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Clinical Cancer Research 13, 4984-4992, September 1, 2007. doi: 10.1158/1078-0432.CCR-07-0946
© 2007 American Association for Cancer Research
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Human Cancer Biology

Is There a Human Homologue to the Murine Proteolysis-Inducing Factor?

Barbara M. Wieland1,2, Grant D. Stewart3, Richard J.E. Skipworth3, Kathryn Sangster3, Kenneth C.H. Fearon3, James A. Ross3, Tony J. Reiman1, Jay Easaw1, Marina Mourtzakis1, Vishesh Kumar1, Brian J. Pak4, Kathryn Calder1, Gerasimos Filippatos5, Dimitris T. Kremastinos5, Monica Palcic2 and Vickie E. Baracos1

Authors' Affiliations: Departments of 1 Oncology and 2 Chemistry, University of Alberta, Edmonton, Alberta, Canada; 3 Tissue Injury and Repair Group, Clinical and Surgical Sciences (Surgery), University of Edinburgh, Edinburgh, United Kingdom; 4 Ciphergen Biosystems, Inc., Fremont, California; and 5 2nd University Department of Cardiology, Atticon University Hospital, Athens, Greece

Requests for reprints: Vickie E. Baracos, Division of Palliative Care Medicine, Department of Oncology, Cross Cancer Institute, University of Alberta, 11560 University Avenue, Edmonton, Canada T6G 1Z2. Phone: 1-780-432-8232; Fax: 1-780-432-8425; E-mail: vickieb{at}cancerboard.ab.ca.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: A tumor-derived proteolysis-inducing factor (PIF) is suggested to be a potent catabolic factor in skeletal muscle of mice and humans. We aimed to establish the clinical significance of PIF in cancer patients and to elucidate its structural features.

Experimental Design: PIF was detected in human urine using a monoclonal antibody (mAb) and related to clinical outcomes. PIF immunoaffinity-purified using the mAb was purified/separated using reverse-phase high-performance liquid chromatography and two-dimensional electrophoresis. Ten human cancer cell lines were tested for expression of mRNA encoding PIF core peptide.

Results: PIF immunoreactivity was present in 160 of 262 patients with advanced cancers of the lung, esophagus/stomach, and other organs. In a Kaplan-Meier survival analysis of 181 lung cancer patients, PIF was unrelated to survival; PIF status was also unrelated to skeletal muscle loss confirmed by computed tomography imaging. PIF was seen in 16 of 24 patients with chronic heart failure and thus is not exclusive to malignant disease. In-gel digestion and mass spectrometric analysis of immunoaffinity purified PIF from cancer patients consistently identified human albumin and immunoglobulins. We showed nonspecific binding of purified albumin and immunoglobulins to the anti-PIF mAb, which is thus not a useful tool for PIF detection or purification in humans. Finally, the human PIF core peptide was detected in human cancer cell lines using reverse transcription-PCR and nucleotide sequencing; however, none of the amplified products had a site for the glycosylation critical to the proteolysis-inducing activity of murine PIF.

Conclusions: A putative human homologue of murine PIF and its role in human cancer cachexia cannot be verified.

A proteolysis-inducing glycoprotein [proteolysis-inducing factor (PIF)] of tumor origin mediates muscle wasting in mice bearing the MAC16 adenocarcinoma (1). PIF elicits intense skeletal muscle catabolism in muscle cells or animals (1–4). Purified PIF has a mass of ~24,000 Da and consists of a short polypeptide containing both N-linked (~10 kDa) and O-linked (~6 kDa) sulfated oligosaccharides (5). Preliminary evidence (6, 7) suggested that an identical factor is associated with weight loss in cancer patients (6–10). Additionally, PIF was absent in cancer patients without weight loss or weight-losing patients with benign disease (7). These results suggested discovery of a critical factor underlying human cancer cachexia, but attempts at further confirmation suggested that PIF was not necessarily associated with clinical outcomes (i.e., survival and weight loss) in cancer patient populations (11, 12).

Regulation of expression of the human PIF core peptide, the sites at which glycosylation occurs to form the functional glycoprotein, and the structure of the oligosaccharides (5, 7), which confer the proteolysis-inducing activity to this unusual molecule, remain unresolved. A peptide sequence (7) and two patents describing the human cachexia-associated protein (HCAP) gene encoding PIF were published (Genbank accession no. AR053250 and U.S. patent 5834192). Other molecules are encoded by the same gene (13–16), which is now named dermcidin (DCD) because it encodes an antimicrobial peptide secreted by eccrine sweat glands (16). The DCD gene has been mapped (Fig. 1A ; ref. 16), but it is unknown how the 110–amino acid transcript is processed into the 30–amino acid PIF peptide and the 47–amino acid antimicrobial peptide. The DCD gene structure potentially allows discrete expression of molecules with various functions; however, the absence of the N-glycosylation site seen in murine PIF (amino acids 13-15, Asn-Pro-Ser) in the human sequence (Asn-Pro-Cys) raises questions as to whether human PIF can be glycosylated. A Ser/Cys substitution mutant expressed in human immortalized cell lines was not glycosylated (17).


