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. 2013 Dec 26;5(6):1650-63.
doi: 10.1016/j.celrep.2013.11.037. Epub 2013 Dec 19.

LATS2 suppresses oncogenic Wnt signaling by disrupting β-catenin/BCL9 interaction

Jiong Li  1 , Xiaohong Chen  2 , Xiangming Ding  1 , Yingduan Cheng  1 , Bin Zhao  3 , Zhi-Chun Lai  4 , Khalid Al Hezaimi  5 , Razqallah Hakem  6 , Kun-Liang Guan  3 , Cun-Yu Wang  7
Affiliations

LATS2 suppresses oncogenic Wnt signaling by disrupting β-catenin/BCL9 interaction

Jiong Li et al. Cell Rep. .

Erratum in

Abstract

Abnormal activation of Wnt/β-catenin-mediated transcription is associated with a variety of human cancers. Here, we report that LATS2 inhibits oncogenic Wnt/β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction. LATS2 directly interacts with β-catenin and is present on Wnt target gene promoters. Mechanistically, LATS2 inhibits the interaction between BCL9 and β-catenin and subsequent recruitment of BCL9, independent of LATS2 kinase activity. LATS2 is downregulated and inversely correlated with the levels of Wnt target genes in human colorectal cancers. Moreover, nocodazole, an antimicrotubule drug, potently induces LATS2 to suppress tumor growth in vivo by targeting β-catenin/BCL9. Our results suggest that LATS2 is not only a key tumor suppressor in human cancer but may also be an important target for anticancer therapy.

