The validation of antibodies with genetic strategy: PP2A inhibitors CIP2A, PME-1 and SET in human triple-negative breast cancer cell lines
Rajaniemi, Suvi (2025-11-13)
The validation of antibodies with genetic strategy: PP2A inhibitors CIP2A, PME-1 and SET in human triple-negative breast cancer cell lines
(13.11.2025)
Julkaisu on tekijänoikeussäännösten alainen. Teosta voi lukea ja tulostaa henkilökohtaista käyttöä varten. Käyttö kaupallisiin tarkoituksiin on kielletty.
avoin
Julkaisun pysyvä osoite on:
https://urn.fi/URN:NBN:fi-fe20251219122774
https://urn.fi/URN:NBN:fi-fe20251219122774
Tiivistelmä
Worldwide, breast cancer is the most common cancer, and the most common cancer leading to death among women. In clinic the diagnostics and treatment of breast cancers is based on biomarkers expressed by these tumors. There are four breast cancer main types and three of them can be treated by targeting these biomarkers i.e., nuclear, or transmembrane receptors expressed in breast cancer cells of each type. The fourth breast cancer main type, triple-negative breast cancer lacks all three clinically important receptors: estrogen receptor, progesterone receptor and HER2 receptor. Triple-negative breast tumors represent a minority of breast cancer cases but unfortunately patients diagnosed with this tumor type have the highest risk of recurrence and the highest mortality rate compared to patients diagnosed with other breast cancer main types. Triple-negative breast tumors are known to be very heterogenous and, there is a crucial need for novel biomarkers to diagnose and treat this complex and aggressive breast cancer type.
The role of endogenous inhibitor proteins of protein phosphatase 2A (PP2A) as potential biomarkers in human cancer are currently under an interest since, the important tumor-suppressive activity of PP2A has evidenced to be inhibited by these inhibitors in the majority of human malignancies. When overexpressed, these inhibitor proteins are known to inhibit the dephosphorylation activity of PP2A by interacting with its dimers or trimers. The target proteins of PP2A remain phosphorylated hence, in active state, which further leads to the activation of downstream signaling pathways related to proliferation of cancer cells. This master’s thesis will concentrate on three PP2A inhibitor proteins: cancerous inhibitor of PP2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and protein SET (SET). For example, the overexpression of CIP2A is linked to poor prognosis among patients with basal-like triple-negative breast cancer, even more aggressive breast tumor compared to plain triple-negative phenotype.
The first aim of this thesis was to validate antibodies targeting PP2A inhibitors CIP2A, PME-1 and SET with genetic approach in western blot. Antibodies are one of the most important tools used universally in the field of molecular biology. Unfortunately, antibodies are causing considerably problems in reproducibility and, there are no standardized methods or reporting practices to assess the validity of these tools. To validate the antibodies with genetic strategy, siRNA technique was used to produce MDA-MB-231 cells knock-down against the PP2A inhibitor of interest. Based on the validation criteria used in this thesis, all three antibodies are trustworthy to use.
The other aim was to examine the expression of CIP2A, PME-1 and SET across TNBC cell lines, both at mRNA and protein level. For this purpose, 18 triple-negative breast cancer cell lines, representing seven transcriptomic subtypes, were studied. The expression studies showed how at mRNA level, all three PP2A inhibitors overlapped since, these were overexpressed across majority of TNBC cell lines and within TNBC subtypes. But interestingly at protein level, CIP2A dominates over PME-1 and SET being only inhibitor protein overexpressed among majority of TNBC cell lines and within all TNBC subtypes. Therefore, the CIP2A protein is the most relevant PP2A inhibitor considering TNBC. In addition, CIP2A seems to be a potential novel biomarker for the invasive mesenchymal stem-like subtype, which expressed CIP2A protein at particularly high level compared to other TNBC subtypes.
The role of endogenous inhibitor proteins of protein phosphatase 2A (PP2A) as potential biomarkers in human cancer are currently under an interest since, the important tumor-suppressive activity of PP2A has evidenced to be inhibited by these inhibitors in the majority of human malignancies. When overexpressed, these inhibitor proteins are known to inhibit the dephosphorylation activity of PP2A by interacting with its dimers or trimers. The target proteins of PP2A remain phosphorylated hence, in active state, which further leads to the activation of downstream signaling pathways related to proliferation of cancer cells. This master’s thesis will concentrate on three PP2A inhibitor proteins: cancerous inhibitor of PP2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and protein SET (SET). For example, the overexpression of CIP2A is linked to poor prognosis among patients with basal-like triple-negative breast cancer, even more aggressive breast tumor compared to plain triple-negative phenotype.
The first aim of this thesis was to validate antibodies targeting PP2A inhibitors CIP2A, PME-1 and SET with genetic approach in western blot. Antibodies are one of the most important tools used universally in the field of molecular biology. Unfortunately, antibodies are causing considerably problems in reproducibility and, there are no standardized methods or reporting practices to assess the validity of these tools. To validate the antibodies with genetic strategy, siRNA technique was used to produce MDA-MB-231 cells knock-down against the PP2A inhibitor of interest. Based on the validation criteria used in this thesis, all three antibodies are trustworthy to use.
The other aim was to examine the expression of CIP2A, PME-1 and SET across TNBC cell lines, both at mRNA and protein level. For this purpose, 18 triple-negative breast cancer cell lines, representing seven transcriptomic subtypes, were studied. The expression studies showed how at mRNA level, all three PP2A inhibitors overlapped since, these were overexpressed across majority of TNBC cell lines and within TNBC subtypes. But interestingly at protein level, CIP2A dominates over PME-1 and SET being only inhibitor protein overexpressed among majority of TNBC cell lines and within all TNBC subtypes. Therefore, the CIP2A protein is the most relevant PP2A inhibitor considering TNBC. In addition, CIP2A seems to be a potential novel biomarker for the invasive mesenchymal stem-like subtype, which expressed CIP2A protein at particularly high level compared to other TNBC subtypes.