Figure 1
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  		Fig. 1. Map and sequence of the DCD polypeptide. A, map of the unprocessed DCD polypeptide. B, DCD nucleotide sequence. PIF core peptide nucleotide sequence (bold). |, site of an intron. Putative O-glycosylation sites of PIF (double underline) and putative N-glycosylation sites for PIF (dotted underline). Start and stop codons are shown in upper case. PIF primer targets (underlined). PIF forward primer: CCTCTTCCTGACAGCTCTGG. PIF reverse primer: CTGGATCTCTGCTTCCTTGG. HCAP primer targets (italics). HCAP forward primer: ACTCTCCTCTTCCTGACAGCTCTGG. HCAP reverse primer: CTGCTGCTCCTGGGTATCATTTCTC.

 
We aimed to establish PIF prevalence in cancer patients, to investigate association of this factor with muscle loss and survival, and to purify human PIF and determine its carbohydrate structure. We also sought evidence for the presence of polymorphic species that may make glycosylation of the resultant PIF core peptide possible.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
PIF quantification and purification. Anti-PIF monoclonal antibody (mAb) was purified from hybridoma cells (provided by M.J. Tisdale, Aston University, Birmingham, United Kingdom) and used for Western blot as described (7, 10) in its biotinylated form (enhanced chemiluminescence protein biotinylation module, Amersham). Proteins were separated by SDS-PAGE (18) on 15% gels or by two-dimensional electrophoresis (19).

A PIF assay based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported (20). Samples were prepared as described (Fig. 2A ; ref. 21) and spectra were obtained using the Voyager Elite MALDI-TOF (Applied Biosystems). Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (22) was also used for antibody-based detection (Ciphergen; Fig. 2B and C). PIF mAb (1 µg) or negative control antibody was covalently linked to preactivated PS20 ProteinChip Arrays (Ciphergen). Spots were then blocked [0.5 mol/L Tris (pH 8.0)]. Six micrograms of total urinary protein in PBS were applied and incubated for 2 h at 23°C. Spots were then washed, dried, and then coated with sinapinic acid matrix. Arrays were read using the PBS-IIC ProteinChip Reader (Ciphergen). Immunoaffinity-purified PIF was also assayed on NP20 (normal phase) arrays (Ciphergen) for mass characterization.


Figure 2
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  		Fig. 2. Purification of PIF from human urine. A, urinary PIF was purified as described (7), with anion exchange chromatography as a first capture step. B, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry of immunoaffinity-purified fraction 4. C, screening of urine samples for PIF immunoreactivity using surface-enhanced laser desorption/ionization. C+, positive control specimen; C–, negative control specimen. D, reverse-phase high-performance liquid chromatography of fraction 4.1 before (dotted line) and after (solid line) tryptic digestion.

 
Urinary PIF was purified as described (Fig. 2A; ref. 7), with anion exchange chromatography as a first capture step (Bio-Rad BioLogic HR fast protein liquid chromatography system, Bio-Rad). Separation by a linear gradient with 20 mmol/L Tris-HCl (pH 8.5) up to 1 mol/L NaCl was improved by a step gradient. Peak fractions identified by Western blot were next purified by immunoaffinity chromatography and reverse-phase high-performance liquid chromatography as described (7).

Immunoglobulin light chains and albumin were removed from affinity-purified 24-kDa positive fractions using the ProteoExtract kit (Calbiochem). Immunoglobulins containing {kappa} light chains were removed from affinity-purified samples by protein L chromatography (Pierce).

Reverse-phase high-performance liquid chromatography–purified PIF (fraction 4.1; Fig. 2A) was deglycosylated using the Enzymatic Deglycosylation kit combined with the Pro-Link Extender (Prozyme). Denaturing conditions were applied as deglycosylation under nondenaturing conditions was incomplete.

Protein identification. In-gel tryptic digestion, peptide extraction, and mass spectrometric analysis were done using a Bruker MALDI-TOF mass spectrometer, Finnigan LCQ Deca ion trap mass spectrometer (Thermo), and MDS Sciex MALDI quadrupole time-of-flight (Applied Biosystems). MS/MS data were searched in the National Center for Biotechnology Information nonredundant database using Mascot Daemon (Matrix Science). Search parameters included carbamidomethylation of cysteine, possible oxidation of methionine, and one missed cleavage/peptide.

