Platelet-induced metastasis and anoikis protection

To examine whether platelet numbers can impact survival of human ovarian cancer cells in the ascites, MDAH-2774 human ovarian cancer cells were injected intraperitoneally into nude mice. Prior to injection of cancer cells, one group of mice received an anti-platelet antibody (APA) to reduce platelet numbers before cell injection, whereas the second group of mice received a control antibody (ctrl IgG). Efficiency of APA at a dose of 0.5 µg g−1 in reducing platelet counts, and specificity of APA for platelets compared to other cell types were demonstrated previously3, 4. Treatment with ctrl IgG or APA was repeated twice weekly for the duration of the experiment. Reducing the number of platelets significantly increased the number of apoptotic cells in ascites, as measured by a higher number of SYTOX Red positivity in the APA as compared to that in the control group (Fig. 1a). To determine whether similar findings would apply to other cancer models, we used the human colon cancer cell line SW620 as a second model system. Here, reduced platelet counts also significantly diminished the number of nodules detected in the liver of mice after intrasplenic injection of SW620 cells (Fig. 1b, c and Supplementary Fig. 1a). No significant bleeding events occurred and mouse weight did not change after reduction in platelet counts (Supplementary Fig. 1b). These results highlight platelets as important contributors to metastasis in ovarian and colorectal cancer in vivo models.

Fig. 1 Platelets facilitate metastasis in vivo and reduce anoikis. a Plot showing percent of SYTOX Red positive EPCAM + MDAH-2774 tumor cells in ascites after treatment with control IgG or anti-platelet antibody (APA, n = 5, two-sided Student’s t-test). b, c Number of liver nodules b and representative bioluminescence imaging pictures c 5 weeks after intrasplenic injection of 2 × 106 SW620 colon cancer cells (n = 10, two-sided Student’s t-test). d Number of dead (SYTOX Red positive, black) and living (SYTOX Red negative, red) HEYA8 cells after 72 hours of low attachment and/or co-incubation with increasing numbers of platelets (n = 3, two-sided Student’s t-test). e Number of dead (SYTOX\ Red positive, black) and living (SYTOX Red negative, red) OVCAR8, SKOV3, MDAH-2774 and SW620 cells after 72 h of low attachment and/or co-incubation with 100 × 106 platelets (n = 3, two-sided Student’s t-test). f Percentage of HEYA8 or OVCAR8 human ovarian cancer cells in sub-G1 phase of the cell cycle after 72 h under low-attachment conditions (n = 3, two-sided Student’s t-test). g Protein analysis and quantification of full length and cleaved PARP in HEYA8 and OVCAR8 cells after 72 h under low-attachment conditions (short exposure of PARP upper panel, long exposure of PARP lower panel, n = 3, two-sided Student’s t-test). GAPDH was used as a loading control. Bars and error bars represent mean values and the corresponding SEMs (*p < 0.05, **p < 0.01, ***p < 0.001) Full size image

To investigate whether platelets can have a direct impact on anoikis rates of ovarian and colon cancer cells in vitro, we co-incubated various human cancer cell lines with platelets under anoikis conditions using ultra-low attachment cell culture plates. After determining the baseline anoikis rate in all of the tested cell lines, we decided to use 72 h as the major endpoint for measuring anoikis levels in vitro (Supplementary Fig. 1c). Increase in the number of co-incubated platelets gradually reduced anoikis rates in HEYA8 human ovarian cancer cells (Fig. 1d). Similar observations were made for the effect of platelets on the anoikis rates of OVCAR8, SKOV3, MDAH-2774 and SW620 cells incubated with 100 × 106 platelets (Fig. 1e), as well as of A2780ip2 and OVCAR4 cells (Supplementary Fig. 1d) in which platelets significantly increased the number of surviving detached cells after 72 h. Moreover, platelet co-incubation with HEYA8 and OVCAR8 cells markedly reduced the percentage of cells in the sub-G1 phase of the cell cycle (Fig. 1f) and reduced protein levels of cleaved PARP (Fig. 1g). Additionally, western blot analysis of HEYA8 ovarian cancer cells co-incubated with platelets for 72 h under low-attachment conditions revealed a significant reduction in cleaved caspase-3 (Supplementary Fig. 1e), confirming a reduction in apoptosis of HEYA8 cells after platelet exposure. In contrast, anoikis rates of OVCA432, OVCAR5 and RKO cells did not change after platelet co-incubation, indicating a cell-specific responsiveness (Supplementary Fig. 1f). In summary, these results indicate that platelets induce anoikis resistance in vitro and enhance metastatic spread of tumor cells in vivo.

