1. m
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-135a
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verexpression
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bled
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ock
U
nstim
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miR-135a overexpression decreases CXCL10 mRNA expression (n=3)
CXCL10expressionrelativetoScrambled
Investigating CXCL10 as a potential target of miRNA-135a in breast cancer
Hannah Cho1,2, Minji Lee1,3, Irene L. Andrulis1,2,3
INTRODUCTION
Patients with axillary lymph node negative (ANN) breast cancer (BC)
generally have a good prognosis, but 20-30% will have recurrence. To
identify biomarkers that prognostically distinguish ANN BC patients, we
have been studying characteristics of breast tumours and their
microenvironment.
Tumour-infiltrating lymphocytes (TILs) that express T-bet (T-box
transcription factor 21) are associated with poor prognostic features such
as high grade, hormone receptor negativity and basal subtype; however,
they are associated with a positive outcome in ANN BC.
To gain insights into the molecular profiles associated with T-bet+ TILs, we
identified various differentially-expressed mRNAs and miRNAs in T-
bet+/high and T-bet-/low breast tumours. One miRNA, miR-135a, was
shown to be downregulated in T-bet+/high tumours (p=3.29x10-5).
Interestingly, in silico target analysis has revealed miR-135a to potentially
target and suppress the expression of the chemokine, CXCL10 (IFNγ-
inducible protein 10). We have previously determined CXCL10 to be
associated with a high level of TILs in breast tumours and to increase in
vitro T cell migration.
RESULTS
HYPOTHESIS & OBJECTIVES
Lunenfeld-Tanenbaum Research Institute, Sinai Health System1, Dept. of Molecular Genetics, University of Toronto2, Dept. of Laboratory Medicine and Pathobiology, University of Toronto3
I would like to acknowledge the members of the Andrulis Lab for allowing
this project to happen, and for the support they have provided.
Dr. Irene Andrulis Nalan Gokgoz Lucine Collins
Dr. Andrew Seto Dr. Gesseca Gos Justin Mayers
Minji Lee Junghyun Nam
ACKNOWLEDGEMENTS
REFERENCES
DISCUSSION
FUTURE STEPS
We hypothesize that miR-135a may target CXCL10 in breast cancer by
examining:
1. The change in CXCL10 mRNA expression upon miR-135a
overexpression
2. The change in CXCL10 protein secretion upon miR-135a
overexpression
Real-Time qPCR
To demonstrate miR-135a
overexpression decreases
CXCL10 mRNA expression
METHODS & MATERIALS
Sandwich ELISA
To determine miR-135a
overexpression decreases CXCL10
protein secretion
T-bet HIGH
T-bet LOW
FIG. 1 (ABOVE) :
A) BC tissues were immunohistochemically-stained and quantified for T-
bet+ TILs.
B) T-bet+ TILs associate with a positive outcome in BC.
(Mulligan et al. 2016 Cancer Immunol Res2)
A B
FIG. 2 (LEFT) :
The top 5 differentially
expressed (DE) miRNAs in T-
bet+/high versus T-bet-/low
ANN BC tumours are shown.
*** p < 0.001
**** p < 0.0001
The data collected at both the mRNA and protein levels show that
miR-135a levels affect CXCL10 secretion.
This suggests that miR-135a may regulate CXCL10 expression in breast
cancer. This relationship may possibly explain why ANN BC patients with
T-bet+/high have better survival, as T-bet+/high patients have elevated
levels of the CXCL10 chemokine, and decreased levels of miR-135a.
The potential miR-135a-CXCL10 axis could be a novel site of prognosis
and treatment for ANN BC patients, and could be applicable to all basal
breast cancers due to the relatively homogenous nature of this subtype.
Since basal breast cancers have distinctive microenvironments and gene
expression, they display unique stromal-epithelial interactions. They
express significantly differential expression of interleukins and
chemokines, including other members of the CXC-chemokine family, as
well as a high correlation with the triple-negative receptor definition.
This results in a necessity for highly specific research to be conducted for
successful immune therapy of this disproportionally fatal breast cancer.
Western Blot
To validate the existence of the miR-135a-CXCL10 axis
at the protein level.
Luciferase Assay
To determine if CXCL10 is the direct target of miR-135a.
Transwell Migration
To examine the role of the miR-135a-CXCL10 axis in
influencing T cell migration.
mRNA expression
miR-135a overexpression resulted in
decreased levels of CXCL10 mRNA
expression.
Protein expression
miR-135a overexpression resulted in
decreased levels of CXCL10 chemokine
secretion.Overexpression of miR-135a decreases CXCL10 secretion
m
iR
-135a
O
verexpression
Scram
bled
M
ock
Unstim
ulated
0.0
0.1
0.2
0.3
0.4
CXCL10(ng/ml)
**
FIG. 3 (ABOVE) :
Real-Time qPCR of the cell line MDA-MB-231
determined that miR-135a overexpression
significantly affected the CXCL10 mRNA
expression as a ratio relative to scrambled.
(p = 0.0142, n = 3)
FIG. 4 (ABOVE) :
Sandwich ELIZA was done complementary
to the Real-Time qPCR experiments. They
determined that miR-135a overexpression
significantly affected the CXCL10
chemokine secretion as a ratio relative to
scrambled.
(p = 0.0096, n = 3)
In vitro study of miR-135a-CXCL10 axis
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in CD4 and CD8 T cells. Science 2012: 338-342.
2.Mulligan AM, et al. Validation of Intratumoral T-bet+ Lymphoid Cells as Predictors of Disease-
Free Survival in Breast Cancer. Cancer Immunology Research 2016; 4(1): 41-48.
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2013;494:361-365
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Immunology 2010; 40(1): 225-231.
8.Chen Y, et al. miRNA-135a promotes breast cancer cell migration and invasion by targeting
HOXA10. BMC Cancer 2012; 12:111.
9. Mulligan AM, et al. Tumoral lymphocytic infiltration and expression of the chemokine CXCL10
in breast cancers from the Ontario Famiial Breast Cancer Registry. Clin Cancer Res
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10. Bertucci F, et al. Basal Breast Cancer: A Complex and Deadly Molecular Subtype. Curr Mol
Med. 2012 Jan; 12(1): 96–110.
FIG 3 (ABOVE) :
In vitro cultures of
the basal cell line
MDA-MB-231.