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PPP2R1A as new biomarker in type II serous endometrial cancer: a pre-clinical study
Belgium (BeMSA) - KU Leuven, Leuven
Department of Cellular and Molecular Medicine Laboratory of Protein Phosphorylation and Proteomics
Prof. Veerle Janssens
drs. Michiel Remmerie
Type of Research Project
- Basic science
What is the background of the project?
Type II (serous) endometrial carcinomas (ECs) are responsible for most EC-related deaths, due to their aggressive nature and therapy resistance. Nevertheless, type II ECs are still treated the same way as the easily treatable type I ECs, albeit with little success. Therefore, there is a need for new treatment options for patients with type II EC. Molecular analysis of EC tumors identified mutations in oncogenes and tumor suppressor genes, thereby revealing potential new therapeutic targets for this cancer. Interestingly, PPP2R1A (Protein Phosphatase 2 Scaffold Subunit Alpha), encoding the Aα subunit of the tumor suppressive phosphatase PP2A (protein phosphatase 2A), is mutated in up to 40% of type II ECs, while lacking in type I EC. More precisely, these are heterozygous missense mutations mainly recurring at three residues; p.P179, R183 and S256, forming so-called hotspot mutations. The host lab found the PI3K/Akt (Phosphoinositide 3-kinase / Protein kinase B ) and mTOR/p70S6K (mammalian target of rapamycin/Ribosomal protein S6 kinase beta-1) signaling pathways to be affected in EC-associated Aα mutants. Thus, specifically targeting these pathways using pharmacologic compounds could have different results when PPP2R1A is mutated compared to wild type PPP2R1A.
What is the aim of the project?
In this project, we aim to reverse the increased oncogenic effects of mutant Aα expressing EC cells with pharmacologic kinase inhibitors targeting PI3K/Akt/mTOR signaling, small molecular activators of PP2A (SMAPs), or pharmacologic modulators of HIPPO signaling (which we believe is also affected by PPP2R1A mutations). Like that, we will establish the predictive biomarker potential of PPP2R1A mutations for different potential anti-cancer therapies in vitro.
What techniques and methods are used?
We are using stable EC cell lines expressing the Aα mutants to assess the effects of the targeted compounds. Cell culture will be a big part of the project and several functional assays will be performed: - 2D-growth: MTT viability assay: MTT assay is a colorimetric assay for assessing cell metabolic activity. - Anchorage-independent (AI) growth. - Migration/invasion assay using Incucyte® S3 Live-Cell Analysis System: This technique allows us to acquire relevant information about our cell culture in real time. Using this assay system it is possible to perform different kinds of assays without having to remove the cell cultures from the incubator. Using this technique we can gain information about the migration and invasion of the tumorous cells. - Oncogenic signaling: Immunoblotting using phospho-specific antibodies, antibodies targeted at the phosphatases of our interest. Meanwhile we are also using the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated protein 9 ) technique to introduce the specific PPP2R1A mutations in the EC cell lines. Depending on the in vitro results, we also plan on testing the therapeutics in vivo, in a xenograft mouse model. PCR (polymerase chain reaction) amplification to determine the mutation status of PPP2R1A in EC tissue from patients. Data analysis will be performed and therefore knowledge of microsoft excel is highly recommended.
What is the role of the student?
- The student will mainly observe
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The student should have a better understanding of the project and should be able to perform some lab tasks individually (cell culture, migration assay, MTT viability assay). The student will perform tasks such as daily cell culture. Furthermore the student will be involved in the migration assay and a viability assay. Besides that, the student will be actively involved in data analysis (via microsoft excel) of the above assays (including statistics). Lab meetings will take place during the research stay of the student , so that the student will have an idea about the implementation of the current project into the broader research performed in the host lab.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
The student should be enthusiastic and eager to learn, be interested in cancer research
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Graduated students (less than 6 months) - Pre-Medical students from the American-British system - Students in biomedical fields
- Remmerie M; Janssens V. Targeted therapies in type II endometrial cancers: too little; but not too late. Int J Mol Sci. (2018) 19:2380. doi: 10.3390/ijms19082380
- Haesen D; Abbasi Asbagh L; Derua R; Hubert A; Schrauwen S; Hoorne Y; et al. Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth. Cancer Res. (2016) 76:5719–31. doi: 10.1158/0008-5472.CAN-15-3342
- Remmerie M and Janssens V (2019) PP2A: A Promising Biomarker and Therapeutic Target in Endometrial Cancer. Front. Oncol. 9:462. doi: 10.3389/fonc.2019.00462
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