Evaluation of skeletal muscle loss by computed tomography. Data are presented for 65 lung cancer patients who had two computed tomography images framing the date of urine collection in their medical record; patients with scans at least 100 days but not >300 days apart were included. The fourth thoracic vertebra was chosen as a landmark; this plane includes pectoralis, trapezius, and scapular muscle group. Images were analyzed (Slice-O-Matic software version 4.2; Tomovision) and tissue cross-sectional area was calculated (23, 24). The change in muscle area occurring over the interval between two scans (gain or loss) was expressed as percentage gain or loss of muscle / day x 100. t test was used to compare rate of muscle changes between the PIF-positive and PIF-negative groups.

Cell culture and transfection of cells with DCD cDNA. Immortalized human cancer cells were cultured under standard conditions. Hormone-sensitive (LNCaP) and hormone-insensitive (PC-3 and DU145) prostate cancer, hepatocellular carcinoma (Huh-7 and Hep G2), and pancreatic adenocarcinoma cell lines (PANC-1, CFPAC, and MIA-Pa-Ca-2) were obtained (European Collection of Cell Cultures, Salisbury, United Kingdom). Hormone-insensitive prostate cell line PC-3M was donated by C. Pettaway (M. D. Anderson Cancer Center, Houston, TX). Malignant melanoma cell line G-361 was donated by M.J. Tisdale.

A robust positive control for DCD reverse transcription-PCR was created by stably transfecting PC-3M cells, which do not express DCD mRNA (25), with a DCD-expressing vector. Transfection was with a pcDNA3.1+DCD vector (Invitrogen) using Fugene 6 (Roche Applied Science). The pcDNA3.1+ mammalian expression vector had full-length DCD cDNA directionally cloned into the multiple cloning site using EcoRI and BamHI. Geneticin selection antibiotic (600 µg/mL; Invitrogen) was used to select transfected cells and eventually create stable transfectants. Sham-transfected cells were also created using an empty pcDNA3.1 vector.

Reverse transcription-PCR. Total RNA was extracted from cells using Trizol (Invitrogen) and quantified by spectrophotometry. One microgram of total RNA was used for reverse transcription, after treatment with RQ1 DNase (Promega). Reverse transcription was done using avian myeloblastosis virus reverse transcriptase (Promega) according to the manufacturer's instructions.

PIF and HCAP primers were used to identify DCD cDNA (Fig. 1B). PIF primers were designed using the Primer3 primer design program6 and used with ß-actin housekeeping primers. The PCR program for PIF and ß-actin was as follows: 94°C for 5 min, then 35 cycles of 94°C for 1 min, and 56°C for 1 min followed by 72°C for 1 min and followed by 72°C for 10 min. HCAP primers (25) were used with ß2-microglobulin (MIC) PCR control for RNA quality and reverse transcription performance. The PCR program for HCAP and MIC was as follows: 80°C for 3 min, then 35 cycles of 95°C for 5 s, and 69°C for 1 min followed by 72°C for 7 min. Primers were manufactured by Sigma.

HCAP (361 bp) and MIC (555 bp) PCR products were separated on 1.6% agarose gel. PIF (174 bp) and ß-actin (535 bp) PCR products were separated on 6% acrylamide gels. Nucleotide sequencing of the PCR products was done by the Medical Research Council Human Genetics Unit (Edinburgh, United Kingdom).


   Results
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 Results
Discussion
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PIF in patient populations. Urine was obtained from 181 patients with a confirmed diagnosis of non–small cell lung cancer (stage IIIb or IV), 40 patients with various metastatic cancers, 41 patients with metastatic adenocarcinoma of the esophagus, gastroesophageal junction, or gastric cardia (11), 7 healthy volunteers, and 24 patients with decompensated chronic heart failure. Approval was provided by the institutional Research Ethics Boards and all subjects gave informed consent. Western blot using a mAb (PIF mAb) is considered the reference method of PIF detection (7, 10). Samples used here as positive control specimens were from cancer patients and were confirmed positive using Western blot (as blinded samples) by M.J. Tisdale. Urine of weight-losing cancer patients, showing positive reaction in Western blot, was treated according to the published purification scheme (Fig. 2A; ref. 7). A species of ~24,000 Da was observed in Western blot, MALDI-TOF MS, and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry in urine of positive control subjects (Figs. 2 and 3 ) and at all steps of purification.


Figure 3
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  		Fig. 3. PIF detection in human urine by Western blot and MALDI-TOF MS. A, representative Western blot of unfractionated urine showing negative control specimens (C–) were and positive specimens (C+). B, negative control specimen assayed by MALDI-TOF MS (20). C, positive specimen assayed by MALDI-TOF-MS; a characteristic peak at 23,762 Da.

 
Overall, 61% of the cancer patients and 75% of the patients with chronic heart failure who lost weight, and 58% of heart failure patients without weight loss were immunoreactive for PIF in Western blot (Table 1 ). Healthy control subjects were uniformly negative in all assays. We did not find a perfect correspondence between Western blot and MALDI-TOF MS of the desalted urine as had been reported by others (8, 9), and 26% of the cancer patients showed discordance between MALDI and Western blot results (Table 1).


