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Expression Analysis of PMP22/Gas3 in Premalignant and Malignant Pancreatic Lesions

Junsheng Li, Jörg Kleeff, Irene Esposito, Hany Kayed, Klaus Felix, Thomas Giese, Markus W. Büchler and Helmut Friess

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

	Summary

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PMP22 is a structural protein of Schwann cells, but it also influences cell proliferation. In the present study, quantitative RT-PCR (QRT-PCR) and immunohistochemistry were used to determine PMP22 mRNA levels and to localize PMP22 in the normal pancreas (n=20), chronic pancreatitis (CP) (n=22), pancreatic ductal adenocarcinoma (PDAC) (n=31), intraductal papillary mucinous neoplasms (IPMN) (n=9), mucinous cystic tumors (MCN) (n=4), and in a panel of PanIN lesions (n=29). PMP22 mRNA levels were significantly higher in CP (3-fold) and PDAC (2.5-fold), compared to normal pancreatic tissues. PMP22 expression was restricted to nerves in the normal pancreas, while in CP and PDAC PMP22 was also expressed in PanIN lesions and in a small percentage of pancreatic cancer cells. PMP22 was weak to absent in the tumor cells of IPMNs and MCNs. PMP22 mRNA was present at different levels in cultured pancreatic cancer cells and up-regulated by transforming growth factor (TGF)-ß1 in 2 of 8 of these cell lines. In conclusion, PMP22 expression is present in both CP and PDAC tissues. Its expression in PanIN lesions and some pancreatic cancer cells in vitro and in vivo suggests a role of PMP22 in the neoplastic transformation process from the normal pancreas to pre-malignant lesions to pancreatic cancer.

(J Histochem Cytochem 53:885–893, 2005)

Key Words: peripheral myelin protein22 • transforming growth factor ß • pancreatic cancer • chronic pancreatitis • nerves • PanIN

	Introduction

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PANCREATIC CANCER IS THE fourth to fifth leading cause of cancer-related mortality in the Western world (Jemal et al. 2004). The pathogenesis of pancreatic cancer involves accumulation of genetic changes, such as k-ras, p53, p16, and Smad4 gene mutations; the disturbance of growth factor pathways; and several other epigenetic alterations, which together lead to defects of cell cycle regulation and apoptosis (Friess et al. 1999; Kleeff et al. 2000). The peripheral myelin protein 22 (PMP22) gene (previously designated PASII, SR13, and gas3 for growth arrest-specific gene-3) codes for a 22-kDa transmembrane glycoprotein and was originally identified as a gene whose expression is downregulated in Schwann cells during nerve regeneration (Welcher et al. 1991; Snipes et al. 1992). PMP22 is highly expressed in these cells and represents 2% to 5% of all myelin proteins (Spreyer et al. 1991; Welcher et al. 1991). It has been established that mutations of PMP22 are responsible for several types of hereditary peripheral neuropathies, such as Charcot-Marie-Tooth syndrome type 1A, which is a peripheral neuropathy characterized by progressive distal muscle atrophy and impaired sensation of the limbs (Ionasescu et al. 1993; Joo et al. 2004; Lupski et al. 1991). Several studies indicate that PMP22 also functions in regulating cell growth. For example, retroviral PMP22 transfer suppresses growth in cultured Schwann cells (Zoidl et al. 1995) and high expression of PMP22 results in growth arrest and apoptosis in fibroblasts (Fabbretti et al. 1995; Karlsson et al. 1999). In addition to its expression in neural cells, PMP22 is also observed in nonneural cells and tissues during development, such as in the epithelial cells of the lung and intestine (Taylor et al. 1995; Wulf et al. 1999), and downregulation of PMP22 expression has been associated with the development of lung cancer in mice (Re et al. 1992). Furthermore, amplification of the chromosome 17p11.2 region and PMP22 expression has been observed in osteosarcoma, as well as in glioblastoma tissues and cell lines (Huhne et al. 1999; Huehne and Rautenstrauss 2001; van Dartel et al. 2002,2003; van Dartel and Hulsebos 2004). PMP22 expression in osteosarcoma cells may result in alternative availability of the PMP22 protein during the cell cycle and aberrant regulation of cell growth (van Dartel and Hulsebos 2004).

