1. The Regulation of APLP2 Intra Cellular Domain on Its Target Genes
Ge Zhou1,2, Samuel E. Harvey2, Chonghui Cheng2
(1) Master of Biotechnology Program of Northwestern University
(2) Robert H. Lurie Comprehensive Cancer Center of Northwestern University
Methods
Results
Conclusion
Objectives
Background
Future Directions
•We could make the stable APLP2 isoform over-expression
cell lines to further test the A2ICD effect on its target genes.
•We need to propose a potential cell signal pathway to explain
how would the A2ICD regulate its target genes and how does
this contribute to breast cancer progression.
•The amyloid precursor protein family includes amyloid
precursor protein (APP), amyloid precursor-like protein1
(APLP1), and amyloid precursor-like protein 2 (APLP2).
This family has been reported to be involved in many
cellular process like development, transcription,
apoptosis, metabolism, and the cell cycle.
•Studies of APP found that it undergoes serial cleavage
by secretases and produces an APP Intra-Cellular
Domain (AICD) that enters the nucleus and is
hypothesized to function as a transcription factor. Some
of these target genes are linked to cancer progression.
Fig.1 The general view of how would AICD be produced in
cells2.
•The mRNA of APP protein family undergoes alternative
splicing. For APLP2 mRNA, the exon 7 could be
retained or spliced, which generates two different
isoforms, the long one with exon 7 and the short one
without.
•Would APLP2 Intracellular Domain (A2ICD) have the
same function as APP; Would different isoforms affect
the function of APLP2 Intra Cellular Domain? How would
APLP2 Intracellular Domain contribute to cancer
progression? These questions still need to be answered.
•To determine which genes would A2ICD regulate.
•To determine if different isoforms of APLP2 regulate
target genes differently.
•To conclude how would the regulation of A2CID on its
target genes affect breast cancer progression.
Breast cancer cell lines:
• APLP2E7 knockdown
•APLP2ΔE7 knockdown
•APLP2 knockdown
RNA Extraction
RT-PCR for cDNA
Analysis of Target Genes Expression
0
1
2
3
4
5
6
7
P2 D1 P4 D1 P6 D1 P2 MME P4 MME P6 MME
shluc
sh7
sh6-8
sh
3'UTR
qPCR
Fig.3 D1, MME expression in different passages (P2, P4, P6) of
APLP2 knockdown MCF10a cell lines. In different passages of
MCF10a cell lines, MME is always up-regulated in the APLP2E7
knockdown cell line and down-regulated in the others, while Cyclin D1 is
always up-regulated in APLP2ΔE7 knockdown and down-regulated in the
others.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
p1 sh7 p1 sh6-8 p1 sh
3'utr
p2 sh7 p2 sh6-8 p2 3'utr p3 sh7 p3 sh6-8 p3 s'utr p4 sh7 p4 sh6-8 p4
shs'utr
p6 sh7 p6 sh6-8 p6
sh3'utr
aplp2 L
aplp2 S
aplp2 T
Fig.2: MCF10a Knockdown Efficiency Check. A. Knockdown efficiency
check of each passage by qPCR. Expression of APLP2E7 and
APLP2ΔE7 recover through the passage. B. Knockdown efficiency
check by western blot for the second passage.
0
0.5
1
1.5
2
2.5
D1 MME
shluc
sh7
sh6-8
sh3'utr
Fig.4 Expression of D1 and MME in HMLE APLP2 knockdown cell
line. In HMLE cell line, expression of Cyclin D1 is consistent with
MCF10a cell line. However, expression of MME is not.
0
0.5
1
1.5
2
2.5
3
3.5
D1 MCF10a D1 HMLE D1 231 MME MCF10a MME HMLE
shluc
sh6-8
Fig.5 D1 and MME expression in different breast cancer cell lines.
The expression of D1 is consistent in different cell lines, but MME varies.
In some cell lines, such as 231 and Hela, the expression of MME is
undetectable.
• By screening all the target genes of AICD in MCF10a
cell lines, we found that only MME and Cyclin D1 shows
differential expression in APLP2E7 and APLP2ΔE7
knockdown cell lines.
• When we test further in other breast cancer cell lines,
we found that only the expression of Cyclin D1 remains
consistent in different cell lines, where APLP2E7 up-
regulates D1 expression while APLP2ΔE7 down-
regulates its expression. Even though, there isn’t a
consistent MME expression trend, we still observe a
significant switch between different APLP2 isoform
knockdowns in different cell lines.
• MME could inhibit angiogenesis in tumor cells4. Cyclin
D1 could activate the cell cycle, which may contribute to
tumor growth5. From the results, we could make the
hypothesis that A2ICD could affect cell cycle and which
may provide a new target for breast cancer treatment.
Reference
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Maeda M, Furuyama H, Kondo Y, Imamura M.Arii, (2000),
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5. Checler Pardossi-Piquard and Frédéric Raphaëlle, (2011), The
physiology of the β-amyloid precursor protein intracellular
domain AICD., Journal of Neurochemistry, 120: 1471-4159.
3
Acknowledgement
This work was supported by Chonghui Cheng’s lab of
Robert H. Lurie Comprehensive Cancer Center as well
as the Master of Biotechnology program of
Northwestern University.
A.
APLP 2
GAPDH
shluc
sh7
Sh6-8
Sh
3’utr
B.