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ACTIONS OF L-THYROXINE (T4)
AND NANO-DIAMINO-TETRAC
(NDAT, NANOTETRAC) ON PD-L1
IN CANCER CELLS
Paul J. Davis, Hung-Yun Lin,
Shaker A. Mousa
Albany Medical College,
Albany, NY, USA;
Pharmaceutical Research
Institute, Albany College of
Pharmacy and Health
Sciences; Taipei Medical
University, Taipei, Taiwan
The PD-1 (programmed death1)/PD-L1 (PD-ligand 1) checkpoint
is a critical regulator of activated T
cell-cancer cell interactions, serving
to defend tumor cells against (T
cell-mediated) immune destruction.
Pharmaceutical interest is high in
PD-L1 antibody use in solid tumor
chemo-therapy to render cancer
cells susceptible to host killer T cell
action. We have developed a nonimmunological strategy for
downregulation of PD-L1 gene
expression and PD-L1 protein
content in tumor cells.
• The non-immunologic strategy is
based on pharmacologic regulation
of a target on the extracellular
domain of plasma membrane
integrin avb3. This target is a
thyroid hormone-tetraiodothyroacetic acid (tetrac) receptor that
controls—from the cell surface—
the expression of a panel of cancer
cell defense genes, including PDL1.
-I
-
-I
3’
HO
3
NH2
CH2-CH-COOH
O
5’
5
-I
-
-I
-
Thyroxine (T4)
-I
-
-I
3’
3
CH2-CH-COOH
O
5’
NH2
5
-I
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3,5,3’-Triiodothyronine (T3)
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3’
HO
3
CH2--COOH
O
5’
5
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Tetrac
Low-grade thyromimetic within cells
TH antagonist at integrin avb3 TH
receptor
I
O
O
N
H
N
H
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O
O
I
I
OH
PLGA
nanoparticle
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O
H
H
N
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O
O
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I
I
O
OH
In Nanotetrac, shown here, tetrac is
covalently bound to a linker (ether bond)
which, in turn, is amide-bonded to a PLGA
nanoparticle. The action of tetrac is
limited in this formulation to the thyroid
hormone-tetrac receptor on the extracellular domain of integrin avb3.
Figure 1
• Human triple-negative breast
cancer (MDA-MB-231) cells and
human colon cancer (HCT116. HT29) cells were cultured in DMEM
(breast) or RPMI-1640 (colon),
each with 10% FBS. Two days
prior to study of cells, 0.25%
charcoal-stripped serum replaced
10% FBS.
• Cells were treated with L-thyroxine
(T4, 10-7 M total hormone, 10-10 M
free), NDAT (10-7 M tetrac
equivalent) or both for 24 h.
• Tumor cell RNA was harvested
and PD-L1 mRNA quantitated
by qPCR.
• PD-L1 protein was measured
by western blotting.
MDA-MB 231 cell mRNA abundance
A.
B.
Figure 2
MDA-MB 231 cell PD-L1 protein content
A.
B.
50 kDa -
◄ PD-L1
36 kDa -
◄ GAPDH 36 kDa -
50 kDa -
2.7-fold increase
25-35% decrease in
content with NDAT
Figure 3
◄ PD-L1
◄ GAPDH
HCT116 cell mRNA
A.
B.
Figure 4
HCT116 cell PD-L1 protein
A.
B.
50 kDa -
◄ PD-L1
36 kDa -
◄ GAPDH
50 kDa -
◄ PD-L1
36 kDa -
◄ GAPDH
25-60% decrease in
basal or stimulated
content with NDAT
Figure 5
HT-29 cell mRNA
A.
B.
Figure 6
HT-29 cell protein
A.
50 kDa -
36 kDa -
B.
◄ PD-L1
50 kDa -
◄ PD-L1
36 kDa -
◄ GAPDH
◄ GAPDH
40% decrease in
basal or stimulated
content with NDAT
Figure 7
Dependence on MAPK of
induction by T4 of PD-L1 in
cultured HCT116 cells
- PD98059
NDAT (10-7 M)
T4 (10-7 M)
-
+
-
-
+
+ PD98059
+
+
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+
-
-
+
+
+
50 kDa -
◄ PD-L1
36 kDa -
◄ GAPDH
Figure 8
SUMMARY
• In MDA-MB-231 breast cancer
cells, T4 significantly stimulated PDL1 gene expression by 40% and
increased PD-L1 protein 2.7-fold;
these effects were blocked by
NDAT.
• In HCT116 and HT-29 colon
carcinoma cells, T4 significantly
increased PD-L1 gene expression
by 20-60% and protein abundance
by 25-65%; these effects were
blocked by NDAT.
• Basal levels of mRNA and protein
were also reduced by NDAT.
• MAPK mediates the T4 effects.
CONCLUSIONS
• The PD-1/PD-L1 defensive
tumor cell checkpoint is T4 responsive. Host patient T4
supports this cancer cell
defense.
• Hormonal effects vary among
cell lines.
• NDAT eliminates the
contribution of T4 to the
checkpoint and also variably
reduces basal levels of PD-L1.