Inhibiting C-Jun to Retard Cell Proliferation Promoted by AP-2β in

the Breast Cancer

Benyu Yang

School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, U.K.

Keywords: AP-2β, Breast Cancer, C-Jun.

Abstract: AP-2β is a molecular marker in breast cancer cells without too much attention before. A tight association

between AP-2β overexpression and breast cancer was reported. This study investigates whether c-jun is the

certain down-stream protein of AP-2β in regulating the cell proliferation in breast cancer. The expression of

AP-2β in human cell line will be measure by Western Blot. Tumor size in vivo will be measured by volume.

The possible results include: the tumor growth of positive control is slower than negative control group, the

tumor is increasingly faster growing compared with negative control, or the tumor is growing in the same

speed in both negative and positive control groups. The result will indicate whether C-jun is a key down-

stream target for mediating the tumor-promoting role of AP-2β.

1 INTRODUCTION

The breast cancer is one of the most threatening type

of cancers for women worldwide. The most

significant four subtypes are: Luminal A or

HR+/HER2- (HR-positive/HER2-negative), Luminal

B or HR+/HER2+ (HR-positive/HER2-positive),

Triple-negative or HR-/HER2- (HR/HER2-negative)

and HER2-positive. (American Cancer Society. 2019)

There are many additional molecular factors

included in developing breast cancer. Among them,

the activator protein-2 family of transcription factor

is very common. They have five members: AP-2α, -

2β, -2γ, -2δ, -2ε. They are encoded by separate genes

(TFAP2A, TFAP2B, TFAP2C, TFAP2D, TFAP2E).

They are thought to bond with specific DNA

sequence as an activator or repressor to stimulate or

terminate the growth process of cells. (Turner et al

1998) Regarding breast cancer, most studies focus on

AP-2α and AP-2γ. The expression of AP-2β in breast

cancer has only started to be noticed and studied in

recent years.

The tight association of AP-2β with breast cancer

has been observed. (Raap et al 2018) AP-2β is a new

mammary epithelial differentiation marker and its

over expression leads to cell proliferation in breast

cancer. With knockdown of AP-2β ， the cell

proliferation is downregulated. But at the same time,

in the previous experiment, proteins included in

developing the breast cancer also get down-regulated

expression such as p75, MMP-2, MMP-9, C-Jun, p-

ERK and STAT3. The expression levels of p75,

MMP-2, MMP-9, C-Jun, p-ERK and STAT3 show

obvious upregulation following overexpression of

AP-2β. (Li, Xu, Luo, Hao, Zhao, Yu et al 2018) To

investigate more about AP-2β underlying mechanism

in developing breast cancer, I hypothesize the c-jun is

a key downstream protein of AP-2β in regulating cell

proliferation. The possible signaling pathway

involving C-Jun is shown in Figure 1. AP-2β is the

transcription factor that contacts the promoter or

enhancer to regulate transcription. C-Jun is supposed

to be a member of downstream pathway in the

hypothesis. The complete downstream pathway is

still not fully understood. AP-2β may be regulate the

process via MEK/ERK/c-jun pathway indicated by

previous study, or maybe through PI3K/Akt pathway

which is also indicated in previous study, or actually

via various pathways together (See Figure 2).

(MAKOTO, Toru, Masaki, Koichi 2011) The

receptors that are responsible for transducing signals

are still under investigation. The estrogen-receptor is

already proven to be independent from expression of

AP-2β. (Raap et al 2018), However, other receptors

included in breast cancer, such as progesterone

receptors, HER2 receptors and EGFR receptors are

still being studied to confirm whether they are

associated with the expression of AP-2β.

302

Yang, B.

Inhibiting C-Jun to Retard Cell Proliferation Promoted by AP-2 in the Breast Cancer.

DOI: 10.5220/0011361100003438

In Proceedings of the 1st International Conference on Health Big Data and Intelligent Healthcare (ICHIH 2022), pages 302-307

ISBN: 978-989-758-596-8

Figure 1: C-Jun Pathway. This figure shows the position and activities of C-Jun in the AP-2β oncogenic pathway.

