Systematic analysis of the Hippo pathway organization and oncogenic alteration in evolution

Chen, Yuxuan; Han, Han; Seo, Gayoung; Vargas, Rebecca Elizabeth; Yang, Bing; Chuc, Kimberly; Zhao, Huabin; Wang, Wenqi

doi:10.1038/s41598-020-60120-4

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Published: 21 February 2020

Systematic analysis of the Hippo pathway organization and oncogenic alteration in evolution

Yuxuan Chen^1,2^na1,
Han Han¹^na1,
Gayoung Seo¹,
Rebecca Elizabeth Vargas¹,
Bing Yang¹,
Kimberly Chuc¹,
Huabin Zhao² &
…
Wenqi Wang¹

Scientific Reports volume 10, Article number: 3173 (2020) Cite this article

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Abstract

The Hippo pathway is a central regulator of organ size and a key tumor suppressor via coordinating cell proliferation and death. Initially discovered in Drosophila, the Hippo pathway has been implicated as an evolutionarily conserved pathway in mammals; however, how this pathway was evolved to be functional from its origin is still largely unknown. In this study, we traced the Hippo pathway in premetazoan species, characterized the intrinsic functions of its ancestor components, and unveiled the evolutionary history of this key signaling pathway from its unicellular origin. In addition, we elucidated the paralogous gene history for the mammalian Hippo pathway components and characterized their cancer-derived somatic mutations from an evolutionary perspective. Taken together, our findings not only traced the conserved function of the Hippo pathway to its unicellular ancestor components, but also provided novel evolutionary insights into the Hippo pathway organization and oncogenic alteration.

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Introduction

A long-standing question in developmental biology is how metazoan species acquired the ability to precisely control their tissue/organ size¹. This intriguing phenomenon could be traced to some non-metazoan organisms, since their aggregative behaviors can be similarly regulated^2,3,4. Initially discovered in Drosophila, the Hippo pathway is a highly conserved pathway known for its crucial role in organ size control via restricting cell proliferation and inducing apoptosis^5,6,7,8,9, shedding light on this fundamental question in evolution.

In Drosophila, the core components of the Hippo pathway comprise two serine/threonine kinases Hippo^{10,11,12,13,14} and Warts^15,16, adaptor proteins Salvador^17,18 and Mats¹⁹, a transcriptional co-activator Yorkie²⁰ and a transcriptional factor Scalloped^21,22,23. Together with Salvador, Hippo phosphorylates and activates Warts, which in turn phosphorylates Yorkie^24,25. Yorkie can shuttle between the cytoplasm and nucleus, a process tightly controlled by the Warts-mediated phosphorylation^24,25. Upon phosphorylation, Yorkie is recognized by 14-3-3 proteins and sequestered in the cytoplasm^24,25. When Hippo signaling is inactivated, Yorkie translocates into the nucleus, where it forms a complex with Scalloped to initiate the transcription of genes involved in proliferation and anti-apoptosis, two critical events required for tissue/organ growth. The Hippo/Salvador-Warts/Mats kinase cascade can be regulated by multiple upstream regulators. Among them, Merlin²⁶, Kibra^27,28,29 and Fat^30,31,32 have been implicated to play conserved roles in the mammalian Hippo pathway. Moreover, Hippo is not absolutely required for the phosphorylation and activation of Warts, since another two serine/threonine kinases Happyhour and Misshapen were also found responsible for Warts activation in both Drosophila and mammals^33,34,35, highlighting the complexity of the Hippo pathway regulation.

