Development and Applications of Novel Biotechnological Tools to Investigate the Physiopathological Role of Receptor-Type Tyrosine Protein Phosphatase Gamma (PTPRG)

ABSTRACT

Background: Protein tyrosine phosphatases (PTPs) have emerged as a new class of signalling molecules that play important roles in the development, regulating cell proliferation, differentiation, migration and transformation. Moreover, deregulation of several PTPs contributes to the pathogenesis of human diseases. As a result, substantial research over the last decade has focused on the structure and function of PTPs, and a number of these enzymes are now being tested as potential pharmaceutical targets. Considering these assumptions, we focused on Receptor-type Tyrosine-Protein Phosphatase Gamma (PTPRG), a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPRG is known to regulate haematopoietic differentiation in a murine embryonic stem cells model and to be involved as a putative tumor suppressor gene in kidney, lung, colon, ovarian and breast cancers. Our studies, supported by the unique tools we developed, led us to recognize new features for this phosphatase, including a possible critical role in the pathogenesis of chronic myeloid leukemia.
Aims:
1) set up or develop new tools for analysis of PTPRG;
2) study PTPRG expression in normal tissues;
3) study PTPRG expression in neoplasia;
4) identification of functional role/s for PTPRG.
Methods:
haematopoietic cells purification and culture, flow cytometry, immunostaining of cells and tissues by immunofluorescence and immunohistochemistry, reverse transcription-polymerase chain reaction (PCR), real-time quantitative reverse transcriptase (RT)-PCR, Western Blot analysis.
Results:
Aim 1. Set up new tools:
We developed a quantitative PCR (QPCR) and semi-quantitative PCR assay, developed a new antibody suitable for flow cytometric detection of PTPRG, set up the conditions for immunohistochemical staining of paraffin embedded tissues by three different antibodies recognizing as many different domains of PTPRG.
Aim 2. PTPRG expression in normal tissues: We demonstrated that:
a) PTPRG is a new biomarker for monocytes, dendritic cells and specialized macrophages;
b) PTPRG is highly expressed by CD34+ circulating precursors and is modulated during differentiation;
c) PTPRG is highly expressed by endocrine cells;
d) PTPRG is highly expressed by epithelial cells, including several endothelium;
e) The isolated PTPRG extracellular domain is expressed in vivo .
Aim 3. PTPRG expression in neoplasia:
a) We demonstrated a marked loss of PTPRG immunoreactivity in subsets of ovarian (21%), breast (56%) and lung (80%) neoplasms. Conversely, cytoplasmic positivity was found in 37% of lymphomas, mainly of high-grade histotypes, while normal lymphocytes were negative. Brain tissue showed PTPRG expression in a few neuronal and glial elements while PTPRG was overexpressed in the majority of high-grade astrocytomas;
b) PTPRG is specifically down modulated in Chronic Myeloid Leukemia (CML) patients in both peripheral blood and bone marrow, including in CD34+ cells, and is re-expressed following molecular remission of the disease.
Aim 4. Functional studies:
a) PTPRG in disease: PTPRG is down-regulated in Chronic Myeloid Leukemia (CML) cell lines where reduced expression correlates with higher clonogenicity and proliferation, while overexpression inhibits both parameters. Clonogenicity is unaffected whereas proliferation is partially inhibited by the expression of a phosphatase inactive mutant;
b) PTPRG is down-regulated in leukocytes of patients affected by CML and its expression is recovered following molecular remission of the disease.
c) PTPRG expression correlates with a tolerogenic phenotype in vitro differentiated dendritic cells isolated from patients affected by pancreatic cancer.

Conclusions:
We propose PTPRG as a new functionally regulated leukocyte marker whose precise role in normal and pathological context deserve further investigation.
We describe particularly high PTPRG expression in endocrine cells and both down- and up-regulation in neoplasia, the latter possibly reflecting the undifferentiated state of the neoplastic cells, suggesting a complex role for this phosphatase in the pathogenesis of cancer in various districts.
We provide experimental evidence that PTPRG might represent a new pharmacological target. Its down-regulation represent an early and critical event in the pathogenesis of CML and the measurement of transcript and protein levels might find clinical application to confirm diagnosis and for the follow up of this disease.
Finally PTPRG expression was found to correlate with a tolerogenic phenotype in monocyte-derived dendritic cells obtained from patients affected by pancreatic cancer. This observation suggest for PTPRG a possible inhibitory role in the regulation of immune response.