Research Article
LATS2 Inhibits Colorectal Cancer Cell Proliferation, Migration and Invasion via
Activating the Hippo Signaling Pathway
Authors: Xing Liu
Publication Date: 21 March, 2025
DOI:
https://doi.org/10.51219/MCCRJ/Xing-Liu/366
Citation:
Liu X. LATS2 Inhibits Colorectal Cancer Cell Proliferation, Migration and Invasion via Activating the Hippo Signaling
Pathway. Medi Clin Case Rep J 2025;3(3):1317-1320.
Copyright:Liu X., This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract
Objective
To
investigate the role of LATS2 (large tumor suppressor 2) in colorectal cancer
(CRC) cell proliferation, migration, invasion and its regulatory effect on the
Hippo signaling pathway.
Methods
LATS2
expression in CRC cell lines (HCT116, SW480) and normal colonic epithelial cell
line (NCM460) was detected by Western blot and qRT-PCR. LATS2 was overexpressed
by plasmid or knocked down by siRNA in HCT116 cells. Cell proliferation was
measured by CCK-8 assay, migration by scratch wound healing assay, invasion by
Transwell invasion assay and expressions of Hippo pathway-related proteins
(YAP1, p-YAP1, TEAD4) by Western blot.
Results
LATS2 was
lowly expressed in CRC cells (P<0.01). LATS2 overexpression decreased HCT116
cell proliferation (OD450 at 72h: 0.65±0.06 vs. 1.30±0.11, P<0.05),
migration rate (24h: 29.8±4.0% vs. 68.5±5.9%, P<0.01), invasion (invasive
cell number: 40±5 vs. 122±9, P<0.01), upregulated p-YAP1 (P<0.05) and
downregulated YAP1 and TEAD4 (P<0.05). LATS2 knockdown showed opposite
effects.
Conclusion
LATS2 exerts
a tumor-suppressive role in CRC via activating the Hippo signaling pathway,
serving as a potential therapeutic target for CRC.
Keywords: Colorectal Cancer; Cell
Proliferation; Transwell; Large Tumor Suppressor 2
Introduction
Colorectal
cancer (CRC) is one of the most prevalent gastrointestinal malignancies
globally, with approximately 1.9 million new cases and 935,000 deaths annually1. The Hippo
signaling pathway is a conserved regulatory cascade that controls cell growth,
tissue homeostasis and tumorigenesis and its dysregulation is closely linked to
CRC progression2,3. LATS2 (large tumor suppressor 2), a core serine/threonine kinase
in the Hippo pathway, functions as a key tumor suppressor by phosphorylating
and inactivating the oncogenic effectors YAP1/TAZ, thereby inhibiting their
nuclear translocation and transcriptional activity4.
Accumulating
evidence indicates that LATS2 is downregulated in multiple cancers, including
liver cancer and ovarian cancer and its low expression correlates with poor
prognosis5,6. In gastrointestinal malignancies, LATS2 deletion has been reported
in gastric cancer, where it contributes to tumor progression by impairing Hippo
pathway activation [7]. However, the expression pattern of LATS2 in CRC and its
functional role in regulating CRC cell malignant behaviors (e.g., invasion, a
critical step in metastasis) remains not fully elucidated. This study aimed to
explore the function of LATS2 in CRC cells and its association with the Hippo
signaling pathway.
Materials and Methods
Cell
lines and culture
Human CRC cell lines HCT116 and SW480 and normal human colonic
epithelial cell line NCM460 were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA). All cells were cultured in RPMI-1640
medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum
(FBS, Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C in a humidified
incubator with 5% CO₂.
Plasmid transfection and SiRNA knockdown
LATS2 overexpression plasmid (pcDNA3.1-LATS2)
and empty vector (pcDNA3.1) were obtained from Addgene (Cambridge, MA, USA).
SiRNA targeting LATS2 (si-LATS2) and negative control siRNA (si-NC) were
purchased from Thermo Fisher Scientific (Waltham, MA, USA). HCT116 cells were
seeded into 6-well plates (5×10⁵ cells/well) and transfected with plasmids or
siRNA using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) when cell
confluency reached 60-70%. LATS2 expression was verified by Western blot and
qRT-PCR 48h post-transfection.
qRT-PCR
and western blot analysis
Total RNA was extracted from cells using TRIzol reagent (Thermo
Fisher Scientific) and cDNA was synthesized with PrimeScript RT Kit (Takara,
Kyoto, Japan). qRT-PCR was performed using SYBR Green Master Mix (Takara) on a
StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). LATS2 primers:
Forward 5'-GCTGCTGCTGCTGTTTCTGA-3', Reverse 5'-CAGCAGCAGCAGCTTCTTCT-3'; GAPDH
primers (internal control): Forward 5'-GAAGGTGAAGGTCGGAGTC-3', Reverse
5'-GAAGATGGTGATGGGATTTC-3'. Relative mRNA expression was calculated using the
2⁻ΔΔCt method.
