chronic cholecystitis;Gallbladder carcinoma;Ptch;Gli1
Summary Sonic hedgehog (Shh) signaling has been extensively studied and is implicated in variousinflammatory diseases and malignant tumors. We summarized the clinicopathological features andperformed immunohistochemistry assays to examine expression of Shh signaling proteins in 10 normalmucosa, 32 gallbladder carcinoma (GBC), and 95 chronic cholecystitis (CC) specimens. The CCspecimens were classified into three groups according to degree of inflammation. Compared withnormal mucosa, CC, and GBC specimens exhibited increased expression of Shh. The immunoreactivescore of Shh in the GBC group was higher than that in the mild to moderate CC groups but lower thanthat in the severe CC group (P b .05). Expression of Patched (Ptch) and Gli1 gradually increased fromnon-malignant cholecystitis to malignant tumors. Compared with CC specimens, GBC specimensshowed higher cytoplasmic and membranous expression for Ptch (P b .05). Gli1 staining showedcytoplasmic expression of Gli1 in both CC (60% for mild, 77% for moderate, and 84% for severe) andGBC specimens (97%). Nuclear expression of Gli1 was detected in 16% of severe CC specimens withmoderate to poor atypical hyperplasia, and in 62.5% of GBC specimens. Shh expression stronglycorrelated with expression of Ptch and Gli1. Furthermore, patients with strongly positive Gli1 staininghad significantly lower survival rates than those with weakly positive staining. Our data indicate that theShh signaling pathway is aberrantly activated in CC and GBC, and altered Shh signaling may beinvolved in the course of development from CC to gallbladder carcinogenesis.© 2013 Elsevier Inc. All rights reserved.
Abbreviations: Shh, Sonic hedgehog; Ptch, Patched; GBC, Gallbladder carcinoma; CC, Chronic cholecystitis.☆ Conflict of Interest: The authors declare no conflict of interest exists.☆☆ Funding disclosures: This work was supported by grants from National Natural Science Foundation (81170354), Guangdong Provincial Science and
echnology Plan Fund (2011B031800195) and Natural Science Foundation of Guangdong Province (S2012010009343).⁎ Corresponding author. Nanfang Hospital, Southern Medical University, Gastroenterology, Guangdong Guangzhou, China.E-mail address: [email protected] (L. Bai).1 These authors contributed equally to this work.
046-8177/$ – see front matter © 2013 Elsevier Inc. All rights reserved.ttp://dx.doi.org/10.1016/j.humpath.2013.10.017
2 F. Xie et al.
1. Introduction
Gallbladder cancer (GBC) accounts for nearly twothirds of biliary tract tumors, and is the fifth most lethalcancer of the gastrointestinal tract [1,2]. GBC generallyoccurs in the sixth to seventh decade of life, and shows astrong female predominance [2]. The prognosis of GBC ispoor, and the 5-year survival rate of patients with GBC isextremely low. This poor prognosis is attributable to thetendency for late presentation, development of early lymph-node metastases, adjacent organ invasion, and poor responseto chemotherapy. To improve the prognosis of GBC, it isimportant to screen out the risk factors or precursor dis-eases relevant to gallbladder carcinogenesis, and establishappropriate prognostic markers, favoring early diagnosisand prevention.
Recent studies have revealed that the development se-quence from chronic cholecystitis (CC) to carcinoma isrelevant for GBC. A mass of clinical data suggests that CCcauses limited or diffused hypertrophy of the gallbladderwall, atypical hyperplasia of the mucous layer, andmetaplasia of the intestinal epithelium. In addition, thepathological changes occurring in CC are commonly seenin GBC tissue. Although it is not proven that CC has adirect carcinogenic effect, an extremely close relationshipbetween CC and GBC has been confirmed [3], similar tothat seen between inflammatory bowel disease and coloncancer [4]; in the chronic viral hepatitis to liver cirrhosis tohepatocellular carcinoma sequence [5]; and betweenchronic pancreatitis and pancreatic cancer [6]. Therefore,in the chronic carcinoma sequence, severe CC is consideredto be a possible precursor lesion for neoplasia under certaincircumstances [7].