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  		Table 1. PIF prevalence in human urine detected by Western blot and MALDI-TOF MS

 
PIF expression has been related previously to weight loss in cancer patients (6–10) and, in turn, this weight loss is known to have important prognostic significance (26–29). The gastric/esophageal patients did not show a relationship between PIF status and weight loss (11). Weight data are not available on all the lung patients, but for 116 of them, there are a total of 970 individual weight records extractable from patient charts or on average 8.3 weight records per patient. When these are analyzed, 26 of 116 (22.4%) were weight losing and lost 13.1 ± 6.1% (SD) of their body weight, during the 6 months encompassing the date that their PIF status was determined; the remaining patients did not lose weight. Although the rate of this loss was substantial, the incidence of PIF immunoreactivity was identical in this group (63%) as it was in the non–weight-losing group (61%). A Kaplan-Meier survival distribution curve for lung cancer patients showed no relationship between PIF status and median survival (Fig. 4A ). Because PIF is proposed to be a muscle-specific catabolic factor, we placed focus on the precise quantification of muscle loss in computed tomography images (Fig. 4B). When we stratified the population into quartiles by intensity of muscle loss/gain (Fig. 4C), the prevalence of PIF immunoreactivity was nearly identical in patients who gained or maintained muscle and in those who showed severe muscle loss. The distribution of muscle loss/gain by PIF status (Fig. 4D) was not different. The lack of concordance between PIF and clinical outcomes led us to examine further the nature of the signal detected by PIF mAb.


Figure 4
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  		Fig. 4. PIF mAb Western blot reaction is unrelated to survival or skeletal muscle atrophy in patients with lung cancer. A, Kaplan-Meier survival distribution curves for patients showing positive and negative response in Western blot using PIF mAb Log-rank P = 0.74. B, representative computed tomography images of a male patient showing generalized loss of skeletal muscle during disease progression. C, rate of change of total thoracic skeletal muscle in cancer patients by quartiles. Mean (SE) for each quartile were as follows: –11.3 ± 1.7, –3.3 ± 0.2, –0.5 ± 0.3, and 4.0 ± 0.8%/100 d. The fraction of patients tested positive in Western blot using mAb to murine PIF was 70% overall and unrelated to muscle loss or gain (n = 65). D, distribution of muscle loss/gain in cancer patients. Patients (n = 45) showing a PIF reaction in Western blot lost muscle –3.39 ± 2.1% (SE)/100 d and PIF-negative patients also lost muscle –2.42 ± 1.7%/100 d (n = 20).

 
Human albumin and immunoglobulins react nonspecifically with the murine anti-PIF mAb. We investigated the proteins purified by immunoaffinity chromatography using the PIF mAb (fraction 4; Fig. 2A), as this fraction should be selectively enriched. This characterization was complicated by low recovery of total protein after immunoaffinity chromatography (0.006-0.012%; Fig. 2A). Representative silver-stained two-dimensional gels (Fig. 5A ) show that the immunoaffinity-purified fraction contains more than 30 proteins with a broad range of sizes and apparent isoelectric points. The corresponding Western blots (Fig. 5B) showed at least five immunoreactive spots, including two >70,000 Da. A high molecular weight protein reacting with the PIF mAb was postulated to be PIF tightly attached to albumin (30). However, the sequence of the >70-kDa immunoreactive spots was consistent with albumin only. Three spots were visualized in the size range reported for PIF (~24,000 Da) on two-dimensional gels, in all cases characterization of these spots by MALDI-TOF MS/MS revealed immunoglobulin fragments (Fig. 5B; Table 2). No peptide corresponding to the reported sequence of PIF peptide was observed in any of the analyses of immunoreactive spots, in any of eight cancer patients evaluated in this manner.


Figure 5
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  		Fig. 5. Human albumin and immunoglobulins react nonspecifically with the murine mAb. A, two-dimensional gel electrophoresis of fraction 4 (affinity purified on PIF mAb column; Fig. 2), from a representative cancer patient. B, Western blot of gel shown in (A), using PIF mAb. The immunoreactive spots, except spot 1, could be identified by in-gel tryptic digestion and MALDI-MS/MS from the corresponding spot eluted tryptic peptides followed by database search in Swiss Prot or National Center for Biotechnology Information (Table 2
Table 2. Details of the immunoreactive spots (Fig. 5B) following a Swiss Prot or National Center for Biotechnology Information database search

Spot no. Identified protein Swiss Prot or NCBI accession no. MW (kDa) Peptides for MALDI-MS/MS
2 Immunoglobulin {kappa} light chain VLJ region (human) gi/21669417/dbj 29.842 FSGSGSGTD; 1302.61
Unknown protein gi/18490211/gb 26.503 FSGSGSGTD; 1302.61
3 Immunoglobulin {kappa} light chain e.g. 33700 25.500 YAASSYLSLTPEQWK; 1743.9
4 Human serum albumin in a complex with myristic acid gi/4389275/pdb 67.988 YLYEIAR; 926.49 ETYGEMADC; 1449.52
5, 6 Unnamed protein gi/28590/emb/CA 71.246 DDNPNLPR; 939.44
Human serum albumin gi/28592/emb/CA 71.316 DLGEENFK; 950.43

Abbreviations: NCBI, National Center for Biotechnology Information; MW, molecular weight.

