Expression Analysis of PMP22/Gas3 in Premalignant and Malignant Pancreatic Lesions
Departments of General Surgery (JL,JK,HK,KF,MWB,HF), Pathology (IE), and Immunology (TG), University of Heidelberg, Heidelberg, Germany
Correspondence to: Jörg Kleeff, MD, Department of General Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany. E-mail: joerg_kleeff{at}med.uni-heidelberg.de
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Summary |
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(J Histochem Cytochem 53:885893, 2005)
Key Words: peripheral myelin protein22 transforming growth factor ß pancreatic cancer chronic pancreatitis nerves PanIN
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Introduction |
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In view of the role of PMP22 in regulating cell growth, in the present study we aimed to investigate the expression of this gene in pancreatic diseases.
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Materials and Methods |
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Patients and Tissue Collection
Pancreatic tissue samples were obtained from patients (median age 62.5 years; range 4178 years) with pancreatic ductal adenocarcinomas (PDACs) (n=31), intraductal papillary mucinous neoplasms (IPMNs) (n=9), and mucinous cystic neoplasms (MCNs) (n=4) at the University Hospital of Bern (Switzerland) and Heidelberg (Germany). PDAC cases were categorized according to tumornodesmetastasis classification (International Union Against Cancer 2003): most cases were T3 (74%) and N+ (70%). Twenty-two chronic pancreatitis samples were obtained from patients who underwent resection for chronic pancreatitis (median age 44 years; range 2266 years). Twenty normal human pancreatic tissue samples were obtained from previously healthy individuals through an organ donor program (median age 45 years; range 2074 years). A panel of paraffin-embedded PanIN lesions 1A (n=6), 1B (n=14), 2 (n=2), and 3 (n=7) was included in the study. Immediately (within 5 min) upon surgical removal, tissue samples were either snap-frozen in liquid nitrogen and then maintained at 80C until use (for RNA extraction) or fixed in 5% formalin and embedded in paraffin after 24 hr. All tissue samples were histologically examined by a pathologist to confirm the diagnosis. The studies were approved by the Ethics Committees of the University of Heidelberg and the University of Bern. Written informed consent was obtained from all patients.
Real-Time Quantitative RT-PCR
All reagents and equipment for mRNA and cDNA preparation were purchased from Roche (Roche Applied Science, Mannheim, Germany). mRNA was prepared via automated isolation using the MagNA Pure LC instrument and isolation Kit I (for cells) and II (for tissues). RNA was reverse-transcribed into cDNA using the 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) according to the manufacturer's instructions. Quantitative RT-PCR was performed with the Light Cycler Fast Start DNA SYBR Green kit as described previously (Li et al. 2004). The number of specific transcripts was normalized to housekeeping genes (cyclophilin B and hypoxanthine guanine phosphoribosyltransferase, HPRT). All primers were obtained from SearchLC (Heidelberg, Germany).
Laser Capture Microdissection
Tissue samples were embedded in OCT (Sakura Finetek, Torrance, CA) by freezing the blocks in an acetone bath within liquid nitrogen and stored at 80C until use. Tissue sections (68 µm thick) were prepared using a Reichard Jung 1800 cryostat. Laser capture microdissection and RNA extraction were performed as described previously in detail (Ketterer et al. 2003).
cDNA Array
The HG-U95Av2 array from Affymetrix (Santa Clara, CA) was used for analysis. Poly(A) + RNA isolation, cDNA synthesis, and cRNA in vitro transcription were performed as reported previously (Friess et al. 2003). The in vitro transcription products were purified and fragmented as previously described (Friess et al. 2003
). Hybridization of the fragmented in vitro transcription products to oligonucleotide arrays was performed as suggested by the manufacturer (Affymetrix).
Immunohistochemistry
Immunohistochemistry was performed using HistoMark Red alkaline phosphatase conjugated reagents (KPL; Gaithersburg, MD). Consecutive paraffin-embedded tissue sections (35 µm thick) were deparaffinized and rehydrated. Antigen retrieval was performed by pretreatment with citrate buffer (pH 6.0) boiling for 20 min. Thereafter, slides were cooled down to room temperature and then placed in deionized water for 5 min. The sections were then incubated for 1 hr at room temperature with normal goat serum before incubation for 1 hr at room temperature with the primary antibody (anti-PMP22, 4 µg/ml; Abcam, Cambridgeshire, UK). Next, the sections were rinsed with washing buffer (Tris-buffered saline with 0.1% BSA) and incubated with biotinylabed goat anti-mouse IgM and streptavidin phosphatase (KPL), followed by reaction with the PhThaloRED activator-buffered substrate mixture (KPL) and counterstaining with Mayer's hemotoxylin. To ensure the specificity of the primary antibody, tissue sections were incubated in the absence of the primary antibody or with control mouse IgM. Under these conditions, no specific immunostaining was detected. All slides were quantified by a qualified pathologist.