The Sonic hedgehog (Shh) signaling pathway is known tobe an essential pathway in the growth and patterning ofvarious tissue types during embryonic development [8,9].Recent studies have confirmed that Shh signaling transduc-tion is mainly mediated by a complex interaction betweenthe Shh, Smoothened (Smo), and Patched (Ptch) proteins,with the two latter proteins functioning as transmembranereceptors. In the absence of Shh, Ptch inhibits the activity ofthe Smo protein, thus repressing activation of the Hedgehogsignaling pathway. When Shh binds to and inactivates Ptch,the inhibitory effect of Ptch on Smo is removed, and Smo isreleased to activate the expression of Gli1 and promote it totranslocate to the nucleus [10,11].
Gli1 is a member of the Gli family, and is a downstreamtranscriptional target molecule of the Shh pathway. In ad-dition, accumulation of full-length Gli1 results in activationof Shh pathway target genes, thus expression of Gli1, andespecially its translocation to the nucleus, is a marker of Shhsignaling activation [10,12]. Moreover, with induction ofdownstream Ptch transcription by Shh–Gli pathway activa-tion, which forms a negative feedback loop to the Shh–Glipathway, various members of the Shh signaling pathway linkinto a coherent circuit. It is widely accepted that aberrant
activation of Shh signaling is implicated in a variety ofcancers and disease states, such as pancreatic intraepithelialneoplasias, which occur in both chronic pancreatitis andpancreatic cancer [13–19]. However, there has been littleresearch into the expression of Shh signaling molecules inthe mucosa of CC and GBC.
In this study, we classified CC into three groups mainlyaccording to the degree of hypertrophic wall, then exploredthe relationship between the expression of Shh signalingproteins and the different stages of CC to GBC. The aim ofthe study was to investigate the role of Shh signaling mole-cules in the development and progression of GBC.
2. Materials and methods
2.1. Ethics approval
This study was approved by the medical ethics committeeof Nanfang Hospital, and specimens were treated anony-mously according to ethical and legal standards.
2.2. Patients and specimens
We obtained 10 normal mucosa, 95 CC, and 32 GBCspecimens. The 95 formalin-fixed and paraffin wax–embedded cholecystectomy specimens taken from patientswith CC were obtained from Nanfang Hospital of SouthernMedical University (Guangzhou, China) during the period2009–2010. All specimens were stained with hematoxylinand eosin and were reviewed by a senior pathologist, whoassessed and defined the degree of CC. Based on the degreeof inflammatory cell infiltration, the degree of hyperplasia,and the thickness of the muscular layer, chronic inflamma-tion was classified as follows: mild cholecystitis (a fewlymphocytes and mononuclear cells present in the mucosaand submucosa, with mild thickening, defined as muscularlayer thickness less than one-third that of the whole wall);moderate cholecystitis (moderate infiltration of lymphocytesand mononuclear cells, and moderate thickening, defined asmuscular layer thickness of between one-third and two-thirdsthat of the wall); and severe cholecystitis (lymphocytes andmononuclear cells present throughout the wall, along withsevere thickening, defined as muscular layer thickness morethan two-thirds that of the wall).
The 32 cases of surgically resected primary GBCs werecollected from April 2006 to June 2010. The AmericanJoint Committee on Cancer primary tumor classificationand the World Health Organization histological typing [20]were applied to all 32 cases, which were staged as follows: 3at stage 0, 2 at stage I, 5 at stage II, 16 at stage III, and 6 atstage IV.
The 10 normal mucosa specimens were collected fromsurgical specimens for comparison. The clinical data on thesepatients were available from hospital records.