Platelet-induced YAP1 gene signature in cancer cells

Next, we sought to identify the dominant signaling events in cancer cells responsible for platelet-mediated anoikis resistance. First, we performed reverse phase protein array (RPPA) analysis to identify proteins and signaling pathways that were significantly altered in tumor cells after addition of platelets (Fig. 2a and Supplementary Data 1). We identified several proteins related to survival and proliferation that were upregulated, including pAktS473, p38T180_Y182 and pSrcY416 (Fig. 2b, Supplementary Fig. 2a). Interestingly, the strongest difference was observed for YAP1, with more than three-fold downregulation in the phosphorylation level at the serine 127 (S127) residue, indicating an activation of YAP1 signaling after platelet incubation (Fig. 2b). YAP1 is a transcriptional co-activator that translocates into the nucleus after S127 dephosphorylation. Hence, to uncover transcriptional changes, we performed unbiased RNA expression analyses and isolated RNA from tumor cells incubated for 24 h with buffer only, or with platelets (Fig. 2c). Gene Set Enrichment Analysis (GSEA) revealed that pathways related to cell cycle and E2F1-signaling were the most upregulated pathways (Fig. 2d and Supplementary Fig. 2b), whereas genes related to hypoxia, oxidative phosphorylation and p53 were downregulated in cancer cells upon platelet incubation (Supplementary Fig. 2c, d). Intriguingly, upstream analysis using Ingenuity Pathway Analysis (IPA) identified YAP1 as the principal and most crucial upstream regulator of gene expression changes seen in HEYA8 cells after platelet co-incubation (Fig. 2e). Additional computational analyses using CCExplorer used the receptor and transcription factor lists as well as background network11 to identify connections between differentially regulated proteins (from RPPA) and transcriptional changes (from microarray analysis). As shown in Fig. 2f, YAP1 was the only differentially regulated protein which significantly connected to differential RNA expression patterns. The red nodes represent signaling nodes that connect YAP1 with differently regulated receptors (marked in yellow), likely to be upstream of YAP1. The green nodes are other genes found by random walk analysis, representing either differentially expressed genes (DEGs) or the ones linking DEGs.

Fig. 2 Platelets induce a YAP1-specific gene signature in cancer cells. a Heat map showing differentially regulated proteins as analyzed by reverse phase protein array (RPPA) in HEYA8 and HEYA8 co-incubated with platelets for two hours (n = 2). b Top 10 down- and upregulated proteins (+plts vs. control) after RPPA analysis. c Heat map depicting differentially regulated genes in HEYA8 or HEYA8 cells co-incubated with 100 × 106 platelets under low-attachment conditions for 24 hours (n = 3, p < 0.001). d Enriched pathways with indicated p-values of upregulated genes in platelet co-incubated HEYA8 cells using Gene Set Enrichment Analysis (GSEA, www.broadinstitute.org/gsea). e Upstream analysis of gene expression changes in HEYA8 ovarian cancer cells co-incubated with platelets using Ingenuity Pathway Analysis (IPA, http://www.ingenuity.com/products/ipa). f Random walk analysis highlighting YAP1 protein as the main connector to transcriptional changes in HEYA8 co-incubated with platelets. g QRT–PCR analysis of YAP1, NDRG1, DDIT4, CDC20, CTGF, MCM5, MCM6, and MCM10 in HEYA8 cells after 24 and 48 h of co-incubation with platelets. 18S was used as the housekeeping gene (n = 3, two-sided Student’s t-test). h QRT–PCR analysis of YAP1, NDRG1, DDIT4, CDC20, CTGF, MCM5, MCM6, and MCM10 after YAP1 knockdown with two different siRNAs (72 hours after transfection, n = 3). 18S was used as the housekeeping gene (n = 3, two-sided Student’s t-test). Bars and error bars represent mean values and the corresponding SEMs (*p < 0.05, **p < 0.01, ***p < 0.001) Full size image