). Bottom, Western blot identical to area outlined in (B), after IgG removal. C, Western blot using PIF mAb. Lanes from left to right, human IgG (Sigma), human serum albumin (Sigma), MW standards (Std), empty lane, C+ (positive) control urine specimen (colon cancer), and C– (negative) control urine specimen. D, MALDI-TOF spectrum of human IgG (Sigma) showing characteristic peak at 23,572 Da.

 
When fraction 4 was treated to remove immunoglobulins, all immunoreactive entities of ~24 kDa in Western blot of the two-dimensional gel were eliminated (Fig. 5B, bottom) as did the MALDI-TOF MS peak at 24,000 Da. Nonspecific binding of the antibody was shown with commercially available human IgG and albumin; both reacted strongly with PIF mAb on Western blot (Fig. 5C and D). Furthermore, purified IgG showed a clear peak in MALDI-TOF MS at ~24,000 Da (Fig. 5D). These results suggest that the apparent PIF signal in human urine is attributable to a nonspecific reaction.

Fraction 4.1 in the PIF purification schema (after reverse-phase high-performance liquid chromatography) was subjected to tryptic digestion. Although published structural data suggest that glycosylated PIF is resistant to trypsin (5), when fraction 4.1 was trypsinized without prior deglycosylation, both the single major protein peak (Fig. 2D) and the MALDI-TOF signal at 23.8 kDa were abolished. Sequence data on the tryptic digests of both the native and deglycosylated fractions were also consistent with immunoglobulin fragments (data not shown).

Expression of DCD mRNA by human cancer cell lines. DCD mRNA expression was consistently detected in the human cancer cells by reverse transcription-PCR. Subsequent PCR product nucleotide sequencing using both the PIF (Fig. 6A ) and HCAP (Fig. 6B) primers confirmed the identity of the amplified products. The PIF primers amplified a band of the appropriate size (174 bp) in 8 of the 10 cell lines (Fig. 6A; Table 3 ): PC-3, PC-3M, DU145, G-361, Hep G2, CFPAC, PANC-1, and MIA-Pa-Ca-2 but not in LNCaP or Huh-7. HCAP primers amplified a band of appropriate size (361 bp) in 7 of the 10 cell lines (Fig. 6B; Table 3): PC-3, PC-3M, DU145, Hep G2, CFPAC, PANC-1, and MIA-Pa-Ca-2 but not in LNCaP, G-361, or Huh-7 cells.


Figure 6
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  		Fig. 6. Reverse transcription-PCR analysis of immortalized human cancer cell lines. A, PIF (upper gel picture) and ß-actin (lower gel picture) primers. DCD cDNA product at 174 bp. B, HCAP (upper gel picture) and MIC primers (lower gel picture). DCD cDNA product at 361 bp. All products were identified as DCD cDNA on nucleotide sequencing.

 

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  		Table 3. Summary of results of reverse transcription-PCR using PIF and HCAP primers

 
The products generated from PIF and HCAP primers were sequenced. DCD cDNA was confirmed in products amplified from PC-3, PC-3M, DU145, G-361, Hep G2, CFPAC, and MIA-Pa-Ca-2 cells (Fig. 6A; Table 3). In all cases, the sequences obtained were those of the sections of the DCD cDNA nucleotide sequence that the primers were designed to amplify. The sequences from PC-3, PC-3M, DU145, G-361, Hep G2, CFPAC, and MIA-Pa-Ca-2 cell lines and the positive control were all identical and exactly matched that of DCD cDNA (Fig. 1). The region of the DCD cDNA, which corresponds to the PIF core peptide cDNA, also had an identical sequence in all of the positive cell lines and positive control, and it exactly matched the published sequence of the putative human homologue of PIF (17). There were no polymorphic forms with mutations at the sites of the putative PIF N-glycosylation and O-glycosylation sites, which would make glycosylation likely to occur. Finally, Western blot on total protein lysates from the cell lines expressing DCD mRNA as well as the DCD-transfected positive control cells was done using the PIF mAb, and in no case was a band at ~24,000 Da observed (data not shown).