3Aberrant Shh signaling in chronic cholecystitis and GBC
2.3. Immunohistochemistry
Indirect immunohistochemistry was performed on forma-lin-fixed, paraffin wax–embedded tissue sections serially cutat 3- to 5-μm thickness. The sections were dewaxed inxylene and rehydrated in a graded alcohol series. Antigenretrieval was achieved by boiling in sodium citrate (0.01 M,pH 6.0) for 10 min. Endogenous peroxidase activity wasblocked by incubation with 3% hydrogen peroxide inmethanol for 10 min at room temperature. After blockingwith normal serum for 20 min, the sections were incubatedovernight with primary antibodies at 4°C. The followingprimary antibodies were used: Shh (Millipore, Massachu-setts, USA), Ptch (Abcam, Cambridge, MA) and Gli1 (SantaCruz Biotechnology, Santa Cruz, CA). After incubation withthe appropriate biotinylated secondary antibodies (Zhong-shanjinqiao, Beijing, China), the labeled antigen–antibodycomplexes were visualized as brown pigments via a standardDAB (Zhongshanjinqiao) protocol. The slides were counter-stained lightly with hematoxylin. Stained sections withoutprimary antibody incubation served as the negative control.
2.4. Immunohistochemical evaluation
Each immunostained section was viewed under lightmicroscopy and evaluated independently by two experiencedpathologists. Immunoreactivity was assessed by a semiquan-titative scoring system, as described previously [5,20]. Ex-pression of Shh signaling target proteins in the membrane,cytoplasm, and nucleus were assessed separately. Theimmunoreactive score (IRS) was obtained by multiplyingthe staining intensity (0, negative; 1, weak; 2, moderate; and3, strong) and the percentage of positive cells (0, b5%; 1,
Table 1 Clinicopathological characteristics of chronic cholecystitis a
NC (n = 10) Cholecystitis
Mild (n = 35)
GenderMale/female 4/6 16/19Age (years) 45 ± 17 56 ± 12 a
Atypical hyperplasiaMild 0 1Moderate to poor 0 0Concurrent diseasesCholecystolithiasis 0 20Gallbladder polyps 0 2Gallbladder adenoma 0 0Differentiation typeWell NA NAModerate to poor NA NA
Abbreviations: NA, not applicable; NC, normal control.a Mild cholecystitis vs GBC, P b .05.b Moderate cholecystitis vs GBC, P b .05.c Severe cholecystitis vs GBC, P b .05.
5%–25%; 2, 26%–50%; 3, 51%–75%; and 4, N75%). Theoverall immunostaining results were classified and definedas follows: negative (−), IRS of 0–1; weakly positive (+),IRS of 1–5; strongly positive (++) , IRS of 5–12.
2.5. Statistical analysis
Results for continuous variables are presented as mean ±SD. A non-parametrically two-tailed Mann-Whitney U testwas used to assess the differences in the Shh, Ptch, and Gli1expression levels. Spearman's rank correlation was used toevaluate the association in the Shh signaling proteinsexpression levels. Cumulative survival scores were calcu-lated by the Kaplan-Meier method and compared using a log-rank test. P b .05 was considered statistically significant.Analyses were performed with SPSS software (version 13.0;SPSS Inc, Chicago, IL, USA).
3. Results
3.1. Clinicopathological features
In total, 95 CC and 35 GBC specimens were analyzed.The clinicopathological features of all cases obtained frompathological reports are shown in Table 1. The age ofpatients with GBC was (mean ± SD) 67 ± 12 years (range45-82 years), and the ages of patients with mild, moderate,and severe CC were 56 ± 12 years (33-71 years), 48 ±18 years (33-72 years), and 53 ± 13 years (35-77 years),respectively (P b .05). In the GBC group, the male-to-female ratio was 1 to 2.2. Mild atypical hyperplasia wasdetected in three mild–moderate CC specimens (4.3%, 3/70),
nd GBC
GBC (n = 32)
Moderate (n = 35) Severe (n = 25)
15/20 12/13 11/2148 ± 18 b 53 ± 13 c 67 ± 12
2 0 00 4 3
27 22 285 6 71 3 5
NA NA 11NA NA 21
Fig. 1 Immunohistochemical staining of Shh, Ptch, and Gli1 in normal mucosa, chronic cholecystitis, and gallbladder carcinoma. A,Immunostaining of Shh, Ptch, and Gli1 proteins in normal mucosa. B-D, Immunostaining of Shh, Ptch, and Gli1 proteins in mild to severechronic cholecystitis. E, Immunostaining of Shh, Ptch, and Gli1 proteins in gallbladder carcinoma.