Double Immunohistochemistry
Paraffin-embedded tissue sections were deparaffnized, rehydrated, and incubated with peroxidase block (DAKO Corporation, Carpinteria, CA) for 5 min. Sections were washed in washing buffer as mentioned before, and nonspecific binding sites were blocked in 3% BSA/TBS for 1 hr. Next, sections were incubated with anti-PMP22 mouse monoclonal IgM (Abcam) overnight at 4C. Tissue sections were washed in washing buffer, then incubated with anti-mouse biotinylated IgM (KPL, Gaithersburg, MD, USA) for 45 min, washed, and incubated with streptavidin phosphatase (KPL) for 40 min. Next, sections were incubated with PhThaloRED-Activator-Buffered substrate mixture (KPL), and incubated in double stain universal block (DAKO Corporation) for 3 min. Anti-CD68 rabbit anti-human IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was applied, and specimens were incubated at 4C overnight. After 24 hr, the secondary biotinylated anti-rabbit antibody (DAKO Corporation) was applied for 45 min. Sections were then washed and incubated with streptavidin peroxidase (KPL) for 40 min and were incubated with a buffered substrate for liquid DAB/liquid DAB chromogen mixture (DAKO Corporation).
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Results |
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Discussion |
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The growth arrestspecific (gas) genes (to which PMP22 belongs) were previously described as a heterogenous group of genes originally detected in NIH3T3 fibroblasts, whose expression was specifically induced under growth arrest conditions (Schneider et al. 1988). Thus, PMP22 expression is induced during cell cycle stop and apoptosis in fibroblasts (Manfioletti et al. 1990
; Zoidl et al. 1997
). Furthermore, decreased expression of PMP22 is associated with the pathogenesis of urethan-induced lung tumors in mice (Re et al. 1992
). In contrast to the observation of Re and coworkers, high PMP22 expression has been observed in osteosarcoma and osteoblastoma, indicating that the role of PMP22 in growth arrest and differentiation is cell and tissue typedependent (Huhne et al. 1999
; Huehne and Rautenstrauss 2001
; van Dartel et al. 2002
,2003
; van Dartel and Hulsebos 2004
). Other gas family members, such as epithelial membrane protein 2, have been shown to inhibit tumor formation, indicating that epithelial membrane protein 2 acts as a functional tumor suppressor (Wang et al. 2001
). In addition, the gene gas1 inhibits cell growth (Del Sal et al. 1992
), and it has been suggested that another member of the gas family, THW, plays a role as a tumor suppressor malignant in melanoma cells (Hildebrandt et al. 2001
). Despite the aforementioned studies, nothing is known about PMP22 expression in pancreatic tissues.
In the present study, the mean PMP22 mRNA levels were significantly higher in chronic pancreatitis and PDAC compared with normal pancreatic tissues. The wide range of PMP22 mRNA expression in CP and PDAC suggests that only certain tissue elements that are characteristic for CP and PDAC are responsible for the overall expression levels. The expression data for microdissected pancreatic ductal and cancer cells demonstrate that PMP22 expression in cancer cells and ductal cells in CP contribute tobut are not solely responsible forthe increased PMP22 mRNA levels observed in whole CP and PDAC tissues. These data were further clarified by immunohistochemistry demonstrating PMP22 expression in tubular complexes and PanIN lesions in CP and PDAC. Tubular complexes are thought to evolve from dedifferentiated acinar cells and have an unknown malignant potential, whereas PanIN lesions are thought to be the premalignant lesions for PDAC (Bockman et al. 2003; Hingorani et al. 2003
). The amount of these two pathological features is highly dependent on the stage of the pancreatic pathological process. This, together with the detection of PMP22 immunostaining in the malignant cells of 10% of pancreatic cancer tissues, explains the wide range of PMP22 mRNA expression in these pathologies. The expression of PMP22 in malignant cells in some cases of pancreatic cancer tissues in vivo was consistent with the cell culture data, demonstrating PMP22 expression in pancreatic cancer cell lines, such as in Su8686 and Panc-1 cells.
