ENDOGLIN/CD105 is expressed in KIT positive cells in the gut and in gastrointestinal stromal tumours

Abstract ENDOGLIN/CD105 (ENG) is a transmembrane glycoprotein and an auxiliary unit of the transforming growth factor-β (TGF-β); receptor, expressed predominantly in vascular endothelium. Noteworthy, Eng mRNA expression has been reported also in Kit+ interstitial cells of Cajal (ICC) in the mouse intestine. Gastrointestinal stromal tumours (GIST) are thought to derive from ICC. Here we have investigated Eng expression in the KitK641E mouse GIST model, in human GIST and in the Ba/F3 cell model. In wild type (WT) mouse antrum, Eng immunoreactivity (-ir) was detected in CD34+/CD31+ endothelium and in Kit+ ICC. In KitK641E mice, hyperplasia of Kit+ cells made Eng-ir even more evident. Quantitative PCR confirmed the increased expression of Eng transcript in KitK641E mice. On human GIST TMA, 26/49 cases stained positive for ENG. Strong ENG staining was associated with malignant and high-risk tumours. ENG negative cases were predominantly of the epithelioid type or harboured PDGFRA mutation. In vitro, Eng mRNA was up-regulated in Ba/F3 cell lines stably expressing various oncogenic Kit mutations (K641E, del559, del814). This effect appeared to be independent of Kit activation, as neither the stimulation of WT Kit by its ligand SCF, nor the inhibition of Kit autophosphorylation by imatinib mesylate in oncogenic mutants, altered Eng expression. Elevated Eng expression in Kit oncogenic mutants appeared rather to be indirectly mediated by DNA hypomethylation, because treatment with the demethylating agent 5-Aza/dC increased Eng mRNA expression in KitWT cells. ENG expression in ICC and in GIST deserves further consideration as ENG is emerging as a potential target for cancer therapy.


ENDOGLIN/CD105 (ENG) is a 180 kD homodimeric transmembrane glycoprotein, expressed predominantly on vascular endothelial and haematopoietic cells. ENG is an auxiliary protein of the transforming growth factor ␤ (TGF-␤) receptor complex involved in cellular proliferation, differentiation and migration
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Two ENG isoforms, differing in the length of their cytoplasmic domain, have been characterized in human and mouse tissues [2,3]. The predominant L-ENG isoform contains in its cytosolic domain a consensus PDZ-binding motive, which is lacking in the barely expressed S-ENG isoform. L-ENG has been shown to be involved in epidermal carcinogenesis, whereas no influence of S-ENG could be demonstrated during carcinogenesis in vivo or in vitro [3]. ENG extracellular domain can undergo proteolytic cleavage and shed from the cell membrane into the bloodstream, giving rise to soluble ENG [4]. Elevated levels of soluble ENG have been reported in pre-eclampsia [5,6] while studies in cancer patients remain conflicting, some studies reporting increased levels [7][8][9] whereas others reported a decrease [4] or no change [10][11][12] in circulating soluble ENG. ENG expression counteracts the potent inhibitory effect of TGF-␤ on cell proliferation in several cell lines [13,14]. Mutations in the ENG gene are responsible for the hereditary haemorrhagic telangiectasia type 1 [HHT1, Osler-Weber-Rendu syndrome (OMIM 187300)]. ENG has been largely investigated in hypoxic responses and as a putative diagnostic, prognostic and therapeutic target in tumoural neoangiogenesis. Conversely, ENG expression in non-endothelial cells has so far attracted little attention [1,15]. Endoglin/CD105 was found to be expressed in non-endothelial cells of various histotypes, including Kit ϩ interstitial cells of Cajal (ICC) in the mouse small intestine [16]. ICC are specialized mesenchymal cells located within the muscularis propria of the gastrointestinal tract, where they play major roles in coordinating peristalsis through intrinsic pacemaker function and network formation [17]. Gastrointestinal stromal tumours (GIST) are the most common sarcoma of the gastrointestinal tract and are thought to derive from the ICC lineage. Approximately 85% of GIST harbour oncogenic KIT mutations and 7% contain oncogenic plateletderived growth factor receptor alpha (PDGFRA) mutations. KIT and PDGFRA are members of the receptor tyrosine kinase family type III. Oncogenic mutations lead to their constitutive autophosphorylation, ligand-independent activation of the downstream signal transduction pathways and subsequent deregulation of cell proliferation, survival and migration [18]. KIT K642E, an oncogenic KIT mutation originally identified in sporadic GIST [19], has also been encountered as germ-line mutation in a family with hyperplasia of the ICC layer and GIST formation [20]. Transgenic mice harbouring Kit K641E, the murine homologue of human KIT K642E, have been generated by a knock-in gene targeting strategy, providing an in vivo GIST model with massive hyperplasia of Kit ϩ cells, especially in antrum and caecum [21,22].
Here we have identified for the first time ENG immunoreactivity (-ir)

Human GIST882 cell line
The human GIST cell line GIST882 [24]

Real-time quantitative PCR (qPCR)
qPCR was performed as described [21]. Briefly [25]. In antrum, the Eng mRNA expression was then normalized by the endothelial marker CD31 mRNA level. Primer sequences are summarized in Table 1.