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.

	Materials and Methods

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Cell Lines and Culture Conditions
MiaPaCa-2, T3M4, Aspc-1, Bxpc-3, Capan-1, Colo-357, Panc-1, SU8686 pancreatic cancer cells were grown in RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Karlsruhe, Germany). Cells were maintained in 37C humidified air with 5% CO₂. For induction experiments, cells were treated with 200 pM transforming growth factor ß1 (R and D Systems Inc.; Minneapolis, MN) for the indicated time.

Patients and Tissue Collection
Pancreatic tissue samples were obtained from patients (median age 62.5 years; range 41–78 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 tumor–nodes–metastasis 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 22–66 years). Twenty normal human pancreatic tissue samples were obtained from previously healthy individuals through an organ donor program (median age 45 years; range 20–74 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 Search–LC (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 (6–8 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 (3–5 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).

	Results

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PMP22 Expression and Localization in Pancreatic Tissues
Quantitative PCR was performed to evaluate the levels of PMP22 mRNA expression in normal pancreatic tissue samples (n=20), chronic pancreatitis (CP) (n=22) and PDAC samples (n=31). Tissue samples from normal tissues had mean PMP22 mRNA levels of 276 ± 37 copies/µl, while PMP22 mRNA levels were increased in both chronic pancreatitis and pancreatic cancer, with mean levels of 874 ± 217 copies/µl in chronic pancreatitis and 681 ± 143 copies/µl in pancreatic cancer (Figure 1A). Thus there was a 2.5-fold increase in pancreatic cancer (p<0.05) and a 3-fold increase in chronic pancreatitis (p<0.05) compared with normal controls. Individual CP and pancreatic cancer tissues exhibited a wide range of variation in PMP22 expression, which overlapped with PMP22 mRNA expression in the normal pancreas (81–613 copies). Specifically, PMP22 mRNA levels were above the normal range in 64% of CP samples and in 50% of pancreatic cancers. To determine whether the overexpression of PMP22 in a subset of CP and PDAC tissues was specifically epithelial (i.e., ductal cell, cancer cells) in origin, a microarray analysis of microdissected pancreatic ductal cells from the normal pancreas and CP, cancer cells as well as of total normal pancreatic tissues was performed. This analysis revealed that only 2 of 5 and 2 of 6 microdissected ductal cells in CP and cancer, respectively, expressed PMP22 above the range of both microdissected normal pancreatic ducts and total normal pancreatic tissues (Figure 1B). Thus, the variable expression of PMP22 among normal, CP, and PDAC tissues may be more due to the morphological differences between the three types of tissues rather than specific overexpression of PMP22 in CP ductal cells or pancreatic cancer cells. To clarify this assumption, immunohistochemistry was performed next to determine the exact distribution of PMP22 in different pancreatic tissues. The normal ductal and acinar cells in all types of pancreatic tissues exhibited no PMP22 immunostaining (Figure 2A). Weak to moderate PMP22 immunoreactivity was observed in the tubular complexes of approximately 60% of CP and PDAC cases (Figure 2B). PanIN lesions also displayed PMP22 immunoreactivity. The intensity of the observed staining was relatively stronger in PanIN 1B and PanIN 2 lesions as compared with PanIN 1A and PanIN 3 lesions (Figures 2C–2F). On the other hand, only faint to occasionally moderate staining of the cancer cells was observed in approximately 10% of PDAC cases (Figures 2G and 2H). In contrast, IPMNs (n=9) and MCNs (n=4) exhibited no PMP22 immunoreactivity (Figures 2K–2L and data not shown). To ensure the specificity of the used antibody, a myelinated human nerve (femoral nerve) was analyzed, demonstrating the expected membranous PMP22 staining pattern in the Schwann cells (Figure 3 A). PMP22 immunostaining was also detected as globular staining in the membranes of the Schwann cells of normal, CP and PDAC tissues (Figures 3A–3M) Interestingly, the percentage of positive-stained nerve cells was higher in the normal pancreas (55%) compared with CP (35%) and PDAC tissues (30% of counted nerves per tissue section). Furthermore, strong PMP22 immunoreactivity was occasionally observed in the histiocytes of some CP cases and pancreatic tumors (Figure 3M). The morphological identification of the PMP22-stained histiocytes was further confirmed by double-staining using CD68 as a histiocyte marker (Holness and Simmons 1993) (Figure 3M, inset).