Interestingly, many of the top up- and downregulated genes are well-known YAP1 target genes, including connective tissue growth factor; CTGF 12, DNA damage inducible transcript 4; DDIT4 13, cell-division cycle protein 20; CDC20 14 and the DNA replication proteins minichromosome maintenance complex components 5/6/10; MCM5, MCM6 and MCM10 15. We validated expression of these genes after co-incubation of HEYA8 cells with platelets after 24 and 48 h (Fig. 2g). Conversely, the same genes were significantly deregulated by knocking down YAP1 with two individual siRNAs (Fig. 2h), indicating that platelets induce a YAP1-dependent gene signature in cancer cells.

It is known that YAP1 and the Hippo signaling pathway are crucial for organ development16, 17 and cancer in various organs such as the liver18, pancreas15, 19 and prostate20, 21, however, the data about the role of the Hippo–YAP pathway in ovarian cancer biology and metastasis is recently emerging. Analyzing TCGA data from high-grade serous ovarian cancer (Supplementary Fig. 2e) indicated that expression of YAP1 or components of the Hippo signaling pathways is mainly regulated by gene amplification and/or mRNA up/downregulation, whereas mutations in these genes are rare, which is consistent with other tumor types22. Moreover, survival analysis evaluating the same set of patient samples indicated that YAP1 protein expression significantly correlated with disease-free survival in ovarian cancer patients (Supplementary Fig. 2f). Finally, we collected baseline platelet counts from 358 stage III and IV high-grade serous ovarian cancer patients whose gene expression patterns were previously analyzed by The Cancer Genome Atlas Research Network23 and applied a verified gene signature for YAP1 activation, which was shown to significantly correlate with patient survival24, 25. When the patients were stratified using this algorithm, tumors of 165 patients showed a YAP1 activation signature. Interestingly, these patients had significantly higher platelet counts compared to patients whose tumors lacked this signature (p = 0.04, Supplementary Fig. 2g).

YAP1 is required for platelet-induced anoikis resistance

YAP1 activity is controlled by S127 and S397 phosphorylation. If hypophosphorylated, YAP1 translocates into the nucleus and binds to other transcription factors such as E2F1 and TEAD2/4, promoting transcription of genes that in turn increase proliferation and inhibit apoptosis15, 26, 27. Next, we analyzed the phosphorylation and intracellular localization of YAP1. Co-incubation of HEYA8 and OVCAR8 ovarian cancer cells with platelets robustly reduced YAP1S127 and YAP1S397 phosphorylation, whereas total YAP1 levels did not change (Fig. 3a, b). These results confirmed phosphorylation changes observed in the RPPA analysis. Reduced S127 phosphorylation was additionally validated in a panel of ovarian and colon cancer cell lines, including SKOV3, MDAH-2774, OVCAR4 and SW620 (Fig. 3c), while OVCAR5, OVCA432 and RKO did not show this effect (Fig. 3d). Interestingly, the latter three cell lines were not protected from anoikis by platelet co-incubation (Supplementary Fig. 1f), implicating an important role of YAP1 in platelet-mediated anoikis protection. Various ovarian cancer cell lines were not different in their ability to activate platelets in vitro (Supplementary Fig. 3a). Intriguingly, our experiment with OVCAR5 ovarian cancer cells confirmed that these cells are not responsive to platelet counts in vivo. We did not observe any reduction in primary ovarian tumor weight or number of metastatic nodules induced by intraovarian injection of OVCAR5 cells into mice treated with APA as compared to those treated with ctrl IgG (Supplementary Fig. 3b). In line with the observed dephosphorylation, co-incubation of HEYA8 and OVCAR8 cells with platelets induced a clear shift of YAP1 protein expression from the cytoplasmic to the nuclear compartment of the cell (Fig. 3e, f and Supplementary Fig. 3c, d). These results together with the observed gene expression signature strongly support the idea that YAP1 activation is responsible for platelet-induced anoikis resistance. To obtain direct evidence supporting this concept, we performed RNAi-mediated knockdown experiments in HEYA8 and OVCAR8 cancer cells using two independent siRNAs that reduced YAP1 at RNA and protein levels (Fig. 3g, i). Twenty four hours after transfection of YAP1 siRNA to ovarian cancer cells, platelets were added to the cells that were kept for an additional 72 h under low-attachment conditions. Analyzing the percentage of apoptotic cells via flow cytometry revealed that YAP1-depleted HEYA8 and OVCAR8 cells did not respond to platelets and showed the same apoptosis rate as the respective controls in which detached tumor cells were grown without platelets (Fig. 3h, j). Importantly, siRNA-mediated YAP1 knockdown alone did not have a significant effect on anoikis rates in cancer cells (Supplementary Fig. 3e, f). Replication of these experiments in the SW620 colon cancer cells generated similar results (Supplementary Fig. 3g, h), suggesting that YAP1 is an essential mediator of platelet-induced anoikis resistance in various types of cancer cells.