   Discussion
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 Materials and Methods
 Results
 Discussion
References
 
The reported detection and purification of PIF has been based entirely on a mAb originally isolated from NMRI mice (7). Here, we show that this antibody is not a useful tool for the study of PIF in humans, due to its lack of specificity and, in particular, strong reactivity toward human albumin and immunoglobulins. All efforts to determine the protein sequences within the immunoaffinity-purified fraction led to the identification of albumin and immunoglobulin fragments. A detection method based on MALDI-TOF MS (8, 20) was also not a valid tool for PIF detection.

PIF was reported to associate specifically with the weight loss of malignant disease; this was based on its absence from healthy controls and 17 weight-losing patients with trauma or sepsis (7). Subsequent results do not support the specificity of PIF to malignancy or association with weight loss or muscle catabolism. PIF immunoreactivity in urine was reported to have a low sensitivity (54%) and specificity (71%) for the detection of pancreatic tumors and a low sensitivity (57%) and specificity (63%) for the distinction of pancreatic cancer from pancreatitis (31). We further show PIF immunoreactivity in chronic heart failure patients. Additionally, mRNA encoding the PIF core peptide was reported recently in nonmalignant tissue (12). The Western blot signal using PIF mAb had no relationship with survival in our or other studies (10, 11). This is an important discrepancy because weight loss predicts survival, especially in cancers of the lung (26–28) and esophagus (29). Computed tomography image analysis allows for a highly precise description of the rate of skeletal muscle loss (23, 24). Our observation that PIF immunoreactivity was not associated with skeletal muscle atrophy is not concordant with the premise that it is a muscle-specific PIF.

We showed that mRNA for DCD is expressed by multiple human cancer cells, with only minor differences from previous results (25), possibly due to clonal variation within cell lines. DCD cDNA was identified by both sets of primers utilized; however, sequencing of the PCR products was consistently possible only from the PIF primers. All PCR products revealed a sequence identical to that described as a human homologue of PIF (7). These results indicate that production of the 110–amino acid DCD polypeptide as well as the 30–amino acid PIF core peptide is theoretically possible by these cell lines.

Glycosylation of PIF core peptide is crucial in the increased muscle proteolysis induced by PIF in mice (i.e., the peptide alone is devoid of proteolysis-inducing activity; ref. 5). There are three putative O-glycosylation sites and two possible N-glycosylation sites on the murine PIF core peptide (5). An Asp-X-Ser/Cys sequon is required for N-glycosylation, in which X may be any amino acid other than proline. One of the putative N-glycosylation sites does possess this Asp-X-Ser/Cys sequence. However, the sequence is Asp-Pro-Cys, and as such, N-glycosylation is unlikely to occur (32). Bioinformatic analysis shows that the PIF core peptide is unlikely to be O-glycosylated at any of the putative sites in the sequence (N-glycosylation and O-glycosylation were modeled, respectively, using Net N-Glyc7 and Net O-Glyc8; ref. 33).

There has been a single report that glycosylated PIF can be produced in human cells, melanoma G-361 (34). However, the PIF core peptide is unlikely to be found in a glycosylated form in these or the cell lines studied here, based on the predicted amino acid sequence. Indeed, Monitto et al. (17) were only able to detect an unglycosylated secreted protein on stable forced expression of human PIF in multiple murine and human cell lines. This group also found that tumor xenografts overexpressing human PIF protein did not induce cachexia in vivo.

The apparent incidence of PIF in cancer patients, as well as lack of a clear relationship with survival, weight loss, and skeletal muscle atrophy, is explained by our work. Immunoglobulin light chain, the major protein in human urine cross-reacting with the PIF mAb, coincidentally migrates in SDS gels near the reported size of PIF. A further coincidence is that immunoglobulin {kappa} light chains are normally absent from the urine of healthy humans but have been shown to represent one of the major species appearing differentially in the urine of lung cancer patients (35). This may not be surprising in the light of intense neurohormonal and immune activation characteristic lung cancer and heart failure.

It may ultimately prove difficult to definitively determine whether PIF exists in humans. No source of authentic murine or human PIF is generally available. The reported sequence encodes a peptide with no apparent site for the critical glycosylation, and the mAb has extensive nonspecific reactivity, precluding its use for either detection or purification. The search for a molecule of such extremely low abundance would seem unlikely to be successful, in the absence of a highly selective means of identification and separation and to these ends novel biological tools and purification methodologies are awaited.


   Acknowledgments
 
We thank Drs. J. Parissis and M. Nikolaou for their assistance in data collection of patients with heart failure.


   Footnotes
 
Grant support: The Royal College of Surgeons of Edinburgh, the Prostate Research Campaign UK, the Association for International Cancer Research, and the Mason Medical Foundation (G.D. Stewart); the Royal College of Surgeons of Edinburgh and the University of Edinburgh (R.J.E. Skipworth); Translational Cancer Research Training Program fellowship (M. Mourtzakis); and the Alberta Cancer Board, the Natural Sciences and Engineering Research Council of Canada, and the Canadian Institutes of Health Research (V.E. Baracos and M. Palcic).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

6 http://frodo.wi.mit.edu/ Back

7 http://www.cbs.dtu.dk/services/NetNGlyc/ Back

8 http://www.cbs.dtu.dk/services/NetOGlyc/ Back

Received 4/20/07; revised 5/28/07; accepted 6/ 7/07.