View larger version (14K): [in this window] [in a new window]   Figure 1 PMP22 mRNA expression in pancreatic tissues and microdissected pancreatic cells. (A) PMP22 mRNA levels in normal pancreatic tissues, chronic pancreatitis (CP), and PDAC tissues (cancer) were determined via real-time quantitative PCR, as described in Materials and Methods. Values were normalized to housekeeping genes (cyclophilin B and hypoxanthine guanine phosphoribosyltransferase) and are presented as copies per microliter. (B) Microarray analysis of the PMP22 in microdissected normal pancreatic ducts (n=3), total normal pancreatic tissues (n=3), ducts from CP tissues (n=5), and cancer cells (n=6). Horizontal lines represent the mean expression level. Values are presented in arbitrary units.

View larger version (190K): [in this window] [in a new window]   Figure 2 PMP22 localization in benign and malignant pancreatic tissues. Immunohistochemistry using a specific PMP22 antibody was performed as described in Materials and Methods. (A) Normal pancreas. (B) Tubular complexes in CP. (C,D) PanIN 1A. (E) PanIN 1B. (F) PanIN 2. (G) PanIN 3. (H) Note the stronger PMP22 staining intensity in a PanIN 2 lesion (arrows) compared with the adjacent PanIN 3 (arrowheads). (I) PDAC tissue. (K) Benign IPMN. (L) Malignant/invasive IPMN.

View larger version (197K): [in this window] [in a new window]   Figure 3 PMP22 localization in pancreatic nerves and histiocytes. Immunohistochemistry using a specific PMP22 antibody was performed as described in Materials and Methods showing PMP22 staining in the nerve tissue of (A) femoral nerve, (B) normal pancreas, (C–F) chronic pancreatitis, and (G–I) pancreatic cancer. Note the specificity of the PMP22 staining in nerves of consecutive pancreatic tissue sections. (K) Anti-PMP22. (L) Negative control. (M) Histiocytes in pancreatic cancer (inset: double-staining of the histiocytes with PMP22 [red] and CD68 [brown]).

View larger version (15K): [in this window] [in a new window]   Figure 4 PMP22 expression and regulation in pancreatic cancer cell lines. (A) PMP22 expression in the indicated pancreatic cancer cell lines. RNA was extracted and real-time quantitative PCR for PMP22 was performed as described in Materials and Methods. The results are presented as the mean ± SEM of three independent experiments. (B) Panc-1 (empty triangle) and Colo-357 (solid square) cells were incubated in the absence (0) or presence of 200 pM TGF-ß1 for the indicated time. RNA was extracted and real-time quantitative PCR for PMP22 was performed as described in Materials and Methods. The respective control mRNA expression levels were set to 100%. Results are presented as the mean ± SEM of three independent experiments.

	Discussion

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Normal cellular growth is regulated by positive and negative factors, and the disruption of this balance can lead to unregulated growth and neoplastic transformation. The pathogenesis of pancreatic cancer involves genetic alterations, such as K-ras proto-oncogene mutations and mutations of p53, p16, and Smad4 tumor suppressor genes, as well as the overexpression of several growth factors and their receptors (Friess et al. 1999; Kleeff et al. 2000). PMP22 (growth arrest-specific gene-3) is known to be localized mainly in the peripheral myelin sheaths as mentioned previously and was identified in Schwann cells of the adult peripheral nervous system (Snipes et al. 1992). However, widespread expression of PMP22 has also been observed in nonneural cells and tissues during development (Taylor et al. 1995; Wulf et al. 1999).

The growth arrest–specific (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 type–dependent (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 to—but are not solely responsible for—the 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.

	Footnotes

 
Received for publication October 9, 2004; accepted January 20, 2005

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