Fig. 3 YAP1 is activated by platelets and is indispensable for platelet-induced anoikis resistance. a, b Western blot analysis of phosphorylated YAP1 (S127 and S397) and total YAP1 in HEYA8 a and OVCAR8 b cells after two hours under low-attachment conditions with or without platelet co-incubation. GAPDH was used as a loading control (n = 5). c, d Western blot analysis of phosphorylated YAP1 (S127) and total YAP1 in SKOV3, MDAH-2774, OVCAR4, SW620 c and OVCAR5, OVCA432 and RKO d GAPDH was used as a loading control (n = 3). e, f Immunofluorescence staining of YAP1 in HEYA8 e and OVCAR8 f cells after two hours under low-attachment conditions with (lower panels) or without (upper panels) platelet co-incubation. Inlets showing higher magnification of cells on the right side of the panels. Nuclear counterstain was done using Hoechst 33342 (n = 3). Scale bars = 20 µm. g QRT–PCR and western blot analysis in HEYA8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs (n = 3). h Bar graphs showing number of dead (SYTOX Red positive, black) and living (SYTOX Red negative, red) HEYA8 cells after 72 h of low attachment and 96 h after siRNA transfection (n = 3, two-sided Student’s t-test). i QRT–PCR and western blot analysis in OVCAR8 cells showing efficiency of YAP1 knockdown on the RNA and protein level using two different siRNAs (n = 3). j Bar graphs showing number of dead (SYTOX Red positive, black) and living (SYTOX Red negative, red) OVCAR8 cells after 72 h of low attachment and 96 h after siRNA transfection (n = 3, two-sided Student’s t-test). Bars and error bars represent mean values and the corresponding SEMs (*p < 0.05, **p < 0.01, ***p < 0.001) Full size image

YAP1 overexpression boosts anoikis resistance and metastasis

Next, we assessed the effects of YAP1 overexpression on anchorage-independent cell growth. Initially, we quantified YAP1 protein levels in various ovarian and colorectal cancer cell lines. This revealed a very low to absent protein expression of YAP1 in OVCA432 ovarian cancer cell line in comparison to a panel of different ovarian and colorectal cancer cells (Fig. 4a). Interestingly, this cell line exhibited very high anoikis rates at baseline levels with more than 50% apoptotic cells after only 24 hours and did not respond to the addition of platelets (Supplementary Fig. 1c, f). We investigated whether overexpression of YAP1 would be sufficient to enhance survival of OVCA432 cells grown under low-attachment conditions. We stably introduced wild-type YAP1 (WT), a constitutively active YAP1S127A mutant or a TEAD-binding defective YAP1 mutantS127A/S94A into OVCA432 cells, and confirmed their expression (Fig. 4b). Overexpression of YAP1 with various constructs did not affect normal 2D cell growth in 96-well plates (Fig. 4c). In contrast, when cells were grown under low-attachment conditions, overexpression of YAP1WT and the YAP1S127A mutant significantly enhanced cell survival, whereas overexpression of the TEAD-binding defective YAP1S127A/S94A double mutant compromised this effect (Fig. 4d). Similar results were obtained in a soft agar colony formation assay that confirmed a higher anchorage-independent growth in YAP1WT, and especially in YAP1S127A overexpressing cells (Fig. 4e). Moreover, co-incubation of YAP1S127A overexpressing OVCA432 cells under low-attachment conditions with platelets for 72 h did not change anoikis rates (Supplementary Fig. 4a), suggesting that YAP1 mediates anoikis resistance even in cells were YAP1 is normally absent. To confirm our in vitro findings, we injected 1 × 106 OVCA432 overexpressed with either control, YAP1WT, YAP1S127A or YAP1S127A/S94A vector plasmids into the left ovary of nude mice. Intriguingly, mice injected with either OVCA432-YAP1WT but especially with constitutive active OVA432-YAP1S127A harbored greatly increased number of metastatic nodules (Fig. 4f, g and Supplementary Fig. 4b) as well as amount of ascites (Supplementary Fig. 4c) compared to control OVCA432 or OVCA432-YAP1S127A/S94A. Primary tumor weight was not significantly different between the different groups despite a slight increase in primary tumor weight in the control group compared to mice injected with OVCA432-YAP1WT (Fig. 4h). YAP1 phosphorylation, in part, is regulated by Hippo and LATS kinases28. To understand the role of the upstream Hippo signaling pathway in the platelet-induced anoikis resistance, we analyzed the expression levels of LATS1, pLATS1, LATS2, MST1, MST2 and MOB1. Co-incubation of platelets with HEYA8 and OVCAR8 cells did not change levels of these proteins significantly (Supplementary Fig. 4d). Moreover, siRNA-mediated knockdown of the major YAP1 kinases LATS1 and LATS2 (Supplementary Fig. 4e) had no effect on anoikis resistance of HEYA8 and OVCAR8 cells compared to control siRNA (Supplementary Fig. 4f). Altogether, these results support the importance of the role of YAP1 in mediating anoikis resistance and anchorage-independent cell growth in vitro as well as metastasis in vivo, which seems to be independent of the upstream Hippo signaling pathway.