For Western blot, cells were lysed with RIPA lysis buffer (Beyotime,
Shanghai, China) containing protease inhibitors. Protein concentration was
measured by BCA assay (Beyotime). Equal amounts of protein (30μg) were
separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, Billerica,
MA, USA), blocked with 5% non-fat milk for 1h at room temperature and incubated
with primary antibodies against LATS2 (1:1000, Abcam, Cambridge, UK), YAP1
(1:1000, Cell Signaling Technology, Danvers, MA, USA), p-YAP1 (Ser127, 1:1000,
Cell Signaling Technology), TEAD4 (1:1000, Cell Signaling Technology) and GAPDH
(1:5000, Beyotime) at 4°C overnight. After washing with TBST, membranes were
incubated with HRP-conjugated secondary antibody (1:5000, Beyotime) for 1h at
room temperature. Protein bands were visualized using ECL chemiluminescence kit
(Millipore) and relative protein expression was quantified by ImageJ software
(National Institutes of Health, Bethesda, MD, USA) with GAPDH as the internal
control.
CCK-8 assay for cell proliferation
Transfected HCT116 cells (2×10³ cells/well)
were seeded into 96-well plates. At 24h, 48h and 72h after transfection, 10μL
of CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well and the
plates were incubated at 37°C for 2h. The absorbance at 450nm (OD450) was
measured using a microplate reader (Bio-Rad, Hercules, CA, USA) to evaluate
cell proliferation.
Scratch wound healing assay for cell migration
Transfected HCT116 cells were seeded into
6-well plates and cultured to full confluency. A scratch was made in the cell
monolayer using a 200μL pipette tip. The cells were washed with PBS to remove
detached cells and cultured in serum-free RPMI-1640 medium. Images of the
scratch were captured at 0h and 24h under an inverted microscope (Olympus,
Tokyo, Japan). The migration rate was calculated as (wound width at 0h - wound
width at 24h)/wound width at 0h × 100%.
Transwell invasion assay
Transwell chambers (8μm pore size, Corning,
Corning, NY, USA) were pre-coated with Matrigel (BD Biosciences, Franklin
Lakes, NJ, USA) and incubated at 37°C for 30min to solidify. Transfected HCT116
cells (2×10⁴ cells/well) resuspended in serum-free RPMI-1640 medium were added
to the upper chamber and RPMI-1640 medium containing 20% FBS was added to the
lower chamber. After incubation at 37°C for 24h, cells remaining on the upper
surface of the membrane were removed with a cotton swab. Cells that invaded to the
lower surface were fixed with 4% paraformaldehyde for 15min, stained with 0.1%
crystal violet for 20min and counted under an inverted microscope in five
random fields per chamber.
Statistical analysis
All experiments were performed in triplicate.
Data were presented as mean ± standard deviation (SD). Statistical analysis was
conducted using SPSS 26.0 software (IBM, Armonk, NY, USA). Differences between
groups were compared using independent samples t-test. P<0.05 was considered
statistically significant.
Results
LATS2 is lowly expressed in CRC cell lines
qRT-PCR results showed that the relative mRNA expression of LATS2
in HCT116 and SW480 cells was 0.29±0.03 and 0.38±0.04 folds of that in NCM460
cells, respectively (P<0.01). Western blot analysis revealed that the
relative gray value of LATS2 protein in HCT116 (0.32±0.04) and SW480
(0.40±0.05) cells was significantly lower than that in NCM460 cells (1.00±0.10,
P<0.01), indicating that LATS2 is downregulated in CRC cell lines compared
with normal colonic epithelial cells.
LATS2 inhibits CRC cell proliferation
After transfection with pcDNA3.1-LATS2, the relative mRNA and
protein expression of LATS2 in HCT116 cells was increased by 3.75±0.35 and
3.42±0.31 folds, respectively (P<0.01). CCK-8 assay showed that the OD450
value in the LATS2 overexpression group was significantly lower than that in
the empty vector group at 48h (0.56±0.07 vs. 0.93±0.09, P<0.05) and 72h
(0.65±0.06 vs. 1.30±0.11, P<0.05). In contrast, LATS2 knockdown (si-LATS2)
reduced the relative mRNA and protein expression of LATS2 by 72.5±5.6% and 68.3±5.1%
(P<0.01) and the OD450 value in the si-LATS2 group was significantly higher
than that in the si-NC group at 48h (1.10±0.10 vs. 0.91±0.08, P<0.05) and
72h (1.42±0.12 vs. 1.28±0.10, P<0.05). These results demonstrated that LATS2
inhibits CRC cell proliferation.
LATS2 suppresses CRC cell migration
Scratch wound healing assay results showed that the migration rate
of HCT116 cells in the LATS2 overexpression group was 29.8±4.0% at 24h, which
was significantly lower than that in the empty vector group (68.5±5.9%,
P<0.01). In the LATS2 knockdown group, the migration rate was 76.3±6.2% at
24h, significantly higher than that in the si-NC group (67.8±5.7%, P<0.01),
indicating that LATS2 suppresses CRC cell migration.
