Daulat AM et al. (2019), ECT2 associated to PRICKLE1 are poor-prognosis ...

XB-ART-55872

Br J Cancer 2019 Apr 01;1209:931-940. doi: 10.1038/s41416-019-0448-z.

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ECT2 associated to PRICKLE1 are poor-prognosis markers in triple-negative breast cancer.

Daulat AM , Finetti P , Revinski D , Silveira Wagner M , Camoin L , Audebert S , Birnbaum D , Kodjabachian L , Borg JP , Bertucci F .

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BACKGROUND: Triple-negative breast cancers (TNBC) are poor-prognosis tumours candidate to chemotherapy as only systemic treatment. We previously found that PRICKLE1, a prometastatic protein involved in planar cell polarity, is upregulated in TNBC. We investigated the protein complex associated with PRICKLE1 in TNBC to identify proteins possibly involved in metastatic dissemination, which might provide new prognostic and/or therapeutic targets. METHODS: We used a proteomic approach to identify protein complexes associated with PRICKLE1. The mRNA expression levels of the corresponding genes were assessed in 8982 patients with invasive primary breast cancer. We then characterised the molecular interaction between PRICKLE1 and the guanine nucleotide exchange factor ECT2. Finally, experiments in Xenopus were carried out to determine their evolutionarily conserved interaction. RESULTS: Among the PRICKLE1 proteins network, we identified several small G-protein regulators. Combined analysis of the expression of PRICKLE1 and small G-protein regulators had a strong prognostic value in TNBC. Notably, the combined expression of ECT2 and PRICKLE1 provided a worst prognosis than PRICKLE1 expression alone in TNBC. PRICKLE1 regulated ECT2 activity and this interaction was evolutionary conserved. CONCLUSIONS: This work supports the idea that an evolutionarily conserved signalling pathway required for embryogenesis and activated in cancer may represent a suitable therapeutic target.

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Species referenced: Xenopus laevis
Genes referenced: ect2 prickle1 rac1 rho tgfb1

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	Fig. 1. Mass spectrometry analysis of the PRICKLE1 protein complex from a TNBC cell line. a Schematic representation of the proteins associated to PRICKLE1 identified by mass spectrometry analysis from MDA-MB-231 cell extracts. Proteins have been classified following their function in several groups: Small G-proteins regulators, cytoskeleton-associated, kinases, membrane proteins, proteins involved in ubiquitination, scaffold proteins, and others. b Volcano plot showing the significance two-sample t-test (−Log p value) vs. fold-change (Log2 (GFP-PRICKLE1 vs. GFP as control)) on the y and x axes, respectively. The full line is indicative of protein hits obtained at a permutation false discovery rate of 1% (pFDR). Data results from two different experiments processed three times. PRICKLE1 (the bait) is represented in red and ECT2, one of the most abundant PRICKLE1-associated partners, is represented in green
	Fig. 2. Prognostic value of PRICKLE1-interacting small G-protein regulators in TNBC and cooperation between PRICKLE1 and ECT2 as poor-prognosis markers. a Boxplot of GEF/GAP regulators expression across breast cancers. b Boxplot of GEF/GAP regulators expression across triple negative (TN) vs. HR+/HER2− or HER2+ breast cancers. c Kaplan–Meier curves of metastasis-free survival among breast cancers patients according to overexpression (up) vs. underexpression (down) of GEF/GAP metagene mRNA. d Kaplan–Meier curves of metastasis-free survival among non-TNBC patients for GEF/GAP metagene mRNA expression. Kaplan–Meier curves of metastasis-free survival among TNBC patients for e GEF/GAP metagene mRNA expression, f PRICKLE1 mRNA expression, g PRICKLE1 mRNA and GEF/GAP metagene expression, h ECT2 mRNA expression, and i PRICKLE1 and ECT2 mRNA expression
	Fig. 3. PRICKLE1 is associated to the Rho-GEF ECT2 and controls its activity. a Immunopurification of GFP-PRICKLE1 from MDA-MB-231 cell lysate using GFP nanobodies coupled to sepharose beads allows the identification of ECT2 associated to PRICKLE1. b Immunofluorescence of MDA-MB-231 cells stably expressing GFP-PRICKLE1 shows that ECT2 (endogenous) is colocalised with PRICKLE1 and enriched in actin structures within the lamellipodia. c Mapping of the PRICKLE1 domain needed for interaction with ECT2. HEK293T cells were co-transfected with the indicated forms of PRICKLE1 (see on the left for topology details) and mCherry-ECT2. After FLAG immunopurification, presence of ECT2 is detected using anti-mCherry antibody. d Downregulation of PRICKLE1 expression using siRNA targeting PRICKLE1 shows an increase of Rac activity in MDA-MB-231 cells. e PRICKLE1 modulates ECT2 activity. Using HEK293T cells, we expressed or co-expressed ECT2 with full length or a deleted version of PRICKLE1 lacking its domain of interaction with ECT2. Overexpression of ECT2 leads to an increase in Rac activity which was inhibited when PRICKLE1 is co-expressed. Co-expression of a mutant form of PRICKLE1 did not modify the gain of function observed by ECT2 overexpression
	Fig. 4. Prickle1 and Ect2 functionally interact in Xenopus during embryonic development. In situ hybridisation against ect2 transcripts at stage 8, 9, and 10. ect2 RNA is detectable in the animal pole (animal view and lateral view) but not in the vegetal pole (vegetal view) at stages 8 and 9, but no longer at stage 10. Schematic representations of embryos at the stages analysed are shown on the right. b Embryos at two-cell stage were injected into two blastomeres with Prickle1 and Ect2 MOs as indicated. In all cases 0.5 ng of mRFP mRNA was injected as control and tracer. Suboptimal doses (10 ng) of either MO did not cause CE problems. However, when both Prickle1 and Ect2 MOs were co-injected at suboptimal doses (5 ng each), embryos displayed CE problems at a rate comparable to high doses of each MO injected separately (40 ng Prickle1-MO or 20 ng Ect2-MO). A total of 60 embryos per condition was analysed in two independent experiments. Pictures illustrate representative phenotypes. SR survival rate, ND percentage of surviving embryos developing normally, CED, percentage of surviving embryos showing convergent-extension defects. Scale bars: a = 0.25 mm; b = 0.5 mm

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