Fig. 4 YAP1 overexpression increases anoikis resistance and metastasis. a Analysis of YAP1 protein expression in ovarian and colon cancer cell lines. GAPDH was used as a loading control (n = 2). b Validation of overexpression of YAP1 with wild-type (WT) YAP1, constitutively active (S127A) YAP1 and TEAD-defective mutant (S127A/S94A) YAP1 in OVCA432 human ovarian cancer cells. GAPDH was used as a loading control (n = 3). c Cell viability analysis (upper panel) and representative crystal violet staining (lower panel) of OVCA432 cells transduced with control or YAP1 overexpressing constructs 72 h after seeding into 96-well plates (n = 6, one-way ANOVA followed by a Tukey’s multiple comparison post hoc test). d Analysis of GFP-positive, viable (SYTOX Red negative) cells after 72 h of low attachment in OVCA432 cells transduced with control or YAP1 overexpressing constructs (n = 6, one-way ANOVA followed by a Tukey’s multiple comparison post hoc test). e Anchorage-independent growth assay in soft agar using OVCA432 transduced with control or YAP1 overexpressing constructs and quantification of colonies after seeding 15,000 cells and incubation for 14 days (n = 3, one-way ANOVA followed by a Tukey’s multiple comparison post-hoc test). f–h Representative necropsy pictures f, number of metastatic nodules g and weight of ovarian primary tumor h after intraovarian injection of OVCA432 overexpressed with either eGFP-control, YAPWT, YAPS127A or YAPS127A/S94A constructs (n = 10, one-way ANOVA followed by a Tukey’s multiple comparison post-hoc test). Bars and error bars represent mean values and the corresponding SEMs (*p < 0.05, **p < 0.01, ***p < 0.001) Full size image