LATS2 inhibits CRC cell invasion
Transwell invasion assay
results showed that the number of invasive HCT116 cells in the LATS2
overexpression group was 40±5, which was significantly lower than that in the
empty vector group (122±9, P<0.01). In the LATS2 knockdown group, the number
of invasive cells was 138±11, significantly higher than that in the si-NC group
(119±8, P<0.01), suggesting that LATS2 inhibits CRC cell invasion.
LATS2
activates the hippo signaling pathway
Western blot analysis showed that the relative gray value of
p-YAP1 in the LATS2 overexpression group was 2.08±0.19, which was significantly
higher than that in the empty vector group (1.00±0.09, P<0.05). In contrast,
the relative gray values of YAP1 and TEAD4 in the LATS2 overexpression group
were 0.40±0.05 and 0.36±0.04, respectively, significantly lower than those in
the empty vector group (1.00±0.10 and 1.00±0.08, P<0.05). LATS2 knockdown
showed the opposite effects: p-YAP1 was downregulated (0.50±0.06 vs. 1.00±0.09,
P<0.05), while YAP1 (1.25±0.11 vs. 1.00±0.10, P<0.05) and TEAD4
(1.32±0.12 vs. 1.00±0.08, P<0.05) were upregulated. These results confirmed
that LATS2 activates the Hippo signaling pathway in CRC cells.
Discussion
This study demonstrated that LATS2 is downregulated in CRC cell
lines (HCT116 and SW480) compared with normal colonic epithelial cells
(NCM460). Functional experiments showed that LATS2 overexpression inhibits CRC
cell proliferation, migration and invasion, while LATS2 knockdown enhances
these malignant behaviors. Mechanistically, LATS2 activates the Hippo signaling
pathway by increasing p-YAP1 expression and decreasing YAP1/TEAD4 expression,
indicating that LATS2 plays a tumor-suppressive role in CRC via the Hippo
signaling pathway.
The downregulation of LATS2 in CRC is consistent with its role in
other cancers. For example, LATS2 was lowly expressed in liver cancer tissues
and cell lines and restoration of LATS2 expression suppressed liver cancer cell
growth and metastasis5. In ovarian cancer, LATS2 deletion was associated with
chemotherapy resistance and poor prognosis6. In gastric cancer, LATS2 downregulation impaired Hippo pathway
activation, leading to increased YAP1 activity and tumor progression7, which is similar to our
findings in CRC, suggesting that LATS2 may play a conserved tumor-suppressive
role in gastrointestinal malignancies.
Mechanistically, LATS2, as a core kinase of the Hippo pathway, is
activated by upstream MST1/2 kinases and MOB1A/B adaptor proteins. Activated
LATS2 directly phosphorylates YAP1 at Ser127, promoting its binding to 14-3-3
proteins and cytoplasmic retention, thereby inhibiting its nuclear
translocation and transcriptional activation of target genes (e.g.,
TEAD4-regulated genes involved in cell proliferation and invasion)4,8. Our results showed that
LATS2 overexpression increases p-YAP1 and decreases YAP1/TEAD4, while LATS2
knockdown has the opposite effect, confirming that LATS2 mediates Hippo pathway
activation in CRC cells. This is supported by Li, et al.9, who reported that LATS2
overexpression inhibits gastric cancer cell invasion via YAP1 phosphorylation.
Notably, LATS2 knockdown only partially enhances CRC malignant
behaviors, which may be due to the redundancy of Hippo pathway components
(e.g., LATS1, a homolog of LATS2) or the involvement of alternative signaling
pathways (e.g., Wnt/β-catenin pathway10) that compensate for LATS2 loss. This suggests that LATS2 acts as
a key but not sole regulator of CRC progression, requiring further exploration
of its crosstalk with other pathways.
Conclusion
This study has several limitations. First, it was only conducted
in CRC cell lines and in vivo experiments (e.g., xenograft mouse models) are
needed to further confirm the tumor-suppressive role of LATS2 in CRC
progression. Second, we only explored the regulation of YAP1/TEAD4 by LATS2 and
the interaction between LATS2 and other Hippo components (e.g., MST1/2,
MOB1A/B) in CRC remains to be investigated. Third, the clinical significance of
LATS2 in CRC (e.g., its correlation with clinicopathological features and
prognosis) needs to be analyzed using clinical CRC tissue samples.
Restoring LATS2 expression or activating its kinase activity may
be a promising therapeutic strategy for CRC. Current approaches, such as gene
therapy to deliver LATS2 or small-molecule activators of LATS2, are in
preclinical development. Our study provides experimental evidence for the
development of LATS2-targeted therapies for CRC, especially in patients with
LATS2 downregulation.
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