There was stronger expression of PMP22 in PanIN 1B and PanIN 2 lesions as compared with PanIN 1A, PanIN 3, and PDAC, suggesting that PMP22 is overexpressed during certain time points in the neoplastic transformation process with low to absent expression in most pancreatic cancer cells. Interestingly, PMP22 seems to be specific to PanINs and PDAC, because other pancreatic tumors such as MCNs and IPMNs were completely devoid of PMP22 staining.
It has been demonstrated previously that there is interregulation between PMP22 and members of the TGF-ß family, which may change the cell fate during development (Hagedorn et al. 1999). TGF-ß1 upregulated PMP22 expression only in Panc-1 and Colo-357 cells, which have a functional TGF-ß pathway (Kleeff and Korc 1998
; Kleeff et al. 1999
), but not in the other pancreatic cancer cell lines that exhibit alterations in this pathway, such as Smad4 mutations (Hahn et al. 1996
) or low levels of TGF-ß receptors (Wagner et al. 1998
). Because pancreatic cancers in vivo frequently exhibit alterations of the TGF-ß signaling pathway (Friess et al. 1993a
,b
; Hahn et al. 1996
; Kleeff et al. 1999
), it could be speculated that the effects of TGF-ß on PMP22 expression are lost in the majority of pancreatic tumors leading to the observed low levels of PMP22 in most of the cancer cells, but not in the PanIN lesions that acquire, for example, Smad4 mutations only at advanced (PanIN 3) stages (Bardeesy and DePinho 2002
).
Overexpression of PMP22 retards proliferation and delays cell cycle progression from G0/G1 to S phase in cultured Schwann cells (Zoidl et al. 1995), Furthermore, overexpression of PMP22 in NIH3T3 fibroblasts induces cell death, which can be blocked by both Bcl-2 and DEVD caspase inhibitors, indicating that it occurs through apoptosis (Fabbretti et al. 1995
; Zoidl et al. 1997
). It can therefore be hypothesized that PMP22 mainly acts to inhibit cancer cell growth and/or to induce apoptosis and that this is lost in the majority of pancreatic cancers as a result of low or absent PMP22 expression. Loss of PMP22 expression might therefore contribute to the aggressive growth behavior of pancreatic cancer.
As another aspect of the present analysis, some histiocytes expressed PMP22 in CP pancreatic cancers, suggesting that this protein is involved in the regulation of the immunological process during the development of CP or pancreatic tumors. PMP22 expression was also observed in the Schwann cells of nerves in the normal pancreas, CP, and PDAC. Interestingly, the percentage of PMP22-positive nerves was lower in pancreatic cancer and chronic pancreatitis compared with the normal pancreas. It has been previously demonstrated that nerve tissues are destroyed in both chronic pancreatitis (CP) and pancreatic cancer. In CP, edema of nerve bundles, damaged individual nerves, and altered peripheral nerve sheaths are present (Bockman et al. 1988). In pancreatic cancer, tumor cells penetrate the perineurium and become intimately associated with Schwann cells and subsequently damage neural elements (Bockman et al. 1994
; Hirai et al. 2002
). These observations could partially explain the observed decreased percentage of positive PMP22 nerves in both CP and pancreatic cancer, inasmuch as the damaged nerves and myelin sheaths might express less PMP22. In addition, these results are also in agreement with the growth inhibitory function of this gene. In both CP and pancreatic cancer, overgrowth of nerves has been observed (Bockman et al. 1988
,1994
), suggesting that downregulation of PMP22 in some nerves in CP and cancer contributes to the nerve changes observed in these conditions.
In conclusion, PMP22 is expressed in the normal pancreas, CP, and PDAC tissues, with a wide range of expression levels. These variable levels of PMP22 expression are likely dependent on the content of tubular complexes and PanIN lesions as well as the responsiveness to endogenous factors such as TGF-ß1. PMP22 may be involved in the transformation process from the normal pancreas to premalignant lesions to pancreatic cancer.
