Cholangiocarcinogenesis Compared with Biliary Ductal ... - NCBI - NIH

American Journal of Pathology, Vol. 151, No. 6, December 1997 Copyright American Society for Investigative Patbology

NEU Overexpression in the Furan Rat Model of Cholangiocarcinogenesis Compared with Biliary Ductal Cell Hyperplasia

Alphonse E. Sirica, Svetlana Radaeva, and Nicole Caran From the Department of Pathology, Virginia Commonwealth University-Medical College of Virginia, Richmond, Virginia

inmmunohistochemical studies have suggested that the tyrosine kinase growth factor receptor pl85nIU is overexpressed in a high percentage of human cholangiocarcinomas. To establish the specificity and temporal relationship between the expression of this receptor in cholangiocarcinogenesis, we investigated c-neu expression in precancerous cholangiofibrotic tissue and subsequently derived primary and transplantable cholangiocarcinomas originated in the livers of furan-treated rats. Proliferated bile ductules formed in rat models of bile ductular hyperplasia and the celi types of normal adult rat liver were also analyzed for c-neu expression. c-neu expression was not detected in normal adult rat liver by either Western blotting, immunohistochemistry, or in situ hybridization. In comparison, all of the cholangiocarcinomas analyzed, which were characterized by intestinal-type mucin-producing neoplastic glands, exhibited a prominent band with a molecular weight 185 kd, corresponding to pl85neu Only the neoplastic glandular epithelia of the cholangiocarcinomas showed a strong immunoreactivity for pl85neu, which was predominantly localized to their cell surface but also observed cytoplasmically. In situ hybridization further revealed the cytoplasm of the tumor glandular epithelial cells to be strongly positive for c-neu mRNA transcripts. Of particular interest was our finding that c-neu is expressed early in furan cholangiocarcinogenesis, being more pronounced in the metaplastic intestinal glands of cholanglofibrotic tissue than in hyperplastic biliary epithelial celis in either the same tissue or in hyperplastic bile ductule tissue. Our results demonstrate that c-neu overexpression is a prominent feature of intestinal-type cholangiocarcinomas as well as of metaplastic intestinal glands that precede their development and is detected at lower levels in hyperplastic biliary epithelia. The overexpression of c-neu in the metaplastic and malignant neoplastic glands also correlated with their increased proliferating cell nuclear antigen (PCNA) labeling indices relative to those of hyperpLastic biliary

ducts and ductules and also appeared to correlate with their intestinal glandular pattern of differentiation. (Am JPathol 1997, 151:1685-1694)

Molecular events associated with the development of intrahepatic cholangiocarcinoma in humans and in experimental animal models have not been extensively investigated, nor have there been any definitive studies to date aimed at identifying critical alterations in the expression of proto-oncogene-encoded growth factor receptors that may be common to both human biliary tumor development/progression and rodent models of cholangiocarcinogenesis. The rat neu proto-oncogene

and its human gene homologue c-erbB-2 (also termed HER-2) encode a 185-kd transmembrane type 1 receptor tyrosine kinase (p 1 85neu) sharing extensive homology with epidermal growth factor receptor (EGF-R). Voravud et al 1 were the first to report positive immunohistochemical staining of neoplastic epithelium in 73% of human cholangiocarcinomas of Thai and English origin when reacted with a polyclonal antiERBB-2 antibody, with many of the tumors seen to have diffuse staining whereas others exhibited strong plasma membrane staining. In this study, immunostaining for p185neu was not detected in the cell types of fetal, immature, and adult human livers. Using a monoclonal antibody, Motojima et a12 also found that 68% of cholangiocarcinomas of Japanese origin were apparently immunoreactive for p185neu and that positive expression of c-erbB-2-encoded receptor correlated with tumor stage and shorter survival. These percentages are markedly higher than the approximately 30% of human breast cancers characterized by c-erbB-2 overexpression associated with poorer prognosis in node-positive patients3.4 and suggest that p185neu expression could be of potential value in distinguishing cholangiocarcinoma from non-

This work was presented in part at the 47th Annual Meeting of The American Association for the Study of Liver Diseases held in Chicago, IL, November 8-12, 1996.37 Supported by grant 5R01 CA39225 to A.E. Sirica from the National Cancer Institute, NIH. Accepted for publication September 21, 1997. Address reprint requests to Dr. Alphonse E. Sirica, Department of Pathology, Virginia Commonwealth University-Medical College of Virginia, P.O. Box 980297, Richmond, VA 23298-0297.