Although multiple Hippo pathway-related studies have been carried out in species from Drosophila to mammals for decades, its components and functions could have deeper conservative roots in evolution. Indeed, several comparative analyses have identified the Hippo pathway components in the ancestor species of cnidarians and bilaterians^36,37,38,39. In addition, a recent study not only further revealed a non-metazoan origin of the Hippo pathway in evolution, but also uncovered a unicellular organism, Capsaspora owczarzaki, where a complete Hippo pathway has already been developed⁴⁰. Notably, several Hippo pathway core components including Mats, Hippo and Warts can be even traced to earlier non-metazoan lineages than Capsaspora owczarzaki⁴⁰. How theses Hippo ancestor components developed their conserved roles in Hippo signaling and how a functional Hippo pathway was evolved from these ancestor components are still largely unknown.

In this study, we traced the evolution of the Hippo pathway organization in both metazoan and non-metazoan species. Specifically, we examined the gene duplication history for the Hippo pathway components from Drosophila to human and functionally characterized the unicellular ancestors for the Hippo pathway core components. Given the crucial role of the Hippo pathway in growth control and cancer inhibition, we also systematically analyzed the evolutionarily conserved residues of the Hippo pathway components in human cancer genomes. Taken together, our study not only illustrated the roadmap for the Hippo pathway evolution from its unicellular origin, but also provided insights into the oncogenic alteration of this key tumor suppressive pathway from a novel perspective.

Results

Whole genome duplication results in the emergence of paralogous genes for the mammalian Hippo pathway components

Compared to the Drosophila Hippo pathway, an interesting phenomenon is the development of paralogous genes for many mammalian Hippo pathway components^8,9. For example, the Drosophila Hippo pathway components Hippo, Warts and Mats were all duplicated into two paralogous genes MST1/MST2, LATS1/LATS2 and MOB1A/MOB1B in mammals, respectively. Some Drosophila Hippo pathway components even have more than two paralogous genes in mammals including Scalloped (TEAD1/TEAD2/TEAD3/TEAD4 in mammals), Kibra (KIBRA or WWC1, WWC2/WWC3 in mammals), Happyhour (MAP4K1/MAP4K2/MAP4K3/MAP4K5 in mammals) and Misshapen (MAP4K4/MAP4K6/MAP4K7 in mammals). However, as for Yorkie (YAP in mammals), Salvador (SAV1 in mammals) and Merlin (NF2 in mammals), they remained as one gene in mammals. Moreover, there is no Drosophila orthologous gene for Taz and its evolutionary history remains largely unknown.

To elucidate the evolutionary history of the Hippo pathway paralogous genes, we examined the Hippo pathway components in the species ranging from Drosophila to human. As shown in Fig. 1A, each Hippo pathway component only has one orthologous gene within the tested organisms of Insecta, Branchiopoda, Polychaeta, Clitellata, Gastropoda, Echinoidea and Leptocardii. Interestingly, the emergence of the Hippo pathway paralogous genes was firstly identified in the fish species and they were remained in amphibians, reptiles and mammals (Fig. 1A). Among the Hippo pathway components, Yorkie and Merlin were actually duplicated in fish too, but their paralogs were lost in amphibians and mammals, respectively (Fig. 1A); Taz was originated in fish and remained as one gene during evolution (Fig. 1A); no paralogous genes of Salvador were identified in all the tested species from Drosophila to human (Fig. 1A). Collectively, these data indicate that fish is the turning point for the Hippo pathway in evolution, where many Hippo pathway components acquired their paralogous genes.

Notably, the Hippo pathway paralogous gene number in fish species was highly correlated with the rounds of whole genome duplication (WGD) (Fig. S1A), including three rounds of WGD happened in different fish ancestors and two in the vertebrate ancestors⁴¹. To determine whether WGD resulted in the emergence of the Hippo pathway paralogous genes in human, we analyzed the statistical confidence q-score for each paralog pair of the Hippo pathway genes in human genome, where several invertebrate species were included as an outgroup. As shown in Fig. 1B, the q scores generated among the Hippo pathway paralogous genes were more significant than the ones generated between these Hippo pathway genes and control genes, suggesting that these Hippo pathway paralogous genes were emerged as a result of WGD. Moreover, compared with the average rate of genes saved after WGD in the human genome (30.1%)⁴², around 72% of the Hippo pathway components retained their duplicated genes after WGD (Fig. 1C). Taken together, these data suggest that the Hippo pathway is highly enriched with paralogous genes after the WGD in vertebrate ancestors, allowing its stable role in regulating tissue/organ homeostasis within the following species such as amphibians, reptiles and mammals in evolution.

