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. 2021 Mar;591(7848):E1-E10.
doi: 10.1038/s41586-020-03131-5. Epub 2021 Mar 3.

Hippo signalling maintains ER expression and ER+ breast cancer growth

Shenghong Ma  1   2 , Zhengming Wu  1   2 , Feng Yang  3 , Jianmin Zhang  4 , Randy L Johnson  5 , Michael G Rosenfeld  3 , Kun-Liang Guan  6   7
Affiliations

Hippo signalling maintains ER expression and ER+ breast cancer growth

Shenghong Ma et al. Nature. 2021 Mar.
No abstract available

PubMed Disclaimer

Conflict of interest statement

Competing interests

K.-L.G. is a co-founder of and has equity interest in Vivace Therapeutics. The other authors declare no competing interests.

Figures

Extended Data Fig. 1 |
Extended Data Fig. 1 |. LATS1/2 are required to maintain ERα and target gene expression.
a, Impaired YAP phosphorylation in LATS1/2 double knockout (dKO) cells. Wild-type (WT) and two MCF-7 LATS1/2 dKO clones were serum starved or treated with 1μM cerivastatin (1 hour), and subjected to immunoblot analysis. b, Increased YAP/TAZ transcriptional activity in LATS1/2 dKO cells. qPCR of YAP/TAZ target genes CTGF and CYR61. c, LATS1/2 deficiency inhibits MCF-7 cell growth. d, YAP signature and estrogen receptor signature are among the top upregulated and down regulated gene sets in LATS1/2 dKO cells, respectively. Gene enrichment analysis of LATS1/2 deficient (n = 3) and WT (n = 3) MCF-7 cells. Circle size represent the relative gene numbers in each set. Red, enriched in LATS1/2 deficient cells; blue, enriched in WT cells. e, Opposite effects of LATS1/2 deletion on the YAP signature genes (red) and estrogen response signature genes (blue) in MCF-7 cells. Significance, false discovery rate-adjusted p-value; Magnitude of difference, fold change; n=3 independent samples. f, YAP/TAZ and ERα localization and intensity in WT or LATS1/2 dKO MCF-7 cells. Scale bar, 20 μm. g, h, Redundancy of LATS1 and LATS2 in regulating YAP activity and ERα expression. MCF-7 clones with different sgRNA targeting LATS1, LATS2, or both were subjected to qPCR analysis for CTGF and ESR1 (g) or immunoblot (h). i-k, Lats1/2 deletion reduces ERα in endometrial organoids. Organoids derived from the endometrial tissues of Lats1+l+/Lats2+/+ and Lats1fl/fl/Lats2fl/fl mice were infected with Cre-encoding adenovirus and subjected to immunohistochemistry (i), immunoblot (j), and qPCR analysis (k). Scale bar, 100 μM for bright-field, 30 μM for IHC. l-n, Lats1/2 deletion reduces ERα in fallopian tube organoids. Organoids derived from the fallopian tube tissues of Lats1+l+/Lats2+/+ and Lats1fl/fl/Lats2fl/fl mice were infected with Cre-encoding adenovirus and subjected to immunohistochemistry (l), immunoblot (m) and qPCR analysis (n). Scale bar, 200 μM for bright-field, 25 μM for IHC staining. ***P < 0.001; mean + s.d.. See Supplementary Fig .1 for gel source data.
Extended Data Fig. 2 |
Extended Data Fig. 2 |. YAP/TAZ mediates Hippo signaling to repress ESR1 expression
a, Repression of ERα level by TAZ. MCF-7 cells transduced with a control vector, Flag-TAZ(4SA) cDNA or Flag-TAZ(4SA/54A) cDNA were subjected to immunoblot. TAZ(4SA) is a constitutively active mutant with mutation of the four LATS phosphorylation sites while TAZ(4SA/54A) is defective in TEAD binding. b, c, YAP reduces ERα level in additional ER+ breast cancer cell lines. T47D (b) and ZR-75-1 (c) cells transduced with a control vector, flag-YAP(5SA) or Flag-YAP(5SA/94A) were analyzed by immunoblot. d, YAP TEAD-binding domain, but not WW domain, is essential for CTGF induction. e, f, YAP and TAZ have redundant role in repressing ERα expression. WT, YAP KO, TAZ KO or YAP/TAZ dKO MCF-7 cells were subjected to immunoblot (e) or qPCR for ESR1 (f). g, YAP/TAZ dKO increases expression of ERα target gene TFF1 and GREB1. h, i, NF2 deficiency decreases ERα expression, YAP phosphorylation, LATS1 phosphorylation without affecting LATS1 protein. Wild-type (WT) or three independent clones of NF2 null MCF-7 cells were subjected to immunoblot (h) or qPCR (i). ***P < 0.001; mean + s.d., n.s., not significant. See Supplementary Fig .1 for gel source data.