4 F. Xie et al.
while moderate–poor atypical hyperplasia, a precursor ofGBC, was found in four severe CC specimens (16%, 4/25).The most frequently observed concurrent disease wascholecystolithiasis, seen in 83.2% of patients with CC and87.5% of patients with GBC. In addition, the occurrence ofconcurrent disease (cholecystolithiasis, polyps, and gallblad-der adenoma) was related to the degree of inflammation inCC.
3.2. Expression of Shh protein in normal mucosa,CC and GBC
Representative immunostaining profiles for Shh, Ptch,and Gli1 are diagrammatically shown in Fig. 1, and thedistribution of staining intensity is presented in Table 2.Compared with normal mucosa, CC and GBC showed in-creased expression of Shh protein. No cytoplasmic or nuclear
Table 2 Immunohistochemical expression of Shh signalingproteins in normal mucosa, chronic cholecystitis and GBC
n Shh Ptch Gli1
− + ++ − + ++ − + ++
Normal 10 10 0 0 9 1 0 10 0 0Mild 35 16 18 1 18 16 1 14 21 0Moderate 35 4 27 4 6 27 2 7 27 1Severe 25 1 2 22 4 12 9 4 15 6GBC 32 2 17 13 1 12 19 1 15 16
expression of Shh was found in normal mucosa, whereasimmunostaining of Shh was detected in the cytoplasm ofboth CC and GBC specimens (Fig. 1). The positive stainingrate of Shh expression in the mild, moderate, and severe CCgroups was 54% (19/35), 89% (31/35), and 96% (24/25),respectively, and that in the GBC group was 97% (31/32).The majority (88%) of severe CC cases showed strongstaining intensity for Shh, which was higher than that of theother groups (3% for mild CC, 11% for moderate CC, and41% for GBC). In addition, as shown in Fig. 2, the IRS ofShh in the severe CC group was significantly higher than thatof the other groups (P b .05).
3.3. Expression of Shh signaling target proteins innormal mucosa, CC and GBC
Based on the positive staining rate and the staining in-tensity, the Ptch expression level gradually increased fromnon-malignant cholecystitis to GBC (Fig. 1; Table 2). Of the10 normal mucosa specimens, only 1 exhibited weak stainingfor Ptch, whereas the positive staining rate for Ptch was 49%(17/35) in mild cholecystitis, 83% (29/35) in moderatecholecystitis, 21/25 (84%) in severe cholecystitis, and 97%(31/32) in GBC. As shown in Figs. 1-3, both CC and GBCdisplayed cytoplasmic and membranous expression of Ptch,and expression of Ptch in the GBC group was significantlyhigher than that of the three CC groups (P b .05).
Analogously, as shown in Figs. 1 and 2, Gli1 expressiondisplayed an increased trend from normal mucosa, throughCC and on to GBC. Gli1 expression was significantly higher
Fig. 2 Immunoreactive scores (IRSs) of Shh, Ptch, and Gli1 in normal mucosa, chronic cholecystitis (CC), and gallbladder carcinoma(GBC). A, IRSs of Shh. Almost all the severe chronic cholecystitis samples expressed Shh, and the IRS of Shh in severe chronic cholecystitiswas higher than that in the normal group, the mild to moderate CC groups and the GBC group (P b .05). B, IRS of Ptch. The IRS of Ptch inGBC group was significantly higher than that of the non-malignant groups (P b .05), and no difference was found between the mild CC groupand the normal group. C, IRS of Gli1. The IRS of Gli1 in the GBC group was significantly higher than that in the non-malignant groups(P b .05), and no difference was found between mild and moderate CC.
5Aberrant Shh signaling in chronic cholecystitis and GBC
in the GBC group compared with other groups (P b .05), andno difference was observed between the mild and moderateCC groups. In the normal mucosa control group, the mucoustissue exhibited no cytoplasmic or nuclear expression of Gli1,whereas 60% (21/35) of mild CC specimens and 77% (27/35)
Fig. 3 Immunohistochemical staining of Ptch in CC and gallbladder carmoderate, and severe CC and gallbladder, respectively.
of moderate CC specimens displayed cytoplasmic expressionof Gli1. Of 25 severe CC tissues, 21 (84%) had cytoplasmicexpression of Gli1, and 4 of the 25 specimens (16%) withmoderate to poor atypical hyperplasia showed nuclearexpression of Gli1. As in the GBC group, positive Gli1
cinoma. Cytoplasmic and membraneous expression of Ptch in mild,
Fig. 4 Immunohistochemical staining of Gli1 in chronic cholecystitis (CC) and gallbladder carcinoma. Mild and moderate CC displayedcytoplasmic expression of Gli1 separately. Severe CC and gallbladder cancer displayed cytoplasmic and nuclear expression of Gli1 separately.