YAP and TAZ share the same evolutionary origin

By analyzing the evolutionary history of the Hippo pathway paralogous genes, we noticed that TAZ was firstly identified in fish (Fig. 1A) and it shares a significant q-score with YAP in the WGD gene analysis (Fig. 1B). Moreover, YAP and TAZ are similarly regulated and can function redundantly in the mammalian Hippo pathway. These evidences suggest that TAZ could be developed from Yorkie during WGD. To test this hypothesis, we performed a syntenic analysis of the surrounding regions of YAP and TAZ genes in both fish and human genomes, where the ancient vertebrate Ciona intestinalis genome was included as an outgroup (Fig. S1B). Interestingly, the genes surrounding Yorkie in Ciona intestinalis genome were similar to the genes near YAP and TAZ in both fish and human genomes (Fig. S1B). Moreover, the genomic regions near YAP and TAZ showed a strong syntenic correlation between fish and human genomes (Fig. 1D). These data are consistent with TAZ and YAP being paralogous genes because of WGD.

In addition to the redundant roles between YAP and TAZ in the Hippo pathway, accumulated evidences also indicated that YAP has a stronger influence than TAZ in cellular physiology-related events such as proliferation, migration⁴³. To elucidate the functional divergence between YAP and TAZ, we examined their coding sequence divergence by using the program DIVERGE 3.0⁴⁴. Interestingly, the YAP and TAZ coding sequences belong to the Type I divergence with the coefficient (θ₁) significantly greater than 0 (Fig. S1C). This finding suggests a potential site-specific rate shift between YAP and TAZ after the gene duplication, which may lead to their partially functional difference.

Roadmap of the Hippo pathway evolution in unicellular species

Next, we further explored how a complete Hippo pathway was evolved from its ancestor components. Consistent with a previous study⁴⁰, most of the Hippo pathway core components can be identified in unicellular species (Fig. 2A). Moreover, a functional and complete Hippo pathway has already emerged in an unicellular organism Capsaspora owczarzaki⁴⁰. Actually, several Hippo ancestor components can be traced to even earlier unicellular species than Capsaspora owczarzaki (Fig. 2A): Mats is the earliest appeared Hippo pathway component that can be identified in the unicellular species such as Tetrahymena thermophile, Thalassiosira pseudonana, Chlamydomonas reinhardtii, Trichomonas vaginalis, Naegleria gruberi, Arabidopsis thaliana; following that, Hippo was the next Hippo pathway component appearing in Amoebozoa (e.g. Acanthamoeba castellanii), Diclyostelia (e.g. Dictyostelium discoideum) and Apusozoa (e.g. Thecamonas trahens); another Hippo pathway kinase cascade component Warts was firstly identified in Fonticula alba as well as the following fungi species (e.g. Saccharomyces cerevisiae), while both of these two species did not contain Hippo. After these three Hippo components, the kinase cascade (i.e. Hippo/Warts/Mats) and transcriptional complex (i.e. Yorkie/Scalloped) in the Hippo pathway became completed in Spizellomyces punctatus and Corallochytrium limacisporum, respectively. Finally, an intact Hippo pathway was eventually developed in Capsaspora owczarzaki. (Fig. 2A).