Extended Data Fig. 3 |
Extended Data Fig. 3 |. LATS deletion inhibits ER+ breast cancer cell growth.
a, Image representative of six biologically independent xenografts for Fig. 2h. Scale bar, 10 mm. b-d, LATS1/2 deficiency inhibits tumor cell proliferation and promotes apoptosis in vivo. Representative images (b) and analysis (c, d) of ERα, p-Histone H3, and cleaved caspase3 immunostaining in LATS1/2 deficient and wild-type MCF-7 xenograft. Scale bar, 100 μM. Box plots indicate median and interquartile range.
Fig. 1 |
Fig. 1 |. LATS is essential to maintain ER expression.
a, LATS1/2 deletion causes an enrichment of YAP gene signature and depletion of ERα gene signature. b, c, LATS1/2 dKO downregulates ESR1 mRNA (b) and ERα protein (c) in MCF-7 cells. Two independent LATS1/2 dKO MCF-7 clones were shown. d, LATS1/2 dKO inhibits ERα target genes. E2 treatment (1 nM E2 for 45 min). e, LATS1/2 deficiency downregulates ERα. T47D and ZR-75-1 cells with lentivirus-mediated CRISPR deletion of LATS1/2 (sgLATS1/2) were subjected to immunoblot analysis with indicated antibodies. f-h, Deletion of Lats1/2 activates YAP/TAZ and downregulates ESR1 expression in mammary organoids. Organoids derived from the mammary tissues of Lats1+l+/Lats2+/+ and Lats1fl/fl/Lats2fl/fl mice were infected with Cre-encoding adenovirus and subjected to immunohistochemistry (f), immunoblot (g) and qPCR analysis (h). Scale bar, 50 μm. i, LATS1 overexpression does not decrease ERα protein. HA-LATS1 or kinase dead mutant (KR) was expressed in WT or LATS1/2 dKO cells of MCF-7 or T47D. j-l, Expression of Lats2 wild-type, but not the kinase-dead mutant, rescued ERα expression in the LATS1/2 dKO cells. Immunoblot (j) or qPCR for ESR1 (k) and CTGF (l). m, n, TEAD-binding is required for YAP mediated ESR1 reduction. MCF-7 cell transduced with a control vector, Flag-YAP(5SA), Flag-YAP(5SA/94A), Flag-YAP(5SA/ΔW1ΔW2), or Flag-YAP(5SA/ΔW1) were subjected to qPCR for ESR1 (m) or immunoblot (n). o, YAP/TAZ mediates the ERα reduction by LATS1/2 deficiency. Numbers denote different cell clones. p, TAZ transgene reduces ERα in vivo. Immunohistochemical staining was performed on mammary tissues from control and MMTV-rtTA TRE-TAZ4SA transgenic (TAZ* Tg) mice. Scale bar, 100 μm. ***P < 0.001, n.s., not significant, Two-sided t-test or ANOVA; mean + s.d.. See Supplementary Fig .1 for gel source data.
Fig. 2 |
Fig. 2 |. LATS knockout inhibits ER+ breast cancer cell growth by abolishing ERα expression.
a, LATS1/2 deletion inhibits growth of ERα positive, but not ERα negative, breast cancer cells. Cells infected with lentivirus encoding CRISPR-cas9 sgRNA targeting both LATS1 and LATS2 (sgLATS1/2) or control vector were grown for 4 days. n=3 independent samples. Two different set of guide sequences targeting LATS1/2 were used and labelled as #1 and #2. b, LATS1/2 is required for estrogen response. Wild-type and LATS1/2 dKO MCF-7 cells were cultured in steroid-free media with or without 0.1 nM E2 added for 5 days. c, LATS1/2 deficient MCF-7 cells are insensitive to growth inhibition by 4-OHT. Wild-type and LATS1/2 dKO MCF7 cells were cultured in absence or presence of 0.5 μM 4-OHT for 4.5 days. d, Re-expression of ERα in LATS1/2 dKO MCF-7 cells. Wild-type, LATS1/2 dKO, and LATS1/2 dKO with Flag-ERα re-expressing cells were subjected to immunoblot analysis. e, f, Ectopic expression of ERα rescues the growth defect caused by LATS1/2 knockout. Soft agar colony formation of wild-type (WT) and LATS1/2 dKO MCF-7 cells, and LATS1/2 dKO Cells re-expressing ERα (e). The colonies were stained with crystal violet for quantification (f). g, LATS1/2 deficiency reduces in vivo xenograft growth. Nude mice were injected with wild-type or LAST1/2 dKO MCF-7 cells and tumor growth was measured at the indicated times. h, Tumour weight on day 28 from (g). See Supplementary Fig .1 for gel source data.
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References

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