Figure 2: RAF/MEK/ERK signalling pathway is shown. Growth factors promoting cell proliferation activate the

RAF/MEK/ERK pathway. MEK regulates the intermediate signalling by phosphorylation and activation of the downstream

ERK molecule. ERK regulates cellular activity, indirect inducers of gene expression and transcription factors of the AP-1

family, such as c-JUN and c-FOS. (MAKOTO, Toru, Masaki, Koichi 2011)

2 METHODS

2.1 Materials

Human breast cancer cell line MDA-MB-231

Xenograft mouse models

C-jun inhibitor JNK-JN-8 (Ebelt ND et al 2017)

2.2 In vitro Cell Culture

The cell line is cultured in RPMI-1640 medium

supplemented with 10% fetal calf serum, 10 μg/ml

bovine insulin, 2.5 g/l glucose, 1 mM sodium

pyruvate, 2 mM glutamine, and 10 mM HEPES, in a

water-saturated atmosphere containing 5% CO2 at

37.5°C. (Raap et al 2018) MDA-MB-231 cells will be

divided into two groups: negative control and positive

control. In the negative control group, the cells will

be injected with saline. In the positive control group,

the cells will be injected with c-jun inhibitor (JNK-

IN-8). The results will be analyzed by observing the

cell proliferation.

2.3 Flow Cytometry

The cell proliferation will be measured through flow

cytometry system by ThermoFisher Scientific. The

Inhibiting C-Jun to Retard Cell Proliferation Promoted by AP-2 in the Breast Cancer

303

cell line sample will be measured every 2 days after

injecting JUN-JN-8.

2.4 AP-2β Western Blot

Total cellular proteins are separated by 12% SDS-

PAGE and transferred to nitrocellulose membranes.

Membranes are probed with anti-AP-2β (H-87,

1:1000, Santa Cruz Biotechnology) (Raap et al 2018).

2.5 Animal Model

The xenograft mice will be divided into two groups

of 4: negative control and positive control. For the

mice in the negative control group, the mice will be

injected with saline. For the positive control group,

MDA-MB-231 cells will be injected subcutaneously

into the left flank of each mouse in the same volume

as the saline injected in the mice in negative control

group. The tumor growth will be monitored every

four days after the tenth day of injecting MDA-MB-

231 cells by measuring the tumor size. The tumor’s

size will be measured in the terms of volume and the

calculation equation is V= (width × length) /2. The

tumors will be weighed after death. (Li, Xu, Luo,

Hao, Zhao, Yu et al 2018)

2.6 Statistics

The western blot and immunohistochemistry will be

repeated three times for each group. To compare data

obtained in the positive and negative control groups,

a student t test will be displayed and P ≤0.05 is

considered significant. (Li, Xu, Luo, Hao, Zhao, Yu

et al 2018)

3 RESULTS (OVERVIEW SHOWN

IN TABLE 1)

3.1 Possible Results 1: Applying

JUN-JN-8 Inhibits the Breast

Cancer Cell Proliferation in

MDA-MB-231 Cell Line and the

Tumor Cells in Mouse Model

Inhibiting c-jun leads to significantly slower cell

proliferation in the breast cancer cells and the stop or

much slower growth of the tumor in the mice of

positive control group.

3.2 Possible Result 2: Applying

JUN-JN-8 Inhibits the Breast

Cancer Cell Proliferation in

MDA-MB-231 Cell Line, But Not

the Tumor Cells in Mouse Model

Inhibiting c-jun leads to no effect on tumor growth

although the cell proliferation is reduced in the In

Vitro cell culture experiment.

3.3 Possible Result 3: Applying

JUN-JN-8 Inhibits the Breast

Cancer Cell Proliferation in

MDA-MB-231 Cell Line, but

Promote the Breast Cancer Cell

Proliferation in Mouse Model

Inhibiting c-jun leads to confusingly much quicker

enlargement of the tumor in the condition that the cell

proliferation is inhibited in the breast cancer cells of

In Vitro conditions.

3.4 Possible Result 4: Applying

JUN-JN-8 Inhibits the Breast

Cancer Cell Proliferation in Mouse

Model, But Not the MDA-MB-231

Cell Line

Injecting c-jun inhibitor (JNK-IN-8) do not show

inhibiting effect on breast cancer cell proliferation in

the cell line. However, when MDA-MB-231 cells that

are injected with JNK-IN-8 are injected into the

mouse, the tumor growth speed is slowing down.

3.5 Possible Result 5: Applying JUN-

JN-K Does Not Inhibit the Breast

Cancer Cell Proliferation in Both

MDA-MB-231 Cell Line and the

Mouse Model

Injecting Both In vitro and In vivo experiments show

that injecting c-jun inhibitor has no effect on

inhibiting the breast cancer cell proliferation and

tumor growth.

ICHIH 2022 - International Conference on Health Big Data and Intelligent Healthcare

304

Table 1: Possible Results.

Cell lines Result 1 Result 2 Result 3 Result 4 Result 5 Result 6

In vivo Model + - ? + - ?