   References
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 Materials and Methods
 Results
 Discussion
 References
 

  1. Lorite MJ, Cariuk P, Tisdale MJ. Induction of muscle protein degradation by a tumour factor. Br J Cancer 1997;76:1035–40.[Medline]
  2. Lorite MJ, Thompson MG, Drake JL, Carling G, Tisdale MJ. Mechanism of muscle protein degradation induced by a cancer cachectic factor. Br J Cancer 1998;78:850–6.[Medline]
  3. Smith HJ, Lorite MJ, Tisdale MJ. Effect of a cancer cachectic factor on protein synthesis/degradation in murine C2C12 myoblasts: modulation by eicosapentaenoic acid. Cancer Res 1999;59:5507–13.[Abstract/Free Full Text]
  4. Wyke SM, Tisdale MJ. NF-{kappa}B mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin-proteasome system in skeletal muscle. Br J Cancer 2005;92:711–21.[CrossRef][Medline]
  5. Todorov PT, Deacon M, Tisdale MJ. Structural analysis of a tumor-produced sulfated glycoprotein capable of initiating muscle protein degradation. J Biol Chem 1997;272:12279–88.[Abstract/Free Full Text]
  6. Cariuk P, Lorite MJ, Todorov PT, Field WN, Wigmore SJ, Tisdale MJ. Induction of cachexia in mice by a product isolated from the urine of cachectic cancer patients. Br J Cancer 1997;76:606–13.[Medline]
  7. Todorov P, Cariuk P, McDevitt T, Coles B, Fearon K, Tisdale M. Characterization of a cancer cachectic factor. Nature 1996;379:739–42.[CrossRef][Medline]
  8. Cabal-Manzano R, Bhargava P, Torres-Duarte A, Marshall J, Bhargava P, Wainer IW. Proteolysis-inducing factor is expressed in tumours of patients with gastrointestinal cancers and correlates with weight loss. Br J Cancer 2001;84:1599–601.[CrossRef][Medline]
  9. Williams ML, Torres-Duarte A, Brant LJ, Bhargava P, Marshall J, Wainer IW. The relationship between a urinary cachectic factor and weight loss in advanced cancer patients. Cancer Invest 2004;22:866–70.[CrossRef][Medline]
  10. Wigmore SJ, Todorov PT, Barber MD, Ross JA, Tisdale MJ, Fearon KC. Characteristics of patients with pancreatic cancer expressing a novel cancer cachectic factor. Br J Surg 2000;87:53–8.[Medline]
  11. Jatoi A, Foster N, Wieland B, et al. The proteolysis-inducing factor: in search of its clinical relevance in patients with metastatic gastric/esophageal cancer. Dis Esophagus 2006;19:241–7.[CrossRef][Medline]
  12. Deans DA, Wigmore SJ, Gilmour H, Tisdale MJ, Fearon KC, Ross JA. Expression of the proteolysis-inducing factor core peptide mRNA is upregulated in both tumour and adjacent normal tissue in gastro-oesophageal malignancy. Br J Cancer 2006;94:731–6.[Medline]
  13. Cunningham TJ, Hodge L, Speicher D, et al. Identification of a survival-promoting peptide in medium conditioned by oxidatively stressed cell lines of nervous system origin. J Neurosci 1998;18:7047–60.[Abstract/Free Full Text]
  14. Cunningham TJ, Jing H, Akerblom I, Morgan R, Fisher TS, Neveu M. Identification of the human cDNA for new survival/evasion peptide (DSEP): studies in vitro and in vivo of overexpression by neural cells. Exp Neurol 2002;177:32–9.[CrossRef][Medline]
  15. Hipfel R, Schittek B, Bodingbauer Y, Garbe C. Specifically regulated genes in malignant melanoma tissues identified by subtractive hybridization. Br J Cancer 2000;82:1149–57.[CrossRef][Medline]
  16. Schittek B, Hipfel R, Sauer B, et al. Dermcidin: a novel human antibiotic peptide secreted by sweat glands. Nat Immunol 2001;2:1133–7.[CrossRef][Medline]
  17. Monitto CL, Dong SM, Jen J, Sidransky D. Characterization of a human homologue of proteolysis-inducing factor and its role in cancer cachexia. Clin Cancer Res 2004;10:5862–9.[Abstract/Free Full Text]
  18. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5.[CrossRef][Medline]
  19. Lafitte D, Dussol B, Andersen S, et al. Optimized preparation of urine samples for two-dimensional electrophoresis and initial application to patient samples. Clin Biochem 2002;35:581–9.[CrossRef][Medline]