1685

1686

Sirica et al

AJP December 1997, Vol. 151, No. 6

neoplastic biliary conditions. The high incidences of human cholangiocarcinoma reported to be immunoreactive for p185neu further suggest that elevated expression of this receptor may be playing a biologically significant role in the development or progression of biliary cell malignancy. However, the studies of Voravud et a1l and of Motojima et a12 are limited by the fact that they did not validate their immunohistochemical findings by Western blot analysis, nor did they determine whether c-erbB-2 mRNA transcripts were also elevated in the human cholangiocarcinomas relative to control livers. Moreover, they did not assess c-erbB-2 expression in cases of non-neoplastic biliary proliferation or in precancerous biliary lesions. Also, in contrast to the findings of Voravud et al1 and of Motojima et a12, Collier et al,5 using a monoclonal antibody (NLC-CB1 1) raised against a synthetic peptide corresponding to the carboxy terminus of the internal domain of the c-erbB-2 receptor were unable to detect plasma membrane immunoreactivity for p1 85neu in tissue sections from 10 archival cases of human cholangiocarcinoma of European origin but noted weak cytoplasmic staining in the neoplastic biliary epithelium of one of the 10 cholangiocarcinomas analyzed. In this same study, Collier et a15 did not detect by Northern blotting c-erbB-2 mRNA in two human hepatocellular carcinomas that showed cytoplasmic staining when reacted with NLC-CB1 1 antibody, confirming that cytoplasmic immunostaining is in itself not a reliable criterion for identifying overexpression of p185neu. Carver et al6 did not detect p185feU by Western blotting in either normal adult rat liver or hepatocyte lysates, nor did they detect by reverse transcriptase polymerase chain reaction analysis mRNA corresponding to c-neu in adult rat liver. Polimeno et al7 also failed to detect c-neu mRNA by Northern blot analysis in rat liver at 12 to 48 hours after two-thirds hepatectomy (PH) but did detect elevated levels of c-neu mRNA from 48 to 96 hours after bile duct ligation (BDL) and at 72 hours after PH. However, the specific liver cell type(s) expressing c-neu mRNA under these conditions was not identified, nor was data given on the production and cytological localization of the p185neu receptor. In an effort to address some of the limitations and conflicts raised above, and to clarify the potential relationship between c-neu and cholangiocarcinoma versus non-neoplastic biliary proliferation, we have used Western blotting, immunohistochemistry, and in situ hybridization to investigate the specificity and temporal pattern of expression of c-neu in the furan rat model of intrahepatic cholangiocarcinogenesis. This model has been well described,8 13 and we have demonstrated a relationship between early-appearing hyperplastic biliary ductal structures and associated development of metaplastic small intestinal-like glands that preferentially form in the right and caudate liver lobes of furan-treated rats.9 10,14 These metaplastic glands precede the development of a stable cholangiofibrosis in these same liver lobes, which then serves as a precursor lesion to the high incidences of intestinal-type cholangiocarcinomas that also preferentially form in the right/caudate liver lobes.11 Specifically, we have analyzed c-neu expression in both primary

and transplantable cholangiocarcinomas that originated in the livers of furan-treated rats, as well as in the hyperplastic biliary and metaplastic intestinal epithelia that characterize early-appearing cholangiofibrotic lesions induced in rat liver by furan. For comparison, we have also analyzed typical hyperplastic bile ductules formed in cholestatic rat livers as well as the cell types of normal adult rat liver for c-neu expression.

Materials and Methods Chemicals and Immunochemical and in Situ Hybridization Reagents Acrylamide/bis-acrylamide (29:1 ratio), 3-aminopropyltriethoxysilane, 3,3'-diaminobenzidine tetrahydrochloride, and hydrogen peroxide were purchased from Sigma Chemical Co. (St. Louis, MO). Hybond ECL nitrocellulose protein blotting membranes and ECL Western blotting detection reagents were purchased from Amersham Life Sciences (Arlington Heights, IL). Peel-A-Way embedding paraffin pellets (melting point 53 to 550C) were obtained from VWR Scientific (Bridgeport, NJ), protein A-agarose from Santa Cruz Biotechnology (Santa Cruz, CA), and bromophenol blue and sodium dodecyl sulfate (SDS)PAGE molecular weight protein standards from Bio-Rad Laboratories (Melville, NY). Paraformaldehyde was purchased from Polysciences (Warrington, PA), and acetic anhydride was purchased from Fisher Scientific (Norcross, GA). Vectastain Elite avidin-biotin-peroxidase (ABC) immunostaining kit and avidin-biotin blocking kit were purchased from Vector Laboratories (Burlingame, CA). Reagents for in situ hybridization were purchased from Boehringer Mannheim Corp. (Indianapolis, IN).