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Figures

Fig. 1
Fig. 1. LATS2 inhibits Wnt/β-catenin-mediated transcription independent of Hippo-YAP signaling
(A) LATS2 inhibited STopflash reporter activities induced by LiCl in 293T cells. Experiments were performed at least three times. Values are mean ± s.d. for triplicate samples from a representative experiment. **P< 0.01, Student’s t-test. (B) LATS2 inhibited STopflash reporter activities induced by Wnt-3a in 293T cells. Values are mean ± s.d. for triplicate samples from a representative experiment. **P < 0.01, Student’s t-test. (C) LATS2 inhibited STopflash reporter activities induced by β-Cat in 293T cells. β-Cat, β-catenin. Values are mean ± s.d. for triplicate samples from a representative experiment. **P< 0.01, Student’s t-test. (D) LATS2, but not LATS1, was knocked down by siRNA in 293T cells. (E) LATS2 knockdown enhanced STopflash reporter activities induced by Wnt-3a. Values are mean ± s.d. for triplicate samples from a representative experiment. **P < 0.01, Student’s t-test. (F) LATS2 knockdown enhanced the expression of AXIN-2 and DKK1 induced by Wnt-3a. Values are mean ± s.d. for triplicate samples from a representative experiment. **P < 0.01, Student’s t-test. (G) LATS2 inhibited β-catenin-mediated transcription independent of its kinase activity. Values are mean ± s.d. for triplicate samples from a representative experiment. **P < 0.01, Student’s t-test. (H) SiRNA knocked down TAZ and YAP. (I) TAZ or YAP knockdown did not affect LATS2 inhibition of β-catenin-mediated transcription. Values are mean ± s.d. for triplicate samples from a representative experiment. **P< 0.01, Student’s t-test. See also Fig. S1.
Fig. 2
Fig. 2. LATS2 interacts with β-catenin
(A) LATS2 and β-catenin interacted when overexpressed in 293T cells. (B) The kinase domain of LATS2 was not required for the interaction between β-catenin and LATS. (C and D) The amino acids 250–575 of LATS2 were required for interaction with β-catenin. (E) β-catenin-133–694 containing arm repeats interacted with LATS2. 293T cells were co-transfected with HA-LATS2 and a variety of β-catenin deletion mutants. (F) Endogenous β-catenin interacted with LATS2 upon LiCl or Wnt-3a stimulation. (G) GST-β-catenin fusion protein pulled down HA-LATS2 protein in vitro. See also Fig. S2.
Fig. 3
Fig. 3. LATS2 expression is inversely associated with human colorectal cancer development and prognosis
(A and B) LATS2 expression was decreased in human colorectal tumor tissues compared with adjacent normal tissues. TMA was stained with anti-LATS2 polyclonal antibodies. (C) Quantitative measurement of LATS2 in human colorectal tumor tissues and adjacent normal tissues. The intensities of immunostaining were quantitatively measured using Image-Pro Plus 6.0 image analysis software. P < 0.01. n = 50. (D) Quantitative measurement of LATS2 in human primary colorectal tumors and metastatic tumors from the lymph node and liver. P < 0.05. n = 30. (E–H) LATS expression was lost in human metastatic colorectal tumors from the lymph node or liver. TMA consisting of primary tumors and metastatic tumors from lymph node and liver was stained with anti-LATS2 polyclonal antibodies. (I) LATS2 expression was inversely associated with the survival times of patients with colorectal cancer. The published colon cancer dataset (GSE17537) containing patient follow-up information was used. Kaplan-Meier survival plot was performed for disease-specific survival and the log-rank test was applied to test the survival differences using SPSS 17.0 software. P < 0.001 (J) LATS2 expression was inversely associated with the recurrence-free survival times of patients with colorectal cancer. P < 0.05. See also Table S1–4 and Fig. S3.
Fig. 4
Fig. 4. LATS2 inhibits β-catenin-mediated recruitment of BCL9 to the Wnt target gene promoter
(A) 293T cells were co-transfected with β-catenin and LATS2 as indicated. The ChIP-enriched DNAs were quantitatively measured using Real-time PCR with AXIN-2- and DKK1-specific primers. A region located in the open reading frame (ORF) was utilized as a negative control. (B) Cells were transfected with LATS2 or control siRNA for 48 hr and then treated with LiCl for 2 hr. ChIP assays were performed as described in (A). (C) Cells were transfected with LATS2 for 24 hr and then treated with Wnt-3a. ChIP assays were performed as described in (A). (D) Cells were transfected with LATS2 or control siRNA and then treated with Wnt-3a. ChIP assays were performed as described in (A). (E) Cells were transfected with LATS2 or control siRNA and ChIPed with anti-LATS2, anti-LATS1 or IgG. Values are mean ± s.d. for triplicate samples from a representative experiment. **P< 0.01, Student’s t-test. (F) Cells were transfected with LATS2 or vector control and then treated with Wnt-3a. ChIP assay were performed using anti-BCL9 antibodies. Values are mean ± s.d. for triplicate samples from a representative experiment. **P< 0.01, Student’s t-test. (G and H) Cells were transfected with LATS2 or control siRNA for 48 hr and then treated with Wnt-3a or LiCl. ChIP assays were performed using anti-BCL9 antibodies. Values are mean ± s.d. for triplicate samples from a representative experiment. **P < 0.01, Student’s t-test.
Fig. 5
Fig. 5. LATS2 inhibits the interaction between β-catenin and BCL9
(A and B) LATS2 inhibited the interaction between β-catenin and BCL9, BCL9L or PYGO2 induced by LiCl and Wnt3a. (C and D) LATS2 knockdown enhanced the interaction between β-catenin and BCL9, BCL9L or PYGO2 induced by LiCl and Wnt3a. (E) LATS2 inhibited the direct interaction between β-catenin and BCL9 in vitro. (F) LATS2 inhibited the direct interaction between β-catenin and BCL9L in vitro. See also Fig. S4.
Figure 6
Figure 6. LATS2 kinase domain deletion mutant inhibits Wnt/β-catenin-mediated transcription Lats2−/−MEFs
(A) Confirmation of LATS2 deletion in LATS2−/− MEFs by Western blot. (B) Lats2−/−MEFs were stably expressed with HA-LATS2N. Nuclear proteins were isolated from Lats2−/−MEFs expressing HA-LATS2N (MEF/LN) or control vector (MEF/V) and probed with anti-HA antibodies. (C) LATS2N inhibited STopflash reporter activities induced by Wnt3a. Values are mean ± s.d. for triplicate samples from a representative experiment. **P < 0.01, Student’s t-test. (D) LATS2N inhibited the expression of Axin2 and Dkk1 induced by Wnt3a. Values are mean ± s.d. for triplicate samples from a representative experiment. **P< 0.01, Student’s t-test. (E and F) Both Lats2−/−MEF/V and Lats2−/−MEF/LATS2N Cells were treated with Wnt-3a and ChIP-ed with anti-LATS2 or anti-β-catenin. The ChIP-enriched DNA was measured using Real-time PCR with the Axin-2-specific primers. (G) LATS2N inhibited the recruitment of BCL9 and PYGO2 to the Axin promoter induced by Wnt3a. Values are mean ± s.d. for triplicate samples from a representative experiment. **P< 0.01, Student’s t-test.
Fig. 7
Fig. 7. Nocodazole induces LATS2 to blockβ-catenin/BCL9-mediated transcription and inhibits tumor growth in vivo
(A) Nocodazole induced LATS2 in HEK293 T cells by Western blot. (B) Nocodazole significantly suppressed β-catenin/Tcf-mediated transcription induced by LiCl or β-Cat overexpression. P < 0.01. (C) Nocodazole induced LATS2, but not LATS1, in a time-dependent manner in HCT116 cells. (D) The knock-down of nocodazole-induced LATS2 by shRNA. Nuclear proteins from HCT116 cells were isolated and probed with anti-LATS2 antibodies. (E) Nocodazole suppressed the expression of AXIN2 and MMP7 dependent of LATS2. Both HCT116/Scrsh and HCT116/LATS2sh cells were treated with nocodazle and the expression of AXIN2 and MMP7 was determined by Real-time RT-PCR.P < 0.01. (F) The restoration of LATS2 or LATS2N suppressed the expression of AXIN2 and MMP7 in HCT116/LATS2sh cells. (G) Nocodazole induced LATS2 binding the promoter of AXIN2 or MMP7 by ChIP assays. Both HCT116/Scrsh and HCT116/LATS2sh cells were treated with nocodazole, and the promoter of AXIN2 or MMP7 was ChIP-ed with anti-LATS2 antibodies. P < 0.05. (H) Nocodazole inhibited the occupancy of BCL-9 on the promoter of AXIN2 or MMP7 via the induction of LATS2. Both HCT116/Scrch and HCT116/LATS2sh cells were treated with nocodazole and the promoter of AXIN2 or BCL9 was ChIP-ed with anti-BCL-9 antibodies. P < 0.05. (I) Nocodazole did not affect β-catenin binding to the promoter of AXIN2 or MMP7. (J) LATS2 was required for nocodazole-mediated inhibition of tumor growth in nude mice. N = 7–8. P < 0.05. (K) Comparisons of actual tumor weights at the end of nocodazole treatment. At the end of experiments, tumors from nude mice were dissected and weighted. * P < 0.05. See also Fig. S5.
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