MDA-MB-231 + + + - - -

Note. "+" represents a a significant decrease in cell proliferation/tumor growth speed of mouse. "-" represents

not significantly different from negative control.

3.6 Possible Result 6: Applying

JUN-JN-8 Does Not Inhibit the

Breast Cancer Cell Proliferation in

MDA-MB-231 and Promote the

Breast Cancer Cell Proliferation in

Mouse Model

In the condition of no difference shown in the

inhibiting cell proliferation compared with negative

control group in the MDA-MB-231, the mouse shows

quicker tumor growth.

4 DISCUSSION

Previous studies have found that silencing AP-2β

leads to downregulation of the expression of a

number of proteins, including c-jun. Possible

downstream pathways are MEK-ERK-c-jun and

MEK/STAT3/MMPs. It is therefore reasonable to

assume that c-jun may be a direct downstream protein

of AP-2β or one of these direct proteins.

Possible Result 1 is consistent with previous

studies that downregulation of AP-2β led to a

downregulation of c-jun protein levels. Previous

studies have shown that the amount of c-jun protein

is influenced by AP-2β. C-jun regulation by AP-2β

most likely promotes tumor cell proliferation through

the MEK/ERK/c-jun signalling pathway, indicated by

previous experiment.

Possible Result 2 contradicts the hypothesis. C-

jun is inhibited by JUN-JN-8 in in vitro human breast

cancer cell line and cell proliferation is also inhibited,

suggesting that c-jun is one of the potential direct

downstream proteins. However, tumor cell

proliferation is not inhibited in the mouse model,

suggesting that there are other signalling pathways at

work in the mouse that disable the inhibition of c-jun

and thus do not inhibit the regulation of AP-2B, or

that there are other AP-2B downstream proteins at

work in the mouse that counteract the proliferative

effects of c-jun inhibition.

Possible Result 3 contradicts the hypothesis. The

inhibition of the cell proliferation process can only

suggest that c-jun can play a role in mediating cell

proliferation, but cannot be characterized as a direct

downstream protein of AP-2β. As there are other

potential direct downstream proteins that mediate the

tumor-promoting effects of AP-2β, the underlying

mechanisms are unclear. In previous studies, when

AP-2β was downregulated, the number of many other

proteins was also reduced, such as MMP9, MMP2

and p75 shown in Figure 3. (Li, Xu, Luo, Hao, Zhao,

Yu et al 2018)

Possible Result 4 are contradictory to the

hypothesis, as they do not provide clear evidence to

identify c-jun as a direct downstream target of AP-2β.

Tumor growth in mice is shown to be inhibited when

no inhibition is shown on MDA-MB-231. This may

demonstrate that c-jun has no direct role in mediating

the cell proliferation pathway, but that it can signal to

other downstream proteins that promote the tumor

pathway of AP-2β.

Possible Result 5 strongly suggests that c-jun is

not a direct downstream protein of AP-2β. and that it

has no therapeutic effect on breast cancer.

Possible Result 6 shows an unlikely result, but it

is also possible that it will emerge in future

experiments. Cell proliferation will not be inhibited

when c-jun inhibitors are injected into breast cancer

cell lines. This is not evidence to determine whether

c-jun is a direct downstream protein of AP-2β, which

may have multiple signalling pathways. However, in

mice injected with c-jun inhibitor-treated MDA-MB-

231, tumor growth will become more pronounced, a

possible outcome that is very strange and if it occurs

more research is needed to explore the mechanisms

involved.

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Figure 3: Expression levels of P75, MMP2, MMP9, C-Jun, STAT3 measured by Western blot in MDA-MB-231 cells

following AP-2β knockdown. (Li, Xu, Luo, Hao, Zhao, Yu et al 2018)

5 CONCLUSIONS

C-jun may be an important protein in the downstream

pathway of AP-2β during tumour growth in breast

cancer as experiments have shown that c-jun

expression is closely associated with AP-2β. Overall,

this article discusses the downstream pathway

component of AP-2β in the mechanisms underlying

the promotion of breast cancer cell proliferation and

uses in vitro cell lines and mouse models to determine

whether c-jun is one of the important downstream

proteins. Our findings will indicate whether c-jun

could be a potential target for blocking breast cancer

tumour growth. Results that contradict the hypothesis

would also indicate the existence of other

downstream protein proteins regulated by AP-2β, in

both respects a confirmation of potential therapeutic

targets in breast cancer in the future. As no previous

attention has been paid to the role of AP-2β in breast

cancer, research into its complete mechanism in

breast cancer progression is limited. More research on

how AP-2β regulates cell proliferation still needs to

be clearly detailed in order to find more effective

targets for the treatment of breast cancer.

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