Primary Antibodies and Antigens Neu (C-18), an affinity-purified rabbit polyclonal antibody raised against an epitope corresponding to amino acids 1 169 to 1 186 mapping at the carboxy terminus of human p185neu, together with the corresponding immunizing peptide (catalog item sc-284 P), were purchased from Santa Cruz Biotechnology. Neu (C-18) antibody reacts with rat p185neu and is non-cross-reactive with EGF-R or with c-erbB-3-encoded proteins.

RNA Probes The clone neu c(t)/SP6400 ligated into the vector pSP65 was purchased from American Type Culture Collection (Rockville, MD). This clone represents a 420-bp segment (BamHI digest) of the neu proto-oncogene derived from the rat neuroblastoma B104 cell line. After linearizing the vector with Hindlil, a digoxigenin (DIG)-labeled antisense cRNA probe was synthesized with SP6 RNA polymerase according to the manufacturer's instructions, using the Boehringer Mannheim DIG RNA labeling kit (SP6/T7). This cRNA probe was then purified by ethanol precipitation and its correct size verified by 1.0% agarose gel

NEU in Rat Biliary Tumors 1687 AJP December 1997, Vol. 151, No. 6

electrophoresis. Typical yields of 8 to 10 ,tg of DIGlabeled neu cRNA probe were obtained. A nonspecific cRNA probe of similar size (760 bp) was generated from control DNA2, pSPT 19-Neo supplied with the DIG RNA labeling kit and was labeled concurrently in exactly the same manner as that described for the neu RNA probe. This nonspecific cRNA probed served as a negative control.

Rat Tissue Specimens The primary and transplantable rat cholangiocarcinomas used in this study originated from the livers of Fischer 344 male rats after carcinogenic exposure to the nongenotoxic agent furan, as described previously.11'12 In all cases, the primary intrahepatic cholangiocarcinomas were classified as mucin-producing intestinal-type (tubular) adenocarcinoma.11-13 These tumors preferentially developed in the right/caudate liver lobes of the furantreated rats,11 they spread intrahepatically but did not show evidence of extrahepatic metastasis,11,12 and they exhibited histological and biological features closely resembling intestinal-type cholangiocarcinomas of the human.15 The transplantable rat cholangiocarcinomas were initially established from 1- to 2-mm viable, non-necrotic primary tumor tissue pieces that were individually transplanted into the inguinal fat pads of young adult Fischer 344 male recipient rats, as described previously.11 These transplantable tumors exhibited rapid autonomous growth at the transplantation site, could be serially passaged in inguinal fat pads, retained histological features that closely resembled those of the parent hepatic tumors, and were invasive but not metastatic, even after 16 passages in vivo. Cholangiofibrotic tissue composed of metaplastic intestinal-like glands and hyperplastic biliary ductal structures in a dense fibrotic stroma was also preferentially induced in the right/caudate liver lobes of Fischer 344 male rats after only 6 weeks of furan treatment, as described previously.10'16 In addition, massive bile ductular hyperplasia in which liver is almost totally replaced with well differentiated bile ductules was induced in the livers of rats subjected to BDL followed by 6 weeks of furan treatment (7-week BDL/6-week furan), also as described previously.12'13'16 Liver samples from untreated Fischer 344 adult male rats weighing -200 g served as normal control tissue. All of the animal experimentation described in this study was conducted after approval from Virginia Commonwealth University's Institutional Animal Care and Use Committee (Animal Protocol Number

9403-1968).