Immunofluorescence staining
Specimens were processed as described [21]. Briefly, slides were brought to RT, rinsed in 10
Proteins were solubilized in sample buffer, heated at 95ЊC for 5 min., separated by SDS-PAGE on 10% polyacrylamide gel and transferred on a 0.2 m nitrocellulose membrane. Primary antibodies raised in different species and secondary antibodies coupled with different fluorochromes (Table 2), were sequentially combined to specifically label one marker in green (800 DyLight), the other in red (680 DyLight). Gapdh was used as loading control. Liver was used as positive control for Eng expression. The Odyssey™ imaging system (LI-COR Biotechnology, Lincoln, NE, USA) was used to quantify the signals.

Human GIST tissue microarrays (TMA)
A cohort of formalin-fixed, paraffin-embedded human GIST samples was retrieved from the pathology files of the Cleveland Clinic, Cleveland, OH.

The collection and analysis of human tissue samples was approved by the Cleveland Clinic Institutional Review Board (IRB 06-977). Consent was not obtained from any participants as it was waived by the IRB because it was a retrospective study involving only collection of tissue cores from paraffin blocks used in the diagnosis of patient tumour samples.
Clinico-pathological characterization of GIST used is summarized in Table 3. Risk assessment was performed using the criteria of Miettinen and Lasota [26], as recommended by the National Cancer Care Network [27]. Mutations in KIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were examined as described previously [28]. This material was used to construct a TMA with 1 mm cores using a TMarrayer TM (P/N 02110016) semi-automated tissue arrayer (Pathology Devices, Westminster, MD, USA). Each case was present in duplicate in this array.   (Coheris, Suresnes, France). These methods provide a graphic representation of the relations between variables, modalities and subjects, close positions indicating similarities [29]. Multiple modalities that could interact with each other were simultaneously analysed and their respective influence was addressed by FAMC, whereas AHC allowed building homogeneous clusters of subjects. By comparing the position of these clusters in the plane with the position of the modalities, subjects belonging to a cluster can be characterized according to these modalities. A test of proportions was performed. Statistical significance was set at a P value of less than 0.05.
A single band at the expected molecular weight of ~90 kD, representing the predominant L-ENG form was detected by WB on protein extracts of P14 Kit K641E/K641E , Kit WT/K641E and Kit WT/WT mice antrum. No statistically significant difference between genotypes was observed. In the liver, used as control, the L-ENG, S-ENG and soluble Eng isoforms were detected by WB (Fig. S1).

Endoglin is expressed in endothelial cells but also in Kit ϩ cells in the mouse gut wall
Eng-ir was observed in CD34 ϩ /CD31 ϩ endothelium, as expected.
Eng-ir was also detected, albeit fainter than in endothelium, in many, but not all, Kit ϩ ICC ( Fig. 2A and C) (Figs S2 and S3). The higher abundance of Kit ϩ cells in Kit K641E heterozygotes and homozygotes made Eng-ir more readily discernible than in the Kit WT/WT antrum ( Fig. 2A). (Fig. S4).

The antibody used for ENG-ir on GIST TMA was validated using the human GIST882 cell line. Two immunoreactive bands of the expected MW for L-ENG and S-ENG were detected by WB. By IF, GIST882 cells stained positively for ENG-ir, whereas omission of the primary antibodies resulted in the absence of signal
In GIST TMA, 26 GIST stained positively for ENG-ir (ENG ϩ/ϩϩ ) and 23
Similarly, FAMC and AHC revealed three clusters of GIST specimens. GIST (Fig. S5).  (Fig. 4A). Efficiency of SCF treatment was confirmed by the increase of pTyr-ir assessed by flow cytometry. SCF had no effect on pTyr-ir in original Ba/F3 (Fig. S6) (Fig. S8).   (Fig. 5). Another possible mechanism for the transcriptional activation of Eng by the oncogenic Kit mutants could be the up-regulation of an upstream transcription factor. Expression of Hypoxia inducible    (Fig. S9).

Discussion
We first focused on Eng-ir in the murine gut as Eng mRNA expression had been reported in Kit ϩ ICC isolated by FACS from mouse small intestine [16].  [21]. Expression of S-Eng transcript was very low and S-Eng protein was hardly detectable on Western blot. Eng antibodies available do not discriminate L-Eng and S-Eng isoforms, precluding study of their respective distribution by IF. S-Eng may play some regulatory role [3], but its distribution could not be resolved in this study.
Next, we analysed ENG expression in human GIST, using TMA and IHC. ENG-ir was detected in half (26/49) [33,34]. The precise mechanism by which Kit oncogenic mutants could lead to alterations of DNA methylation remains however to be determined.