A soluble form of the giant cadherin Fat1 is released from pancreatic cancer cells by ADAM10 mediated ectodomain shedding

In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells in vitro. Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by in silico analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells in vitro, a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated in vivo leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior

Fat1 and ADAM10 are overexpressed in pancreatic cancers.

<p>Oncomine analysis of five mRNA datasets indicate overexpression of Fat1 and of its sheddase ADAM10 in pancreatic cancer. Copyright 2008–11 Compendia Bioscience, Inc. Oncomine (Compendia Bioscience, Ann Arbor, MI) was used for analysis and visualization.</p

Western blotting indicates the two known forms of Fat1 and a new released form.

<p>Cell lysates (75 µg each) and a representative secretome (8 µg) were blotted together with a protein marker (M). The same blot was probed first, with an antiserum raised against the cytoplasmic domain (CTD) of Fat1 and subsequently with an antiserum raised against the extracellular domain (ECD 2). Beta-Tubulin, finally, was used as a loading control for cell lysate. The extracellular domain specific antibodies, only, detect the processed cellular form and the released Fat1 in the secretome.</p

Fat1 cadherin is a major component of the secretome of pancreatic cancer cell lines.

<p>Abundance of Fat1 derived peptides in the secretome fraction of five pancreatic cancer cell lines (A818-4, BxPc3, MiaPaCa2, Panc1, PaCa44) compared to normal immortalized pancreatic cells (HDPE) determined using MS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090461#pone.0090461.s013" target="_blank">materials and methods S1</a>). The table shows the number of identified Fat1 peptides for each cell line analyzed as well as the rank and the percentage of all identified matches.</p

Serum level of soluble Fat1 in patients with pancreatic cancer.

<p>A) Levels of soluble Fat1 in serum as determined by ELISA (see Methods). Serum samples from 28 unaffected individuals are compared to the levels of Fat1 in 31 patients with pancreatic cancer (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090461#pone.0090461.s010" target="_blank">table S4</a>). B) Comparison of circulating Fat1 with CA19-9 levels in patients with pancreatic cancer. Samples are the subset of 26/30 patients with CA19-9 levels available.</p

ADAM10 mediates Fat1 ectodomain shedding.

<p>Western blot analysis of secretome fractions (8 µg) from PaCa44 and Panc1 cells after ADAM10 knockdown (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090461#pone-0090461-g006" target="_blank">Figure 6</a>) showed significant reductions in Fat1 ectodomain shedding. Again, the two experiments provided similar results as indicated by the normalized quantification.</p

Mass spectrometric analysis of secretomes exclusively indicates peptides derived from the extracellular domain of Fat1.

<p>Mass spectrometric analyses were performed with all six secretomes and Fat1 specific peptides were detected beginning near the amino terminus down to and including the EGF domain (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090461#pone.0090461.s012" target="_blank">file S1</a> for details). Shown here are alignments of Fat1 specific peptides from three exemplary results with high sequence coverage in the section of amino acids 3500 down to the C-terminus at AA4588 (according to HPRD).</p

Metalloproteases are involved in Fat1 ectodomain shedding.

<p>The figure in A) shows a representative Western blot. PaCa44 and Panc1 cells were incubated in serum free medium for two days with the addition of either the broad spectrum metalloprotease inhibitor Batimastat (B, 10 µM), or the ADAM10-specific inhibitor GI245023X (GI, 5 µM) or solvent as a control (C, DMSO). Secretomes, 8 µg protein per sample, were analyzed by Western blot analysis using the Fat1 ECD2 polyclonal antibody. Blotting against transferrin was used as a loading control. (B) Fat1 specific signals from three independent experiments were quantified and normalized with the transferrin signals. The bar graphs show the mean values +/− S.E.M. from three experiments.</p

Alternative Fat1 processing in pancreatic cancer cells.

<p>Full-length Fat1 can be expressed on the cell surface; alternatively, the full length protein can be cut by a furin-type protease to yield the membrane-integrated heterodimer as described by Sadeqzadeh et al. and designated as “classical processing”. Another alternative is ectodomain shedding as described in this work, where the full-length protein is cut by ADAM10 into a p60 C-terminal remnant fragment (which may be further processed) and a gp515 soluble form released into the secretome.</p

Fat1 cadherin in secretome and lysate fractions of pancreatic cancer cell lines.

<p>Western blot analysis of Fat1 in the respective secretome (S; 8 µg loaded) and lysate (L; 50 µg loaded) fractions from four pancreatic cell lines and HPDE. The blot was probed with an antiserum raised against the extracellular domain of Fat1 (ECD1).</p