Immunoprecipitation and Western Blot Analysis Rat tissue specimens used for Western blotting were snap-frozen on removal and then stored at -800C until analyzed. Immunoprecipitation of p185neu was carried out on tissue lysates prepared by homogenizing 0.5 g of individual rat tissue samples (tumor or normal liver) described above in 1.5 ml of RIPA buffer on ice, pH 7.4 (9.1

mmol/L Na2HPO4, 1.7 mmol/L NaH2PO4, 150 mmol/L NaCI, 1.0% Nonidet P-40. 0.05% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), containing 1.1 mmol/L phenylmethylsulfonyl fluoride, 0.45 to 0.9 TIU (trypsin inhibitor units) of aprotinin, and 1 mmol/L sodium orthovanadate). Clarified lysates were immunoprecipitated with Neu (C-1 8) at 4°C for 1 hour. The samples were then allowed to complex with protein A-agarose at 4°C overnight. The resulting immunocomplexes were solubilized by boiling in SDS sample buffer (0.2 mol/L Tris, pH 6.8, containing 5.0% SDS, 8.0% P-mercaptoethanol, 17% glycerol, and 0.002% bromophenol blue). They were then subjected to SDS-polyacrylamide gel electrophoresis on 7.5% acrylamide gels along with a cocktail of molecular weight standards. Western blot analysis of the immunoprecipitated proteins was performed according to the procedure of Towbin et al.17 The blots were probed with Neu (C-18) at a 1:500 dilution and then incubated with horseradish-peroxidase-conjugated goat anti-rabbit IgG at 1:50,000 dilution (Bio-Rad Laboratories). Enhanced chemiluminescence (ECL) was used to detect positive bands according to instructions provided in the Amersham ECL kit. Additional details are given in the figure legends.

Immunohistochemistry Rat tissue specimens used for immunohistochemistry were fixed in 99 parts 95% ethanol/i part 1.0% glacial acetic acid and embedded in low melting point paraffin as described previously. 11'16 Immunohistochemistry was performed on 5-,um-thick tissue sections, with a routine histological examination also being conducted for each specimen on sections stained with hematoxylin and eosin (H&E). Immunohistochemical staining for p185neu was performed according to the standard avidin-biotin-peroxidase complex (ABC) method of Hsu et al,18 as described previously.11'16 Briefly, deparaffinized tissue sections were treated for 15 minutes with 0.1% H202 in 100% methanol to inhibit endogenous peroxidase activity. The tissue sections were then incubated overnight at 4°C with Neu (C-18) primary antibody (dilution = 1:150). Next, the sections were incubated for 30 minutes at room temperature with biotinylated secondary antibody, followed by treatment with Vectastain Elite ABC reagent. 3.3'-Diaminobenzidine tetrahydrochloride was used as the chromogen, and the tissue sections were counterstained with Harris hematoxylin. Negative control sections were incubated with Neu (C-18) antibody that had been neutralized by pretreatment with the immunizing peptide sc-284 P. Some sections were also pretreated with avidin-biotin blocking reagent before incubation with the primary antibody to produce optimal minimization of nonspecific

background staining.

In Situ Hybridization Tissue specimens fixed overnight in 4.0% paraformaldehyde in phosphate-buffered saline, pH 7.2, were routinely processed, embedded in paraffin, sectioned at 5 um,

1688

Sirica et al

AJP December 1997, Vol. 151, No. 6

and mounted on silanized (3-aminopropyltriethoxysilanetreated) glass microscope slides. After deparaffinization and rehydration, the sections were immersed in 0.2% HCI, followed by incubation with 10 ,ug/ml proteinase K for 30 minutes at 370C. They were then post-fixed in 4.0% paraformaldehyde and acetylated in triethanolamine containing 0.25% acetic anhydride. The sections were prehybridized in 50% formamide/2X SSC (20X SSC stock contains 3.0 mol/L NaCI and 0.3 mol/L citric acid/trisodium salt, pH 7.0) for 2 hours at 37°C and then hybridized in buffer containing 0.01 mol/L Tris/HCI, pH 7.5, 12.5% Denhardt's solution (0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 2X SSC, 50% formamide, 0.5% SDS, 250 gg/ml salmon sperm DNA, 5 mg/ml sodium pyrophosphate, and cRNA probe (final concentration, 5 ,tg/ml) for 16 hours at 420C. The following day, the sections were washed twice in 2X SSC for 15 minutes at room temperature and then treated with RNAse A for 30 minutes at room temperature. Next, the sections were washed twice in 0.1 X SSC for 20 minutes at 420C, followed by a 5-minute wash in 0.1 mol/L Tris/HCI, pH 7.5, containing 0.15 mol/L NaCI. Immunological detection was performed according to the manufacturer's instructions, with the optimal concentration of the antiDIG antibody (coupled to alkaline phosphatase) being 1:1500. Detection of bound antibody was done using Fast Red tablets as the substrate. To inhibit endogenous nonspecific alkaline phosphatase activity, levamisole at a final concentration of 240 ,ug/ml was added with the Fast Red substrate. Development was carried out for 10 to 18 hours. The reaction was stopped by washing the slides in TE buffer (0.01 mol/L Tris/HCI containing 0.001 mol/L EDTA, pH 8.0), and the slides were then mounted in glycergel. Negative controls included 1) replacement of the specific neu cRNA probe by nonspecific cRNA probe, 2) omission of the cRNA probe or antibody, and 3) previous treatment of the tissue section with RNAse A.