  20. Choudhary G, Chakel J, Hancock W, Torres-Duarte A, McMahon G, Wainer I. Investigation of the potential of capillary electrophoresis with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for clinical analysis: examination of a glycoprotein factor associated with cancer cachexia. Anal Chem 1999;71:855–9.[Medline]
  21. Dai Y, Whittal RM, Li L. Two-layer sample preparation: a method for MALDI-MS analysis of complex peptide and protein mixtures. Anal Chem 1999;71:1087–91.[Medline]
  22. Cadieux PA, Beiko DT, Watterson JD, et al. Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS): a new proteomic urinary test for patients with urolithiasis. J Clin Lab Anal 2004;18:170–5.[CrossRef][Medline]
  23. Mitsiopoulos N, Baumgartner RN, Heymsfield SB, Lyons W, Gallagher D, Ross R. Cadaver validation of skeletal muscle measurement by magnetic resonance imaging and computerized tomography. J Appl Physiol 1998;85:115–22.[Abstract/Free Full Text]
  24. Shen W, Punyanitya M, Wang Z, et al. Total body skeletal muscle and adipose tissue volumes: estimation from a single abdominal cross-sectional image. J Appl Physiol 2004;97:2333–8.[Abstract/Free Full Text]
  25. Wang Z, Corey E, Hass GM, et al. Expression of the human cachexia-associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia. Int J Cancer 2003;105:123–9.[CrossRef][Medline]
  26. DeWys WD, Begg C, Lavin PT, et al. Prognostic effect of weight loss prior to chemotherapy in cancer patients. Eastern Cooperative Oncology Group. Am J Med 1980;69:491–7.[CrossRef][Medline]
  27. Vigano A, Bruera E, Jhangri GS, Newman SC, Fields AL, Suarez-Almazor ME. Clinical survival predictors in patients with advanced cancer. Arch Intern Med 2000;160:861–8.[Abstract/Free Full Text]
  28. Palomares MR, Sayre JW, Shekar KC, Lillington LM, Chlebowski RT. Gender influence on weight-loss pattern and survival of nonsmall cell lung carcinoma patients. Cancer 1996;78:2119–26.[CrossRef][Medline]
  29. Kelsen DP, Ginsberg R, Pajak TF, et al. Chemotherapy followed by surgery compared with surgery alone for localized esophageal cancer. N Engl J Med 1998;339:1979–84.[Abstract/Free Full Text]
  30. Todorov PT, McDevitt TM, Cariuk P, Coles B, Deacon M, Tisdale MJ. Induction of muscle protein degradation and weight loss by a tumor product. Cancer Res 1996;56:1256–61.[Abstract/Free Full Text]
  31. Teich N, Kleeff J, Lochs H, et al. The presence of the proteolysis-inducing factor in urine does not predict the malignancy of a pancreatic tumour. BMC Gastroenterol 2005;5:20.[CrossRef][Medline]
  32. Sato C, Kim JH, Abe Y, Saito K, Yokoyama S, Kohda D. Characterization of the N-oligosaccharides attached to the atypical Asn-X-Cys sequence of recombinant human epidermal growth factor receptor. J Biochem (Tokyo) 2000;127:65–72.[Abstract/Free Full Text]
  33. Lowrie AG, Wigmore SJ, Wright DJ, Waddell ID, Ross JA. Dermcidin expression in hepatic cells improves survival without N-glycosylation, but requires asparagine residues. Br J Cancer 2006;94:1663–71.[Medline]
  34. Todorov PT, Field WN, Tisdale MJ. Role of a proteolysis-inducing factor (PIF) in cachexia induced by a human melanoma (G361). Br J Cancer 1999;80:1734–7.[CrossRef][Medline]
  35. Tantipaiboonwong P, Sinchaikul S, Sriyam S, Phutrakul S, Chen ST. Different techniques for urinary protein analysis of normal and lung cancer patients. Proteomics 2005;5:1140–9.[CrossRef][Medline]
  36. Anker SD, Negassa A, Coats AJ, et al. Prognostic importance of weight loss in chronic heart failure and the effect of treatment with angiotensin-converting-enzyme inhibitors: an observational study. Lancet 2003;361:1077–83.[CrossRef][Medline]
  37. Luo Y, Zhang LL, Ning L, He DL, Fan JH, Hou HL. [Establishment of bone metastasis model of prostate cancer in nude mice by intratibia injection of human prostate cancer cell line Du145]. Zhonghua Nan Ke Xue 2006;12:133–6.[Medline]



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Re: Wieland BM, et al. Is There a Human Homologue to the Murine Proteolysis-Inducing Factor?
Clin. Cancer Res., April 1, 2008; 14(7): 2245 - 2245.
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