6 F. Xie et al.
expression was detected in 97% of GBC specimens (31/32),and 62.5% of those specimens (20/32) showed nuclearexpression (Figs. 1 and 4).
3.4. Correlation of Shh overexpression with Ptchand Gli1 proteins
To investigate correlations between expression of Shhand that of the other two target proteins (Ptch and Gli1) inthe Shh signaling pathway, we applied Spearman's rankcorrelation analysis to the 95 CC cases and 32 GBC cases.The results for the correlations between Shh expression andthat of Ptch and Gli1 are shown in Table 3. Shh accumulationwas associated with overexpression of both Ptch (ρ = 0.351,P b .001) and Gli1 (ρ = 0.343, P b .001), and expression ofPtch also correlated with that of Gli1 (ρ = 0.357, P b .001).
Table 3 Correlations of Shh expression with Ptch and Gli1expression
Shh Ptch Gli1
γs P γs P γs P
Shh 1.000 .000Ptch 0.351 ⁎⁎ .000 1.000 .000Gli1 0.343 ⁎⁎ .000 0.357 ⁎⁎ .000 1.000 0.000
⁎⁎ Correlation significant at the .01 level (2-tailed).
3.5. Survival analysis
Based on the immunohistochemistry results, survivalcurves were used to analyze whether Shh, Ptch, and Gli1expression in GBC has an influence on clinical prognoses.Using the Kaplan–Meier method and the log-rank test(median follow-up 20 months), we found that patients withhigh Gli1-expressing tumors (IRS 5–12) had a less favorableoutcome than patients with low Gli1-expressing tumors (IRS0–5) in a univariate survival analysis (P = .040). By con-trast, overexpression of Shh and Ptch did not correlate withthe clinical prognosis of patients with GBC (Fig. 5).
4. Discussion
In this study, we evaluated the clinicopathologicalfeatures and the expression of Shh signaling proteins innormal gallbladder epithelium, CC, and GBC. Based on thedegree of inflammatory cell infiltration, the degree ofhyperplasia, and the thickness of the muscular layer, CCwas classified into three groups (mild, moderate, and severe),consistent with previous reports [3,21]. Considering theaverage age of patients with CC (52 ± 15 years) and of thosewith GBC (67 ± 12 years), it is possible that there is a periodof continuous inflammation preceding the development of
Fig. 5 Survival curves of patients with gallbladder cancer, based on expression of Shh, Ptch and Gli1. A and B, Prognosis did not correlatewith Shh and Ptch expression separately. C, Patients with high Gli1 expression (IRS = 5–12) displayed a poorer overall survival estimation(P = .04; univariate log-rank analysis).
7Aberrant Shh signaling in chronic cholecystitis and GBC
gallbladder tumors. In the present study, we found that theoccurrence rate of concurrent disease (cholecystothiasis,polyps and gallbladder adenoma, which are closely related togallbladder carcinogenesis) increases in line with theincrease in degree of inflammation in CC, and poor atypicalhyperplasia was detected in four specimens of severe CC.Aberrant activation of Shh signaling proteins is observed inCC and GBC, and a positive correlation between expressionof Shh and that of Ptch and Gli1 was also confirmed in thisstudy. We further found that Shh protein expression wascorrelated with inflammatory levels, and severe CC ex-hibited higher Shh protein expression compared with mild tomoderate CC. Interestingly, the level of Shh expression inGBC was lower than that in severe CC. We speculate that,during repeated damage in the chronic inflammation →regeneration → epithelial atypical hyperplasia → carcino-genesis sequence, Shh expression may initially increase andthen decrease during the progression to GBC. It has beenwidely reported that overexpression of inflammatory cyto-kines in chronic pancreatitis can activate Shh expression, andthus result in an acceleration of pancreatic cancer cellproliferation, which indicates that chronic pancreatitis may bean important precursor in pancreatic carcinogenesis [11,22].Therefore, we suggest that in a similar manner, CC, especiallysevere CC, may be a precursor of GBC. Such a findingwarrants further and more extensive studies for confirmation.