Results Western Blot Analysis The results of our Western blot analysis of p185neu immunoprecipitated from lysates of various primary and transplanted rat cholangiocarcinomas relative to that of normal adult rat liver are shown in Figures 1 and 2. As demonstrated, p185neu was not detected by Western blotting in any of the normal adult rat liver samples analyzed nor in the respective RIPA buffer (no tissue) control samples. However, Western blotting revealed the presence of a prominent band with a molecular weight of 185 kd being expressed in each of the primary (H6A, H6B, and H7A) as well as different in vivo passages of transplanted

cholangiocarcinomas (17Tpl, E5Ap3, E5Ap7, ESApl1, E5Ap12, E5Ap14, and E5Ap16) analyzed (Figures 1 and 2). As also shown in Figure 2, A and B, neutralizing the Neu (C-18) antibody with the immunizing peptide before immunoprecipitation specifically blocked immunodetection of the tumor p185neu protein in the Western blots.

a:

p185_

Z

wlu

I-_

200 kD

; r w ,.~.

F

F

--

t16kD 97.4 kD

66 kD -

A

45kD

Cw

Z3

p185

200 kD

-b -4-4-0-

116 kD 97.4 kD 68 kD

45 kD

B Figure 1. Results of two separate Western blot analyses (A and B) demonstrating overexpression of pl85... in rat primary and in serially transplanted furan-induced cholangiocarcinomas relative to normal rat liver. The immunoreactive thick band at -55 kd corresponds to the heavy chain of the primary antibody separated from protein A agarose used in the immunoprecipitation procedure. RIPA, no tissue buffer control; NL, normal adult rat liver samples; H6A, H6B, and H7A, primary rat cholangiocarcinoma samples; 17Tpl, passage 1 tumor transplant derived from primary cholangiocarcinoma designated 3SHF1N; E5Ap3 (passage 3), E5Ap7 (passage 7), E5Ap1l (passage 11), and E5Ap12 (passage 12), serially-transplanted tumor samples originated from primary cholangiocarcinoma designated E5A.

Immunohistochemistry Our immunohistochemical findings for p185neu expression are shown in Table 1 and in Figures 3 and 4. As exemplified by Figure 3A, immunohistochemical staining for p185neu was not detected in the intrahepatic biliary epithelia nor in the hepatocytes or in any of the other cell types of pormal adult rat liver. In agreement with our previous published findings, 1213,16 tissue sections from the right liver lobe of 7-week BDL/6-week furan-treated rats were typically observed to be composed entirely of well differentiated hyperplastic bile ductules supported by a fine fibrotic stroma. All of the hyperplastic bile ductules in these tissue sections were weakly positive for p185neu immunoreactivity (Figure 3B). Likewise, proliferated bile ductules in liver sections from 7-week BDL rats without furan treatment exhibited weakly positive immunohistochemical staining for p185neu (data not shown). In comparison, metaplastic intestinal glands of cholangiofibrotic tissue, which, as already noted, form preferentially in the right/caudate liver lobes of unoperated rats after short-term furan treatment only, were characterized by a more strongly positive immunohistochemical staining for p185neu than that exhibited by hyperplastic biliary ductal structures in the same tissue section (Figure 3, C and D; Table 1). These early-appearing metaplastic glands also showed a more strongly positive immunoreactivity for

NEU in Rat Biliary Tumors 1689 AJP December 1997, Vol. 151, No. 6

+

w

_~

'-

'-