Essay
practicumverslag pCB
- Instelling
- Universiteit Leiden (UL)
Verslag van het practicum cellulaire biochemie 2020/2021
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AXL as an possible indicator of EGFR-inhibitor resistance in Basal-B-like Triple Negative Breast Cancer Cells
Author:
Co-authors:
Abstract
Breast cancer is the most occurring cancer in women and is subdivided in four categories
with triple negative breast cancer (TNBC) being the worst prognosis because they lack
HER2- and hormone receptors which makes them unsuitable for targeted therapy. Lapatinib,
a dual inhibitor of EGFR and HER2 is often used for the treatment, however some TNBC are
resistant to lapatinib. This study investigates the resistance to TNBC2 cells by analysing the
effects on the cell viability, proliferation, protein activation and gene expression of the
inhibitors: lapatinib and MK-2206 used as single treatments or in a combination treatment.
The most important finding was the overexpression of AXL in TNBC2 cells after treatment
with MK-2206 which suggests that AXL possibly mediates the resistant role against lapatinib
in TNBC2 cells.
Introduction
Breast cancer is the most occurring cancer many cells develop or are resistant to
in women and the second most occurring EGFR inhibitors. Preliminary data confirms
cancer overall with 2 million new cases in the intrinsic resistance in TNBC2, an
20181. Breast cancer is subdivided into humane basal-B-like cell, against lapatinib
HER2-positive (HER2+), triple positive while TNBC1, an humane basal-like cell,
breast cancer (TPBC), estrogen-positive remains sensitive to this treatment8.
(ER+) and/or progesterone-positive (PR+) Mechanisms mediating TNBC2 resistance
and triple negative breast cancer remain unknown and affect lapatinib
(TNBC)2,3. HER2+ and TPBC cancers treatment efficacy. One study has found
have an overexpression of receptor that lapatinib mainly acts through HER2
tyrosine kinases RTKs, mainly the HER2 and cells with higher EGFR levels
RTK as the HER2+ already implies4. This compared to HER2 are more resistant to
makes RTKs an important target for the lapatinib7. Another study has shown that
treatment for breast cancer. Another AXL-overexpressing cells get
possibility for targeted treatment is transactivated by EGFR and thus
hormone therapy by inhibiting the effect of activating downstream pathways beyond
estrogen and progesterone or by lowering the EGFR-pathway9. Activation of AXL
their levels which is effective for hormone inhibits apoptosis, increases migration and
positive cells PR+ and/or ER+ and also for growth through multiple downstream
TPBC cancers5. However, TNBCs lack pathways10. The activation of PI3K and
HER2-receptor overexpression and downstream targets such as AKT is a
hormone-receptors overexpression. This central regulator for the survival of the cell
type of breast cancer accounts for 15-20% in the AXL-signal transduction10. To shut
of all breast cancers and is considered as down this central signal transduction
a bad prognosis due to limited treatment regulator, an AKT-inhibitor such as MK-
options6 because it lacks the HER2- and 2206 can be used. Preliminary data shows
hormone receptors. Lapatinib, a dual that MK-2206 has similar effects on the
inhibitor of EGFR and HER2 tyrosine cell viability in both cell lines8. MK-2206
kinase, is used for treatment7. However, may be a possible candidate for a
, combination treatment against lapatinib proliferation. We also performed a western
resistant cells. Preliminary data shows that blot analysis on pERK and pAKT and
TNBC2 cells have higher EGFR- qPCR analysis for AXL, HER3, EGF and
expression levels compared to TNBC1 AKT3 to examine the effect in pathways
while HER2-expression levels remain the that the inhibitors cause. We expect to see
same8, suggesting that the resistance acts an overexpression of AXL in TNBC2 cells
through one of the mechanisms described which causes an alternative pathway for
by these prior studies. The aim of this the mechanism behind the resistance as
study is to investigate the resistance to described in the study A. S. Meyer et al.7
lapatinib in TNBC2 cells by researching By inhibiting the central AKT regulator with
the effects of the combination treatment MK-2206 we expect to shut down the
lapatinib + MK-2206 in lapatinib-sensitive AXL/PI3K/AKT pathway resulting in a
TNBC1 cells and lapatinib-resistant lower survivability and possibly feedback
TNBC2 cells. In order to investigate this, loops which restore sensitivity to lapatinib
we examined the effect on viability and in the resistant TNBC2 cells.
Materials & Methods
Cell Culture and Reagents
Two TNBC cell lines: TNBC1 and TNBC2, MK-2206 and lapatinib 1 mM stock-
were obtained from the division of Drug solution were obtained from Selleckchem
Discovery and Safety from Leiden and were both stored in aliquots at -20° C.
Academic Centre for Drug Research and The stock solutions of the two inhibitors
maintained in RPMI 1640 medium were diluted to 2 µM. This dilution was
(ThermoFisher Scientific) supplemented once again diluted by adding 0.1% DMSO
with 10% fetal bovine serum (Sigma to each well, resulting in a 1 µM inhibitor-
Aldrich) and penicillin-streptomycin 10,000 concentration. Both plates were put back
U/mL (ThermoFisher Scientific) in an in the incubator.
atmosphere containing 5% CO2 at 37° C.
SRB viability assay
Cell plating
50% trichloroacetic acid was added to the
The medium of the two cultured cell lines wells containing cells and the plate was
was removed and were washed with PBS. put in the fridge at 4° C for an hour. The
0.25% trypsin was added. Flasks were TCA solution was removed from the wells
returned to the incubator for 5 minutes. and washed with demi-water.
Complete RPMI medium was added to
0.4% SRB solution was added in each well
both flasks. 10 µL of both cell suspensions
and brought to a shake for 30 minutes. The
were added to a TC10 slide and put into a plate was washed with 1% acetic acid. 10
cell counter (BIO-RAD TC20). The mM Tris was added and brought to a shake
solutions were diluted with medium to for 10 minutes after which the absorbance
1500 cells/well (TNBC1) and 1000 was measured with Powerwave XS2
cells/well (TNBC2). The final solution was (Biotek).
added to the wells in both plates. PBS was
added to the empty wells. The plates were KI67 proliferation assay
returned to the incubator for one night. Medium from the plate was removed and
Inhibitor exposure wells were washed with PBS. 4%
PFA/0.01% Triton-X solution was added to
the wells and left to incubate for 15 minutes.
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