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A-27 Phospholipase Cγ1 represses colorectal cancer growth

by inhibiting the Wnt/β-catenin signaling axis
Hyun-Jun Jang
Department of Biological Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919,
Republic of Korea
Abstract
As essential phospholipid signaling regulators, phospholipase C (PLC)s are activated by various extracellular ligands and mediate intracellular signal
transduction. PLCγ1 is involved in regulating various cancer cell functions. However, the precise in vivo link between PLCγ1 and cancer behavior remains
undefined. To investigate the role of PLCγ1 in colorectal carcinogenesis, we generated an intestinal tissue-specific Plcg1 knock out (KO) in adenomatous
polyposis coli (Apc) Min/+ mice. Plcg1 deficiency in ApcMin/+ mice showed earlier death, with a higher colorectal tumor incidence in both number and size than
in wild-type mice. Mechanistically, inhibition of PLCγ1 increased the levels of its substrate phosphoinositol 4,5-bisphosphate (PIP2) at the plasma membrane
and promoted the activation of Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) to enhance β-
catenin signaling. Enhanced cell proliferation and Wnt/β-catenin signaling were observed in colon tumors from Plcg1 KO mice. Furthermore, low PLCγ1
expression was associated with a poor prognosis of colon cancer patients. Collectively, we demonstrated the role of PLCγ1 in vivo as a tumor suppressor
relationship between the regulation of the PIP2 level and Wnt/β-catenin-dependent intestinal tumor formation.the National Research Foundation of Korea (NRF)
grant funded by the Korea government (2020R1I1A1A01074940)
Results

Fig. 3. PLCγ1 depletion enhances cell proliferation in cancer cells with high
β-catenin expression. (a) Correlation between the PLCγ1 (PLCG1) and β-
catenin (CTNNB1) mRNA expression levels in human CRC cell line using
the CCLE database. (b) Proliferation rate of four CRC cell lines, which
Fig. 1. Intestine-specific knockout of Plcg1 promotes tumor development show high β-catenin expression (red circle in A), treated with PLCγ1 siRNA.
in Apc min/+ mice. (a) Schematic showing the strategy to generate a Plcg1 conditional The proliferation rate was measured relative to the control siRNA-treated
knockout in Apc min/+ mice. (b) The mRNA expression levels of Plcg1 and Plcg2 in the cells (siCtrl). (c) Western blot assay to confirm the absence of PLCγ1
intestinal adenomas from Apc min/+ ;Plcg1 +/+ and Apc min/+ ;Plcg1 −/− mice were measured by protein in extracts from the Plcg1 KO MEF cell line compared with those
qRT-PCR. The values are represented as the fold change vs. Apc min/+ ;Plcg1 +/+ . from the WT MEF cell line. GAPDH was used as the loading
(c) Representative images of the small intestine and colon control. (d) Colonies were analyzed by crystal violet staining after 10 days.
from Apc min/+ ;Plcg1 +/+ and Apc min/+ ;Plcg1 −/− mice for 15 weeks. (d and e) Number of small The right panels represent the average number of colonies.
intestinal and colonic adenomas in 15-week-
old Apc min/+ ;Plcg1 +/+ and Apc min/+ ;Plcg1 −/− mice. (f) H&E staining of intestines from 15-
month-old mice of the indicated genotype. Scale Bar, 100 μm. (g) Kaplan–Meier survival
curves of Apc min/+ ;Plcg1 +/+ and Apc min/+ ;Plcg1 −/− mice. The p-value is based on the log-
rank test.

Fig. 4. Disrupted Plcg1 enhances the activation of Wnt/β-catenin signaling
by increasing PIP2 generation. (a) WT and Plcg1 KO MEF cells were
Fi g. 2. Loss of Plcg 1 i nduces W nt/β-cateni n signali ng. (a) Ki-67 and treated with Wnt3a for the indicated times and analyzed by western
β-cateni n immuno histochemi cal stai ni ng of i ntesti nes obtai ned blotting with the indicated antibodies. (b) FRET-based analysis of Wnt3a-
from Apc m in/+ ;Plcg 1 +/+ and Apc m in/+ ;Plcg1 − /− mal e mi c e. Scal e bar, stimulated PIP2 accumulation in CT26 cells following PLCγ1 knockdown.
100 μm (b) Quantifi cati on of Ki-67-positive cell s and β-cateni n The time lapse of FRET images obtained before and after treatment with
i ntensity . (c) Heat map of microarray anal ysi s i n i ntesti nal Wnt3a. The quantitative data expressed as a change in the normalized
adenomas derived from Apc m in/+ ;Plcg 1 +/+ and Apc m in/+ ;Plcg1 −/− mice. FRET ratio in response to Wnt3a over time, reflecting the abundance of
The scal e bar i ndi cates the gene expression l ev el. Red, high PIP2 at the plasma membrane (bottom). (c) Western blot analysis of total
expressi on l evel compared with the means; Green, l ow expression and phosphorylated forms of the indicated proteins in WT and Plcg1 KO
level compared with the means. (d) qRT-PCR confi rmation of MEF after treatment with CHIR99021 at 3 μM for 24 h. (d) Colony formation
i nterested W nt target genes identifi ed by the mi croarray. assay using WT or Plcg1 KO MEF cells. MEF cells were seeded on 6-well
(e) W estern bl ot assays were performed to demonstrate the plates and treated with 1 μM CHIR99021 for 5 days. The clonogenic
i ncreased expressi on of β -cateni n, Cycli n D1, and c -Myc, whi ch are capacity was determined by quantifying well areas covered by crystal
W nt signali ng -rel ated protei ns, i n Apc m in/+ ;Plcg1 − /− mi ce compared violet-stained colonies (bottom). (e) Kaplan–Meier survival plots of the
with that in Apc m in/+ ;Plcg1 + /+ mice. overall survival and disease-free survival in CRC patients, according to the
expression levels of PLCγ1.