To investigate the activation of Shh signaling in CC andGBCs, we also examined the positive staining rate and thestaining intensity of two Shh signaling target proteins, Ptchand Gli1. GBC specimens displayed significantly higherlevels of Ptch and Gli1 proteins compared with that of non-malignant groups. Ptch is a known tumor suppressor with aregulatory feedback effect on Shh signaling, and is impli-cated in many human tumors [12,16,18,23]. Because it is atransmembrane receptor, both membranous and cytoplasmicexpression of Ptch was detected in CC and GBC. Gli1, an
important nuclear transcriptional regulator in the Shhsignaling pathway, has also been reported to be implicatedin the development of various human tumors. It is wellknown that nuclear accumulation of full-length Gli1can promote the transcription of Shh target genes [24–29].In the present study, we found nuclear expression of Gli1 in16% of severe CC specimens with moderate to poor atypicalhyperplasia and in 62.5% of GBC specimens, further sup-porting that Shh signaling is abnormally activated in GBCand its potential precursor, severe CC. As mentioned above,Shh accumulation commonly has a positive correlation withthe levels of its target molecules. Thus, these results indicatethat altered Shh signaling may contribute to gallbladdercarcinogenesis by functioning as a trigger in the CC tocarcinoma sequence. Intriguingly, in the present study, somecases in the GBC group that displayed Ptch or Gli1 over-expression did not have high Shh expression, suggesting thatin GBC, up-regulation of Shh may not be the only reasonfor activation of Ptch or Gli1, and that additional proteinsmight be involved in regulating these two members of theShh signaling pathway, as previously reported in colorectalcarcinomas [30,31].
We also performed a survival analysis based on weak orstrong staining for Shh, Ptch, and Gli1 expression. We foundthat high Gli1 expression correlated with unfavorable out-come for patients with GBC, whereas expression of Shh andPtch had no significant effects. These results indicate thatGli1 overexpression might be a prognostic marker for diag-nosis of GBC, which is in accordance with earlier findings inhuman breast cancer, esophageal squamous carcinoma, andprostate cancer [8,15,26].
In addition, it is well known that two important modes ofShh pathway activation exist in various cancers [32]. Thefirst is the Shh ligand-dependent mode; overexpression ofShh responds to Ptch through the autocrine or paracrinepathway, thus leading to constitutive activation of the Shh
8 F. Xie et al.
pathway. The second is the Shh ligand-independent mode;loss-of-function mutations in Ptch1 or gain-of-functionmutations in Smo results in the aberrant activation of theShh pathway. Because overexpression of Shh/Gli1 ormutation of Ptch/Smo lead to aberrant activation of theShh pathway and thus result in tumorgenesis and metas-tasis, inhibition of the Shh pathway is assumed to be apotential therapeutic option to reduce tumor burden. Manyreports have confirmed that small molecules, such ascyclopamine and GDC-0449, are effective in inhibitingdevelopment and invasiveness of basal cell carcinoma byrepressing Smo activation [33,34]. SANT1 and Cur-61414,which bind to Smo and result in repression of Smo, have aninhibitory effect in activation of Shh signaling pathway[35]. Current research suggests that compounds andderivations from natural products have also been shownto be effective Shh pathway inhibitors. For example,epigallocatechin-3-gallate, a compound of green tea, in-hibits growth of prostate cancer cells by suppressing Gli1expression [36]. Similarly, zerumbone was demonstrated toinhibit the activation of the Shh signaling pathway in HeLacells by suppressing Gli expression [37]. These resultssuggest that small molecule inhibitors and natural com-pounds may be useful in clinical treatment of cancers.Considering the complicated mechanism of Shh pathwayactivation, special attention should be paid to thetherapeutic effect of inhibitors.