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Footnotes |
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Literature Cited |
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Bardeesy N, DePinho RA (2002) Pancreatic cancer biology and genetics. Nat Rev Cancer 2:897909[CrossRef][Medline]
Bockman DE, Buchler M, Beger HG (1994) Interaction of pancreatic ductal carcinoma with nerves leads to nerve damage. Gastroenterology 107:219230[Medline]
Bockman DE, Buchler M, Malfertheiner P, Beger HG (1988) Analysis of nerves in chronic pancreatitis. Gastroenterology 94:14591469[Medline]
Bockman DE, Guo J, Buchler P, Muller MW, Bergmann F, Friess H (2003) Origin and development of the precursor lesions in experimental pancreatic cancer in rats. Lab Invest 83:853859[Medline]
Del Sal G, Ruaro ME, Philipson L, Schneider C (1992) The growth arrest-specific gene, gas1, is involved in growth suppression. Cell 70:595607[CrossRef][Medline]
Fabbretti E, Edomi P, Brancolini C, Schneider C (1995) Apoptotic phenotype induced by overexpression of wild-type gas3/PMP22: its relation to the demyelinating peripheral neuropathy CMT1A. Genes Dev 9:18461856[Abstract]
Friess H, Ding J, Kleeff J, Fenkell L, Rosinski JA, Guweidhi A, Reidhaar-Olson JF, et al. (2003) Microarray-based identification of differentially expressed growth- and metastasis-associated genes in pancreatic cancer. Cell Mol Life Sci 60:11801199[Medline]
Friess H, Kleeff J, Korc M, Buchler MW (1999) Molecular aspects of pancreatic cancer and future perspectives. Dig Surg 16:281290[CrossRef][Medline]
Friess H, Yamanaka Y, Buchler M, Berger HG, Kobrin MS, Baldwin RL, Korc M (1993a) Enhanced expression of the type II transforming growth factor beta receptor in human pancreatic cancer cells without alteration of type III receptor expression. Cancer Res 53:27042707[Abstract]
Friess H, Yamanaka Y, Buchler M, Ebert M, Beger HG, Gold LI, Korc M (1993b) Enhanced expression of transforming growth factor beta isoforms in pancreatic cancer correlates with decreased survival. Gastroenterology 105:18461856[Medline]
Hagedorn L, Suter U, Sommer L (1999) P0 and PMP22 mark a multipotent neural crest-derived cell type that displays community effects in response to TGF-beta family factors. Development 126:37813794
Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, et al. (1996) DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271:350353[Abstract]
Hildebrandt T, van Dijk MC, van Muijen GN, Weidle UH (2001) Loss of heterozygosity of gene THW is frequently found in melanoma metastases. Anticancer Res 21:10711080[Medline]
Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, Ross S, et al. (2003) Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4:437450[CrossRef][Medline]
Hirai I, Kimura W, Ozawa K, Kudo S, Suto K, Kuzu H, Fuse A (2002) Perineural invasion in pancreatic cancer. Pancreas 24:1525[CrossRef][Medline]
Holness CL, Simmons DL (1993) Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood 81:16071613[Abstract]
Huehne K, Rautenstrauss B (2001) Transcriptional startpoints and methylation patterns in the PMP22 promoters of peripheral nerve, leukocytes and tumor cell lines. Int J Mol Med 7:669675[Medline]
Huhne K, Park O, Liehr T, Rautenstrauss B (1999) Expression analysis of the PMP22 gene in glioma and osteogenic sarcoma cell lines. J Neurosci Res 58:624631[CrossRef][Medline]
Ionasescu VV, Ionasescu R, Searby C, Barker DF (1993) Charcot-Marie-Tooth neuropathy type 1A with both duplication and non-duplication. Hum Mol Genet 2:405410[Abstract]
Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ, et al. (2004) Cancer statistics, 2004. CA Cancer J Clin 54:829
Joo IS, Ki CS, Joo SY, Huh K, Kim JW (2004) A novel point mutation in PMP22 gene associated with a familial case of Charcot-Marie-Tooth disease type 1A with sensorineural deafness. Neuromuscul Disord 14:325328[CrossRef][Medline]
Karlsson C, Afrakhte M, Westermark B, Paulsson Y (1999) Elevated level of gas3 gene expression is correlated with G0 growth arrest in human fibroblasts. Cell Biol Int 23:351358[CrossRef][Medline]