Thrombocytosis-induced metastasis is regulated by YAP1

Most patients with newly diagnosed ovarian cancer have widespread metastases, which represents a challenge for every-day clinical management29, 30. Increased nuclear YAP expression was found to be associated with poor patient prognosis17. However, the specific role of YAP1 in ovarian cancer metastasis has not been evaluated so far. By analyzing protein expression and quantifying nuclear versus cytoplasmic levels of YAP1 protein in 21 matched primary high-grade serous ovarian carcinomas and metastatic nodules (Supplementary Table 1), we found that primary tumors showed cytoplasmic YAP1 protein expression in almost 50% of tumor cells. In contrast, more than 70% of tumor cells in metastatic tumor nodules showed primarily nuclear YAP1 protein expression (Fig. 5a, b). Thrombocytosis significantly correlates with increased metastasis31, 32 and is a hallmark of many solid tumors including ovarian cancer. We have recently shown that transfused platelets infiltrate into tumor tissue and increase tumor weight after intraperitoneal injections of cancer cells4. To investigate the role of platelets and YAP1 in ovarian cancer metastasis, we injected 2 × 105 HEYA8 human ovarian cancer cells into the left ovary of nude mice and generated thrombocytosis in mice by platelet transfusions twice weekly. Five days after tumor cell injections, treatments with the well-characterized 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes2, 33, 34 carrying either non-targeting siRNA (siCTRL) or YAP1 siRNA alone, or in conjunction with platelet transfusions were started and repeated twice weekly for 4 weeks. Intriguingly, our results indicate that platelet transfusion in siCTRL-DOPC-treated mice only slightly increased primary tumor growth in the ovary. This trend was, however, not significant (Fig. 5c). In stark contrast, the number of metastatic nodules (Fig. 5d, e and Supplementary Fig. 5a) and their aggregate tumor weight (Supplementary Fig. 5b) increased by up to 140% in platelet-transfused mice. Importantly, this effect of thrombocytosis on the number and total weight of metastatic nodules was completely abolished by simultaneous depletion of YAP1. Moreover, primary tumor weight, number of nodules and aggregate total tumor weight in the control and both YAP1 siRNA-DOPC groups were comparable, irrespective of platelet transfusions (Fig. 5c, d and Supplementary Fig. 5b). Representative IVIS images are shown in Supplementary Fig. 5c. Mouse weights at the time of necropsy were not significantly different, suggesting that neither the injection of platelets nor YAP1 siRNA-DOPC treatment had any obvious harmful effects in vivo (Supplementary Fig. 5d). To verify the efficiency of intraperitoneally injected YAP1 siRNA in reducing YAP1 in vivo, we performed protein analysis on resected tumors using Western blotting for total YAP1 and pYAPS127 (Fig. 5f and Supplementary Fig. 5e, f) and immunohistochemistry for total YAP1 (Fig. 5g). These results confirmed efficient knockdown of YAP1 in whole tumor lysates and histological tumor sections. Evaluation of nuclear versus cytoplasmic YAP1 in primary and metastatic tumor nodules receiving platelet transfusions or vehicle treatment revealed an up to 2.3-fold increase in the number of nuclear YAP1-positive cells in metastatic nodules in mice with thrombocytosis (Fig. 5h) compared to primary tumors and metastatic nodules from mice without thrombocytosis, further supporting the role of YAP1 in metastatic spread of ovarian cancer. In addition, immunohistochemical analysis of tumors using antibodies against cleaved caspase 3 and Ki67 showed decreased apoptosis (Fig. 5i) and increased proliferation (Supplementary Fig. 5g) after platelet transfusion, whereas knockdown of YAP1 in tumors reversed these effects.

Fig. 5 Inhibition of YAP1 in vivo impedes thrombocytosis-enhanced metastasis. a, b Immunohistochemical staining and quantification of nuclear versus cytoplasmic YAP1 protein expression in 21 matched primary high-grade serous ovarian carcinomas and metastatic nodules. N > C: nuclear YAP1 > cytoplasmic YAP1; N = C: nuclear YAP1 = cytoplasmic YAP1; N < C: nuclear YAP1 < cytoplasmic YAP1 (n = 21, two-sided Student’s t-test). c–e Aggregate tumor weight of ovarian primary tumor c, number of metastatic tumor nodules d and representative necropsy pictures e in mice receiving control (siCTRL) or YAP1 (siYAP) siRNA with or without platelet transfusion (PLTS TRANS) twice weekly, respectively (n = 10, one-way ANOVA followed by a Tukey’s multiple comparison post hoc test). f, g Validation of YAP1 knockdown using western blot analysis of whole tumor lysates f or immunohistochemical staining g with antibodies against YAP1 (for WB and IHC) and phosphorylated YAP1 (S127, for WB). GAPDH was used as a loading control (for WB). Representative immunohistochemical images and western blot after processing of tumors from 7 mice. h Immunohistochemical analysis and quantification of nuclear versus cytoplasmic YAP1 positive tumor cells in tumor sections of primary and metastatic nodules. N > C: nuclear YAP1 > cytoplasmic YAP1; N = C: nuclear YAP1 = cytoplasmic YAP1; N < C: nuclear YAP1 < cytoplasmic YAP1 (n = 7, two-sided Student’s t-test). Representative immunohistochemical images after processing of primary and metastatic nodules from 7 mice. i Immunohistochemical staining for cleaved caspase 3 (CC3) and quantification of the number of CC3-positive cells per high power field (HPF) in tumors treated with control (siCTRL) or YAP1 (siYAP) siRNA with or without platelet transfusions twice weekly (n = 7, one-way ANOVA followed by a Tukey’s multiple comparison post-hoc test). Representative immunohistochemical images after processing of tumors from seven mice. Bars and error bars represent mean values and the corresponding SEMs (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bars = 50 µm Full size image

