Cancer causes 1.5 million deaths a year in Europe, accounting for a quarter of all deaths. The incidence of cancer is increasing, with 20 million new cases and 10 million deaths expected in the next 20 years. Approximately 90% of deaths are due to metastases rather than primary tumours. Genetic mutations and genomic instability allow tumour cells to invade and form metastases.
Colorectal cancer can be non-hereditary or hereditary, such as familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC). Genes involved in colorectal neoplasia include 'gatekeepers' (e.g. APC) and 'caretakers' (e.g. p53) that control cell proliferation and genomic stability. Mutations in these genes lead to the formation of benign and malignant tumours. The process of metastasis is complex and involves genes such as RhoC and Asef, which regulate tumour cell migration and invasion. Protein tyrosine phosphatases (PTPs), in particular the PRL family, play an essential role in colorectal cancer metastasis. PRL-3 is overexpressed in colorectal metastases and has potential as a molecular marker for metastasis, but further studies are needed on subcellular localisation, the effect of mutations and levels in different tumour types.
There are three key questions for future research:
(1) Where is PRL-3 located subcellularly? - Localisation may influence the physiological role, but current data are conflicting, suggesting the presence of PRL-3 at the plasma membrane, early endosomes, endoplasmic reticulum, centrosomes and mitotic spindle.
(2) To what extent are the enzymatic properties of PRL-3 affected by mutations or truncations of the polypeptide chain? - Certain mutations can have dramatic effects on catalytic activity, and the primary sequence of PRL-3 has unique features that may affect enzyme efficiency.
(3) What is the concentration of PRL-3 in primary and metastatic tumour cells? - It is important to determine whether there is a threshold concentration that indicates the metastatic state of the cells.
Head of Department
Prof. Dr. Stefan Szedlacsek is the Head of the Enzymology Department at the Institute of Biochemistry of the Romanian Academy. He holds a PhD degree in Biotechnology from Polytechnical University of Bucharest as well as a MSc in Organic Synthesis (Polytechnical University, Bucharest) and MSc in Mathematics (University of Bucharest). As a visiting scientist, he performed research in the field of cholesterol metabolism at the University of Illinois at Urbana-Champaign (USA), where he succeeded to evidence a new pathway in the metabolism of oxysterols. He is an “Alexander von Humboldt” fellow and worked in Germany, in the Institute of Biochem More...
Project coordinator: Dr.Stefan SZEDLACSEK
Partner 1 Centre for Bioanalysis - National Research Institute for Biological Sciences: Project manager: Prof. Dr. Ing. G.L. Radu
Partner 2 Foundation for Liver Surgery, Fundeni Clinical Institute: Project manager: Prof. Univ. Dr. Irinel Popescu
(1) To obtain molecular truncations and a specific mutation on the enzymatic activity of PRL-3.
(2) To analyse the truncated and mutant forms on the subcellular localisation of PRL-3.
(3) Qualitative analysis of PRL-3 from colorectal tissue, primary tumour and metastatic tumour.
(4) To develop a laboratory method for the quantitative determination of PRL-3 in colorectal tissue as a potential marker of colorectal metastasis.
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ȘtefanSzedlacsek Geogiana Petrăreanu Mihaela Pascaru Andrei Văcaru Maria Drăgan |
Project coordinator Research assistant Research assistant PhD Student Assistant |
PRL3 is a protein tyrosine phosphatase that is overexpressed in colorectal cancer metastases to the liver. Its C-terminal prenylation motif is essential for its localisation to the plasma membrane and early endosomal compartment. Inhibition of prenylation leads to nuclear localisation of PRL3. To evaluate whether truncation of the polypeptide chain affects the catalytic activity of PRL3, two truncation mutants were obtained, one by removing the first 10 N-terminal amino acids and another by removing the 4 C-terminal amino acids. The specificity constant of the N-terminal truncated protein was similar to that of the native form of PRL3, whereas the specificity constant of the C-terminal truncated protein was 2.4 times higher than that of the native form.
These results suggest that the last 4 amino acids have a modulating role on the catalytic activity. To determine whether a potential nuclear localisation signal near the C-terminus plays an effective role in targeting PRL3 to the nucleus, 3 EGFP-PRL-3 mutants were constructed in which 3 basic amino acids from the potential localisation signal were substituted with neutral amino acids, in the presence and absence of the prenylation motif. This mutation did not significantly affect the subcellular localisation of PRL3, suggesting that other amino acids in the potential localisation signal may also be involved in nuclear localisation, or that PRL3 is transported to the nucleus by association with other targeted proteins.
Using the RT-PCR technique, it was possible to highlight PRL3 in metastatic cells from the liver and in primary tumour tissue, but the presence of PRL3 was also observed in normal tissue. Two specific assays based on immunoblotting and ELISA were developed to identify PRL3 in tissue samples. In the metastatic tissues analysed, PRL3 was highlighted in one of the samples by the immunoblotting method and in 5 of the 8 samples analysed by the ELISA method.
The ELISA method proved to be the most sensitive for the identification of PRL3. This method showed that metastatic tissue contained between 0.8-8ng PRL3/mg total protein.
The use of the data obtained, including the two methods developed in the present work, in the analysis of a large number of metastatic and tumour tissues will allow the consideration of PRL3 as a molecular marker of colorectal liver metastasis.
PRL3 protein in metastatic tissues was highlighted using primary anti-PRL3 antibodies. (Figure 1).
Quantitative PRL3 could be determined by the development of an ELISA type method, which was able to determine between 0.8 - 8ng PRL3/mg total protein. (Figure 2).
Oral presentation:
(1) Seminars of the Institute of Biochemistry of the Romanian Academy, 2004: "PRL-3, a protein tyrosine phosphatase involved in colorectal cancer", PhD student Andrei Vacaru
(2) Annual Scientific Meeting of the Institute of Biochemistry of the Romanian Academy, 2004: "Enzymes involved in cellular signalling processes. Expression, characterisation, applications", Dr. Stefan Szedlacsek.
Poster:
(1) Workshop, "Glycosylation and Disease" - 3-5 June 2004 - Bucharest, "Molecular analysis of the protein tyrosine phosphatase PRL-3, a protein with dual subcellular localisation", Carmen A. Tanase, Andrei Vacaru, Stefan Szedlacsek
(2) VIASAN National C-D Programme Scientific Symposium "From Molecular and Cellular Medicine to Clinical Medicine", 2006: "Molecular Analysis of the Involvement of Protein Tyrosine Phosphatase PRL-3 in Colorectal Cancer Metastasis", Mihaela Pascaru, Carmen Tanase, Andrei Vacaru , Elena Neagu, Patricia Boeti, Vlad Herlea, Irinel Popescu, Stefan Szedlacsek
Article
(1) Analysis of Molecular Determinants of PRL-3”: Pascaru M, Tanase C, Vacaru AM, Boeti P, Neagu E, Popescu I, Szedlacsek SE., J Cell Mol Med. 13(9B), 3141-50; 2009; https://pubmed.ncbi.nlm.nih.gov/19040419/
Romanian Academy