In conclusion, our data suggest that expression of Shh,Ptch1, and Gli1 is significantly upregulated in GBC. Futurein vitro and in vivo studies will be needed to address whetherinhibitors of Shh signaling are effective in treating GBC.
References
[1] Yang Y, Xian X, Xie X, Zhuang Y, Wu W, Wang W. Expression andregulation of hedgehog signaling pathway in pancreatic cancer.Langenbecks Arch Surg 2010;395:515-25.
[2] Priya TP, Kapoor VK, Krishnani N, Agrawal V, Agrawal S. Role of E-cadherin gene in gall bladder cancer and its precursor lesions.Virchows Arch 2010;456:507-14.
[3] Yanagisawa N, Mikami T, Koike M, Okayasu I. Enhanced cellkinetics, p53 accumulation and high p21WAF1 expression in chroniccholecystitis: comparison with background mucosa of gallbladdercarcinomas. Histopathology 2000;36:54-61.
[4] Jawad N, Direkze N, Leedham SJ. Inflammatory bowel disease andcolon cancer. Recent Results Cancer Res 2011;185:99-115.
[5] Yoshihara H, Noda K, Kamada T. Interrelationship between alcoholintake, hepatitis C, liver cirrhosis, and hepatocellular carcinoma.Recent Dev Alcohol 1998;14:457-69.
[6] Guerra C, Schuhmacher AJ, Canamero M, et al. Chronic pancreatitis isessential for induction of pancreatic ductal adenocarcinoma by K-Rasoncogenes in adult mice. Cancer Cell 2007;11:291-302.
[7] Yanagisawa N, Yamashita K, Kuba T, Okayasu I. Sporadic TP53transition mutations in chronic cholecystitis are possibly linked togallbladder carcinogenesis. Anticancer Res 2010;30:4443-9.
[8] Ingham PW, McMahon AP. Hedgehog signaling in animal develop-ment: paradigms and principles. Genes Dev 2001;15:3059-87.
[9] Gilbert-Barness E, Debich-Spicer D, Cohen Jr MM, Opitz JM.Evidence for the “midline” hypothesis in associated defects of lateral-
ity formation and multiple midline anomalies. Am J Med Genet 2001;101:382-7.
[10] Chen X, Horiuchi A, Kikuchi N, et al. Hedgehog signal pathway isactivated in ovarian carcinomas, correlating with cell proliferation: It'sinhibition leads to growth suppression and apoptosis. Cancer Sci2007;98:68-76.
[11] Nakashima H, Nakamura M, Yamaguchi H, et al. Nuclear factor-Bcontributes to hedgehog signaling pathway activation through sonichedgehog induction in pancreatic cancer. Cancer Res 2006;66:7041-9.
[12] Cui W, Wang L-H, Wen Y-Y, et al. Expression and regulation mech-anisms of Sonic Hedgehog in breast cancer. Cancer Sci 2010;101:927-33.
[13] Williams JA, Guicherit OM, Zaharian BI, et al. Identification of asmall molecule inhibitor of the hedgehog signaling pathway: effects onbasal cell carcinoma-like lesions. Proc Natl Acad Sci U S A 2003;100:4616-21.
[14] Berman DM, Karhadkar SS, Hallahan AR, et al. Medulloblastomagrowth inhibition by hedgehog pathway blockade. Science 2002;297:1559-61.
[15] Xu L, Kwon Y-J, Frolova N, et al. Gli1 promotes cell survival and ispredictive of a poor outcome in ERα-negative breast cancer. BreastCancer Res Treat 2010;123:59-71.
[16] Thayer SP, di Magliano MP, Heiser PW, et al. Matthias Hebrok:Hedgehog is an early and late mediator of pancreatic cancertumorigenesis. Nature 2003;425:851-6.
[17] Kim JH, Choi YJ, Lee SH, et al. Effect of Helicobacter pylori infectionon the sonic hedgehog signaling pathway in gastric cancer cells. OncolRep 2010;23:1523-8.