Platelet-induced RhoA increases MYPT1–YAP1 interaction

Detachment of cancer cells from the extracellular matrix alters cellular architecture, focal adhesion formation and cytoskeletal arrangement35. The Rho family small GTPases play a key role in actin cytoskeleton organization, and RhoA has been shown to play a critical role in mediating the effect of cell attachment on YAP1 phosphorylation, likely through actin cytoskeleton organization36. To assess whether RhoA might also be involved in the platelet-induced dephosphorylation of YAP1 in detached ovarian cancer cells, we performed RhoA pulldown activation assays in HEYA8 and OVCAR8 cells. Interestingly, platelets strongly increased RhoA activation in these tumor cells (Fig. 6a). Moreover, treatment of cells with the RhoA inhibitor botulinum toxin C3 (1.0 µg ml−1) inhibited Rho activity (Supplementary Fig. 6a) and YAP1 dephosphorylation induced by platelet co-incubation while leaving total YAP1 unchanged (Fig. 6b). More importantly, subsequent analysis of anoikis levels in the same set of cells revealed that RhoA inhibition abolished the protective effect of platelets and increased anoikis levels compared to cells exposed to platelets alone (Fig. 6c). This effect was specific to the platelet-induced anoikis resistance since treatment of detached cells with the Rho inhibitor alone did not change anoikis rates compared to untreated cells, both in HEYA8 and OVCAR8 cells (Supplementary Fig. 6b, c). In contrast, inhibition of Rho kinase (ROCK) with the small-molecule inhibitor Y-27632 did not prevent YAP1 dephosphorylation induced by platelet co-incubation in HEYA8 and OVCAR8 (Supplementary Fig. 6d) and had no influence on platelet-induced anoikis resistance (Supplementary Fig. 6e, f). These results suggest that platelets enhance RhoA activation in detached cancer cells, which in turn activates YAP1 by promoting its dephosphorylation and leading to higher anoikis resistance.

Fig. 6 Regulation of YAP1 activity by RhoA-MYPT1-PP1 axis controls anoikis. a Analysis of active RhoA using RhoA-GTP pulldown assay in HEYA8 and OVCAR8 cells with or without platelet co-incubation for two hours. RhoA expression in input samples was used as control for quantification and GAPDH as loading control (n = 2, two-sided Student’s t-test). b Protein analysis of phosphorylated (S127) and total YAP1 in HEYA8 and OVCAR8 cells after platelet co-incubation for two hours with or without 1.0 µg ml−1 of Rho inhibitor C3 transferase (n = 3). c Bar graphs showing the number of dead (SYTOX Red positive, black) and living (SYTOX Red negative, red) HEYA8 or OVCAR8 ovarian cancer cells after growing for 72 hours in low attachment with or without platelet co-incubation and treatment with 1.0 µg ml−1 of Rho inhibitor C3 transferase (n = 3, two-sided Student’s t-test). d Protein analysis for phosphorylated MYPT1 (T853, T696 and S507), total MYPT1, phosphorylated YAP1 (S127) and total YAP1 in HEYA8 and OVCAR8 ovarian cancer cells after two hours of low attachment with platelet co-incubation or treatment of 1.0 U ml−1 Rho activator (n = 3). e YAP1 co-immunoprecipitation assays showing MYPT1 interaction with YAP1 after platelet co-incubation or treatment with 1.0 U ml−1 Rho activator for two hours under low-attachment conditions. Similar pulldown of YAP1 protein was confirmed. GAPDH was used as loading control (n = 3). f, g Protein analysis for phosphorylated (S127) and total YAP1 after platelet co-incubation for two hours with or without pre-treatment with 30 nM phosphatase inhibitor Calyculin A (CA) for 30 minutes in HEYA8 f and OVCAR8 g cells (left panels, n = 3). Bar graphs representing the number of dead (SYTOX Red positive, black) and living (SYTOX Red negative, red) HEYA8 f or OVCAR8 g ovarian cancer cells after platelet co-incubation for 72 h with or without pre-treatment with 30 nM phosphatase inhibitor Calyculin A (CA) for 30 min (right panels, n = 3, two-sided Student’s t-test). h Proposed model of YAP1 activation, anoikis resistance and metastasis in cancer cells by platelets Full size image

