Practical Cellular Biochemistry
Increment of SOX9 and JAK in gene expression is
associated with resistance development against the
combinatorial therapy of Lapatinib and Rapamycin
Bio-pharmaceutical science, Leiden University, 2333 AL Leiden, The Netherlands
Submitted on December 13, 2022.
1
,Abstract
Triple-negative breast cancer (TNBC) is one of the small subtypes of breast cancer that
causes the majority of breast-cancer related deaths. The clinical efficacy has been limited due
to resistance development of the tumor cells. In order to comprehend this resistance
conundrum, we investigated the role of lapatinib and rapamycin on cell proliferation
promoted by the MAPK/ERK- and PI3K/AKT pathways in the TNBC cell lines.
TNBC1 and TNBC2 cell lines were used in this study to investigate the role of lapatinib and
rapamycin as a mono- and combinatorial treatment. Sulforhodamine B (SRB) assay was
executed to determine the cell viability, and the cell proliferation was measured by the
immunofluorescence (IF) assay. A western blot assay was performed to inspect the inhibition
of phosphorylation of AKT and ERK by the combinatorial treatment. Followed by the qPCR
assay that investigates the relative gene expression of AKT3, AXL, JAK and SOX9.
The analysis revealed that although the phosphorylated AKT and ERK were inhibited by the
combinatorial treatment, the cell proliferation of TNBC1 remained the same in comparison
with the control group. Our results showed that JAK and SOX9 were highly expressed when
the combinatorial treatment was used in both cell lines. Rapamycin inhibits the mTOR in the
PI3K/AKT pathway and lapatinib inhibits the receptor tyrosine kinase HER2 and EGFR.
Combining the two inhibitors resulted in a negative effect of cell viability, but the cell
proliferation remained unchanged in one of the TNBC cell lines. Increment of relative gene
expression of JAK and SOX9 may represent a mechanism of developed resistance to the
inhibitors.
Introduction
Breast cancer is the world’s most common malignancy and the most common invasive
cancer in women, responsible for over 40,000 deaths in the United States p.a. [1,2].
Breast cancer has several subtypes, of which triple-negative breast cancer (TNBC) is one of
the small subtypes that constitutes for 10-20% of all the different subtypes of breast cancer.
But regardless of being a small percentage of all subtypes, TNBC causes the majority of
breast-cancer related deaths. [3,4].
Epidermal growth factor receptor (EGFR) and human epidermal growth factor 2 (HER2)
are transmembrane receptor tyrosine kinases (RTKs) and are part of the ErbB family amongst
all RTKs (HER1/EGFR; HER2; HER3; HER4) [5]. The EGFR and HER2, activated by
epidermal growth factor (EGF), heterodimerizes with other RTKs of the ErbB family which
leads to cross-phosphorylation of the c-terminus tyrosine residues, that results in a cascade of
downstream signaling and end in an accrued cell growth and proliferation [5-9]. The RTKs in
breast cancer cells are usually overexpressed, which is a crucial hallmark to keep proliferating
the breast cancer cells. For instance, the overexpression of HER2 or amplification of the
ERBB2 gene often occurs in other human malignancies, like ovarion, stomach and uterine
cancers, and results in a poor clinical outcome [10, 12, 13].
There are many different signaling pathways a cancer cell can activate, when exposed to a
growth factor, such as EGF, Heparin-binding EGF (HB-EGF) or Amphiregulin (AREG). One
of the pathways is the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of the
rapamycin (mTOR) signaling pathway. This pathway is used in different types of cancer and
regulates a variety of cellular mechanisms, such as proliferation, growth, survival,
metabolism, metastasis and angiogenesis [14-17]. An HER2-HER3 dimer is an essential
dimer to activate the PI3K/AKT signaling pathway, which in other solid tumors, like the
breast cancer tumor, is an important signaling unit to utilize in order to generate oncogenic
activities [11]. For HER3 and EGFR is HER2, the desired RTK to form a dimer receptor
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, complex, which amplifies the signal for the downstream cascade [7]. Among all the
intracellular signaling pathways, the mitogen-activated protein kinase (MAPK) plays a more
important role in controlling a variety of cellular mechanisms like cell growth, development,
division and death. The extracellular signal-regulated kinase (ERK) is a conserved, ubiquitous
serine/threonine kinase, that is a member of the MAPK family. It regulates cellular signaling
and forms the core of the signaling network of the MAPK/ERK pathway [18,19].
Lapatinib is an FDA-approved, small-molecule dual inhibitor of the HER2 and EGFR and
is used for advanced-stage HER2+ breast cancers [20]. Lapatinib has been an effective
treatment of HER2-positive breast cancer cells, but the clinical effectiveness of it has been
limited due to the inevitable acquired resistance of the breast tumor cells [20,21]. The
mechanisms of the acquired resistance are reported in a number of articles [5, 22-28].
The mammalian target of rapamycin (mTOR) on the other hand, is a central regulator of
cell growth and proliferation. The mTOR is a conserved serine/threonine protein kinase, and it
regulates growth and proliferation by receiving multiple signals of growth factors and
nutrients signals [29,30]. Even though mTOR-targeted inhibitors, like rapamycin, do delay
progression and extend survival of tumor cells, TNBC patients will ultimately develop
resistance to the mTOR inhibitors with unsought clinical outcome [31,32] There are a few
articles that shows that rapalog treatment has the possibility to release mTOR negative
feedback on upstream kinases and uses other signaling pathways, like the PI3K/AKT and
MAPK/ERK pathways, to bypass mTOR inhibition [33-35].
Even though the mono-targeted therapies above and more have been proven to be effective
for the treatment of breast cancer, there is a great possibility of developing intrinsic or
acquired resistance. Since TNBC cell lines utilizes multiple signaling pathways and axes to
stimulate cell proliferation, thus mono-targeted therapies are not sufficient in the
long-term to control TNBC. [21,33,36] Therefore, there must be an alternative combinatorial
treatment for TNBC. To comprehend the resistance conundrum, it is critical to understand the
role of the combination treatment of lapatinib and rapamycin on cell proliferation promoted
by the MAPK/ERK and PI3K/AKT pathway in TNBC cell lines.
For this reason, a combinatorial treatment for TNBC, which includes lapatinib and
rapamycin that specifically inhibits parts of the MAPK/ERK and PI3K/AKT pathway, was
made to reduce tumor growth, cell proliferation and cell viability. In order to comprehend the
effect of the combinatorial treatment, it is crucial to investigate the reaction of the TNBC cells
on the treatment on a gene expression level of AKT3, AXL, JAK and SOX9.
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