THE INHIBITING EFFECT OF THE COMBINATION TREATMENT OF SELUMETINIB AND MK-2206 ON AKT AND ERK SIGNALING IN TWO TYPES OF TRIPLE-NEGATIVE BREAST CANCER CELLS COMPARED TO THE MONO-TREATMENTS.
THE INHIBITING EFFECT OF
THE COMBINATION
TREATMENT OF
SELUMETINIB AND MK-
2206 ON AKT AND ERK
SIGNALING IN TWO TYPES
OF TRIPLE-NEGATIVE
BREAST CANCER CELLS
COMPARED TO THE MONO-
Author: Zheng Xuan Su
Co-Authors: Ö. Baser, R. Kiliç, N. Soebedar,
Student number: 2488841 / Leiden University
, ABSTRACT
The purpose of this research was finding the effect on proliferation by inhibiting
phosphorylated AKT and ERK and their signaling by using a combination therapy of
Selumetinib and MK-2206 on Triple Negative Cancer Cells compared to the mono-
treatments. In prior findings it was found that Selumetinib had a significant effect on TNBC1
cells, resulting in a total inhibition of p-AKT and for MK-2206 on TNBC2 cells on p-ERK. The
results were found by using SDS-PAGE Western Blot, qPCR, IF and SRB. The primers that
were used in qPCR are SOX9, PIM2, AREG and HB-EGF. And KI67 was used to determine
the proliferation inhibition. There were statistics used to determine the significance of the
combination treatments compared to the mono-treatments. It is verified that the combination
inhibited Akt and ERK phosphorylation, and thereby inhibition of both proliferation and cell
viability. This research has not been done before thus it can provide critical information for
future clinical research.
INTRODUCTION
Breast cancer was rated the second most common cancer in overall according to the World
Cancer Research Fund (WCRF) in 2018. Importantly, it was the most frequent type of cancer
diagnosed in women and was the leading cause of death in over 100 countries1. With its
most aggressive cancer known being Triple-Negative Breast Cancer (TNBC)2. TNBC cells do
not respond to HER2- and hormone targeted therapy and can develop drug-resistance which
results in a poor prognosis3,4. Although that there are possible therapies for TNBC left,
(chemotherapy and immunotherapy4) the metastasis rate was higher than non-TNBC tumors.
In this research two types of TNBC were used. TNBC1 is a humane basal-like cell line
originated from a rare and very aggressive hormone receptor negative breast carcinoma.
They characterize themselves by developing acquired resistance. The cells have various
mutations in their signaling proteins such as p53. TNBC2 is a humane basal-B-like cell line
with fibroblast-like characteristics (mesenchymal morphology) originated of a hormone
receptor negative breast carcinoma. The same applies to TNBC2 referring to the acquired
resistance and mutations in the signaling protein p53 and tumor suppressor NF1. The two
types distinguish themselves in the morphology as well as sub-types because of their gene
expression.
Due to recent studies of two TNBC cell lines when exposed to various treatments at Leiden
University (accessed via pCB cohort 2019, resultaten SRB, IF, WB en qPCR, Labbuddy
theoretische kader, Figuur 1) two kinase-inhibitors were chosen to investigate Selumetinib,
an Akt-inhibitor and MK-2206, a MEK-inhibitor. From this previous study is interpreted that
both TNBC cell lines are resistant to inhibition of p-ERK induced by MK-2206. And p-Akt was
resistant to Selumetinib in both cell lines. However, these therapies do show significant
results as single agents in inhibiting the phosphorylation ERK and Akt in both TNBC cell
lines. There have been no earlier studies of this approach with these treatments and the two
TNBC cell lines which make this research of great importance for future research.
The aim of this research is to evaluate the effect of the combination treatment compared to
the mono-treatments on the signaling of the ERK and Akt and their role in proliferation in the
two types of TNBC cells. It is believed that the combination therapy proffers a more effective
impact than the singular treatments, at least in one of the cell lines. Hence, there were four
experiments done to obtain explicit data in order to confirm this. Firstly, using Western Blot
(WB) analysis it was determined if ERK and Akt phosphorylation were inhibited by the
treatments. Secondly, a Sulforhodamine B (SRB) was carried out to decide whether the cell
viablity was inhibited or not. Additionally, an (Immunofluorescence) IF was performed with Ki-
67, marker of cell proliferation, to attain if the proliferation was inhibited5. It is critical to define
the impact of the treatments on hallmarks of cancer such as proliferation and apoptosis.
Consequently, a real-time quantitative PCR (qPCR) was performed with four primers that
play a critical role in the proliferation of TNBC cells: AREG, HB-EGF, PIM2 and SOX9. And
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