PP1 and PP2 are two protein phosphatase family members that have been shown to dephosphorylate YAP1/237, 38. The myosin phosphatase target subunit 1 (MYPT1 or also called PPP1R12A) is a crucial regulatory subunit of myosin phosphatase PP1, and has been shown to interact with activated RhoA39. MYPT1 is a well-known PP1-interacting protein, which enhances the specificity of PP1 for various substrates40, 41. MYPT1-PP1 activity is controlled through inhibitory phosphorylation of MYPT1 at threonine 696 (T696) and 853 (T853)42. Interestingly, MYPT1-PP1 was previously shown to regulate NF2/Merlin phosphorylation thereby modulating the Hippo signaling pathway43, 44. Hence, we hypothesized that the platelet-induced YAP1 dephosphorylation might be due to a RhoA-stimulated activation of the MYPT1-PP1 phosphatase leading to an enhanced interaction with YAP1. We investigated whether platelets alone or a direct activation of the RhoA signaling pathway would modulate MYPT1 phosphorylation in detached HEYA8 and OVCAR8 cells. We either co-incubated the cancer cells with platelets or treated cancer cells with 1 U ml−1 of calpeptin, an activator of Rho family GTPases45. Treatment of cells with calpeptin induced a robust RhoA activation (Supplementary Fig. 6g). After 2 h, protein lysates were collected and immunoblot analysis indicated that similarly to platelet co-incubation, activation of RhoA by calpeptin reduced YAP1S127 and the inhibitory phosphorylation of MYPT1 at T696 and T853 while leaving the phosphorylation of serine 507 in MYPT1, used as a negative control, unchanged (Fig. 6d). This suggests that MYPT1-PP1 activity is increased by platelet incubation. Interestingly, immunoprecipitation of YAP1 in HEYA8 and OVCAR8 cells after platelet co-incubation or RhoA activation revealed a substantial increase in YAP1-MYPT1 interaction (Fig. 6e). These data suggest that platelets can induce the intracellular phosphatase activity causing dephosphorylation of YAP1, which in turn activates its downstream signaling to enhance anoikis resistance. Consequently, we predicted that blocking of phosphatase activity would abolish the platelet effects and restore anoikis rates. In fact, pre-treatment of HEYA8 and OVCAR8 cells with 30 nM of the serine and threonine phosphatase inhibitor Calyculin A (CA) for 30 min prior to incubation for two hours with 100 × 106 platelets in low-attachment plates kept YAP1 in a hyperphosphorylated state (Fig. 6f, g, left panels). Importantly, analysis of anoikis rates after 72 hours revealed that phosphatase inhibitors diminished the protective effect of platelets against anoikis in cancer cells (Fig. 6c, d, right panels). This further indicates that platelets induce an active dephosphorylation of YAP1 in cancer cells, which in turn activates YAP1 signaling allowing survival under low-attachment conditions.

In conclusion, we have identified platelets as major mediators for survival of detached cancer cell in vitro and in vivo, and provided evidence that YAP1 activation is critical for metastasis in ovarian and colorectal cancer models, at least partly due to its essential role in mediating anoikis resistance. Mechanistically, we identified a function for platelets in regulating MYPT1-PP1 activity, and we detected a platelet-induced YAP1-MYPT1 interaction leading to an enhanced dephosphorylation of YAP1 causing its nuclear trans-localization, and subsequent gene expression changes leading to inhibition of apoptosis and increase in cell survival. Inhibition of YAP1 or phosphatase activity holds potential to reduce metastatic spread and could have substantial clinical implications (Fig. 6h).