[18] Yoo YA, Kang MH, Kim JS, Oh SC. Sonic hedgehog signalingpromotes motility and invasiveness of gastric cancer cells throughTGF-beta-mediated activation of the ALK5-Smad 3 pathway.Carcinogenesis 2008;29:480-90.
[19] Strobel O, Rosow DE, Rakhlin EY, et al. Pancreatic duct glands aredistinct ductal compartments that react to chronic injury and mediateShh-induced metaplasia. Gastroenterology 2010;138:1166-77.
[20] Edge SB, Byrd DR, Compton CC. AJCC Cancer Staging Manual.7th ed. New York: Springer; 2009. p. 211-7.
[21] Barcia J. Histologic analysis of chronic inflammatory patterns in thegallbladder: diagnostic criteria for reporting cholecystitis. Ann DiagnPathol 2003;7:147-53.
[22] Garcea G, Dennison AR, Steward WP, Berry DP. Role of inflam-mation in pancreatic carcinogenesis and the implications for futuretherapy. Pancreatology 2005;5:514-29.
[23] Yoshikawa K, Shimada M, Miyamoto H, et al. Sonic hedgehog relatesto colorectal carcinogenesis. J Gastroenterol 2009;44:1113-7.
[24] Ten Haaf A, Bektas N, von Serenyi S, et al. Expression of theglioma-associated oncogene homolog (GLI) 1 in human breast canceris associated with unfavourable overall survival. BMC Cancer 2009;9:298.
[25] Zhu W, You Z, Li T, et al. Correlation of hedgehog signal activationwith chemoradiotherapy sensitivity and survival in esophagealsquamous cell carcinomas. Jpn J Clin Oncol 2011;41:386-93.
[26] Mori Y, Okumura T, Tsunoda S, Sakai Y, Shimada Y. Gli-1 ex-pression is associated with lymph node metastasis and tumor pro-gression in esophageal squamous cell carcinoma. Oncology 2006;70:378-89.
[27] Ruiz i Altaba A, Stecca B, Sanchez P. Hedgehog–Gli signaling inbrain tumors: stem cells and paradevelopmental programs in cancer.Cancer Lett 2004;204:145-57.
[28] Wang K, Pan L, Che X, Cui D, Li C. Sonic Hedgehog/GLI1 signalingpathway inhibition restricts cell migration and invasion in humangliomas. Neurol Res 2010;32:975-80.
[29] Shigemura K, Huang WC, Li X, et al. Active sonic hedgehog signalingbetween androgen independent human prostate cancer cells andnormal/benign but not cancer-associated prostate stromal cells.Prostate 2011;71:1711-22.
9Aberrant Shh signaling in chronic cholecystitis and GBC
[30] Bian YH, Huang SH, Yang L, Ma XL, Xie JW, Zhang HW. Sonichedgehog-Gli1 pathway in colorectal adenocarcinomas. World JGastroenterol 2007;13:1659-65.
[31] Ji Z, Mei FC, Xie J, Cheng X. Oncogenic KRAS activates hedgehogsignaling pathway in pancreatic cancer cells. J Biol Chem 2007;282:14048-55.
[32] Rubin Lee L, de Sauvage Frederic J. Targeting the Hedgehog pathwayin cancer. Nat Rev Drug Discov 2006;5:1026-33.
[33] Chen JK, Taipale J, Cooper MK, Beachy PA. Inhibition of Hedgehogsignaling by direct binding of cyclopamine to Smoothened. Genes Dev2002;16:2743-8.
[34] Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL, TibesR. Inhibition of the hedgehog pathway in advanced basal-cellcarcinoma. N Engl J Med 2009;361:1164-72.
[35] Stanton BZ, Peng LF. Small-molecule modulators of the SonicHedgehog signaling pathway. Mol Biosyst 2010;6:44-54.
[36] Slusarz A, Shenouda NS, Sakla MS, et al. Common botanicalcompounds inhibit the hedgehog signaling pathway in prostate cancer.Cancer Res 2010;70:3382-90.
[37] Hosoya T, Arai MA, Koyano T, Kowithayakorn T, Ishibashi M.Naturally occurring small-molecule inhibitors of Hedgehog/GLI-mediated transcription. Chembiochem 2008;9:1082-92.