Ketterer K, Rao S, Friess H, Weiss J, Buchler MW, Korc M (2003) Reverse transcription-PCR analysis of laser-captured cells points to potential paracrine and autocrine actions of neurotrophins in pancreatic cancer. Clin Cancer Res 9:51275136
Kleeff J, Friess H, Berberat PO, Martignoni ME, Z'Graggen K, Buchler MW (2000) Pancreatic cancernew aspects of molecular biology research. Swiss Surg 6:231234[Medline]
Kleeff J, Ishiwata T, Maruyama H, Friess H, Truong P, Buchler MW, Falb D, et al. (1999) The TGF-beta signaling inhibitor Smad7 enhances tumorigenicity in pancreatic cancer. Oncogene 18:53635372[CrossRef][Medline]
Kleeff J, Korc M (1998) Up-regulation of transforming growth factor (TGF)-beta receptors by TGF-beta1 in COLO-357 cells. J Biol Chem 273:74957500
Li J, Kleeff J, Guweidhi A, Esposito I, Berberat PO, Giese T, Buchler MW, et al. (2004) RUNX3 expression in primary and metastatic pancreatic cancer. J Clin Pathol 57:294299
Lupski JR, de Oca-Luna RM, Slaugenhaupt S, Pentao L, Guzzetta V, Trask BJ, Saucedo-Cardenas O, et al. (1991) DNA duplication associated with Charcot-Marie-Tooth disease type 1A. Cell 66:219232[CrossRef][Medline]
Manfioletti G, Ruaro ME, Del Sal G, Philipson L, Schneider C (1990) A growth arrest-specific (gas) gene codes for a membrane protein. Mol Cell Biol 10:29242930[Medline]
Re FC, Manenti G, Borrello MG, Colombo MP, Fisher JH, Pierotti MA, Della Porta G, et al. (1992) Multiple molecular alterations in mouse lung tumors. Mol Carcinog 5:155160[Medline]
Schneider C, King RM, Philipson L (1988) Genes specifically expressed at growth arrest of mammalian cells. Cell 54:787793[CrossRef][Medline]
Snipes GJ, Suter U, Welcher AA, Shooter EM (1992) Characterization of a novel peripheral nervous system myelin protein (PMP-22/SR13). J Cell Biol 117:225238[Abstract]
Spreyer P, Kuhn G, Hanemann CO, Gillen C, Schaal H, Kuhn R, Lemke G, et al. (1991) Axon-regulated expression of a Schwann cell transcript that is homologous to a "growth arrest-specific" gene. EMBO J 10:36613668[Abstract]
Taylor V, Welcher AA, Program AE, Suter U (1995) Epithelial membrane protein-1, peripheral myelin protein 22, and lens membrane protein 20 define a novel gene family. J Biol Chem 270:2882428833
van Dartel M, Cornelissen PW, Redeker S, Tarkkanen M, Knuutila S, Hogendoorn PC, Westerveld A, et al. (2002) Amplification of 17p11.2 approximately p12, including PMP22, TOP3A, and MAPK7, in high-grade osteosarcoma. Cancer Genet Cytogenet 139:9196[CrossRef][Medline]
van Dartel M, Hulsebos TJ (2004) Amplification and overexpression of genes in 17p11.2p12 in osteosarcoma. Cancer Genet Cytogenet 153:7780[CrossRef][Medline]
van Dartel M, Leenstra S, Troost D, Hulsebos TJ (2003) Infrequent but high-level amplification of 17p11.2 approximately p12 in human glioma. Cancer Genet Cytogenet 140:162166[CrossRef][Medline]
Wagner M, Kleeff J, Lopez ME, Bockman I, Massaque J, Korc M (1998) Transfection of the type I TGF-beta receptor restores TGF-beta responsiveness in pancreatic cancer. Int J Cancer 78:255260[CrossRef][Medline]
Wang CX, Wadehra M, Fisk BC, Goodglick L, Braun J (2001) Epithelial membrane protein 2, a 4-transmembrane protein that suppresses B-cell lymphoma tumorigenicity. Blood 97:38903895
Welcher AA, Suter U, De Leon M, Snipes GJ, Shooter EM (1991) A myelin protein is encoded by the homologue of a growth arrest-specific gene. Proc Natl Acad Sci USA 88:71957199
Wulf P, Bernhardt RR, Suter U (1999) Characterization of peripheral myelin protein 22 in zebrafish (zPMP22) suggests an early role in the development of the peripheral nervous system. J Neurosci Res 57:467478[CrossRef][Medline]
Zoidl G, Blass-Kampmann S, D'Urso D, Schmalenbach C, Muller HW (1995) Retroviral-mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth. EMBO J 14:11221128[Abstract]
Zoidl G, D'Urso D, Blass-Kampmann S, Schmalenbach C, Kuhn R, Muller HW (1997) Influence of elevated expression of rat wild-type PMP22 and its mutant PMP22Trembler on cell growth of NIH3T3 fibroblasts. Cell Tissue Res 287:459470[CrossRef][Medline]