We also examined the co-evolutionary relationship among the Hippo pathway core components by analyzing their gene sequences from unicellular organisms to human. Interestingly, the earliest emerged Hippo pathway components Mats/MOB1, Warts/LATS and Hippo/MST showed a relatively weak correlation in between and with other Hippo pathway components as indicated by the low multiple R value (Fig. 2B). In contrast, strong coevolution was identified among the Hippo nuclear components (Scalloped/TEAD and Yorkie/YAP), one kinase cascade adaptor (Salvador/SAV1) and two upstream regulators (Merlin/NF2 and Kibra/KIBRA). This result indicated that the intrinsic function of Mats/MOB1, Warts/LATS and Hippo/MST could be established independently at their evolutionary origins, whose upstream and downstream Hippo pathway components were then coevolved, allowing the development of a regulatory network and a functional output for these three Hippo pathway ancestor components.

To test this hypothesis, we characterized the roles of the Mats, Hippo and Warts ancestor proteins in Tetrahymena thermophile (hereafter called its Mats as “Tetra-Mats” in short), Acanthamoeba castellanii (hereafter called its Hippo as “Acan-Hippo” in short) and Fonticula alba (hereafter called its Warts as “Fonti-Warts” in short), respectively, in their corresponding human knockout (KO) cells. Interestingly, both Tetra-Mats and Acan-Hippo were able to partially rescue YAP’s cytoplasmic localization in the MOB1A/B double KO (DKO) (Fig. 2C) and MST/MAP4K-8KO HEK293A cells (Fig. 2D), respectively. Surprisingly, Fonti-Warts has already acquired a full or even a stronger ability to rescue YAP’s cytoplasmic localization in the LATS1/2 DKO HEK293A cells as compared to its human othologous protein LATS1 (Fig. 2E). As controls, the MOB1 LATS-unbinding mutant (MOB1-E55A) (Fig. 2C), MST1 kinase dead mutant (MST1-KR) (Fig. 2D) and LATS1 kinase dead mutant (LATS1-KR) (Fig. 2E) all failed to rescue YAP’s cytoplasmic localization in their corresponding KO cells. Collectively, these results not only suggested the intrinsically conserved Hippo pathway activity for these three Hippo ancestor components, but also indicated a different alteration of their activities in evolution.

Opposite evolutionary trends exist among the three Hippo pathway ancestor components

Next, we further assessed the role of Fonti-Warts by taking human LATS1 as a control in MDA-MB-231, a human triple negative breast cancer cell line with the impaired Hippo signaling activity due to NF2 deficiency⁴⁵ (Fig. 3A). Consistent with our reconstitution study (Fig. 2E), Fonti-Warts showed a stronger ability than human LATS1 in inducing YAP phosphorylation at S127 (Fig. 3A), promoting YAP’s cytoplasmic translocation (Fig. 3B) and inhibiting YAP downstream gene transcription (Fig. 3C). Warts/LATS activation requires its association with adaptor Mats/MOB1^46,47 and the NF2-mediated its membrane translocation and the followed Hippo/MST-induced phosphorylation⁴⁸. Interestingly, Fonti-Warts interacted with human MOB1 (Fig. 3D) and showed membrane localization even in the NF2-deficient MDA-MB-231 cells (Fig. 3B), suggesting that Fonti-Warts has already acquired the basic properties required for its activation. Moreover, Fonti-Warts displayed much higher kinase activity than human LATS1 by comparing their abilities to phosphorylate the bacterially purified GST-YAP (Fig. 3E) and induce their autophosphorylation (Fig. 3F). These data suggested a decrease of Warts/LATS kinase activity from its unicellular ancestor in evolution.

Since Mats/MOB1 and Hippo/MST are two key regulators for Warts/LATS, we also characterized their unicellular ancestor proteins Tetra-Mats and Acan-Hippo by taking their human orthologs MOB1 and MST1 as controls, respectively. In contrast to human MOB1, we hardly detected the interaction between Tetra-Mats and human LATS1 (Fig. 3G), which may explain why Tetra-Mats only partially rescued YAP’ cytoplasmic localization in the MOB1A/B DKO cells (Fig. 2C). Moreover, Tetra-Mats failed to bind Fonti-Warts (Fig. 3D). On the other hand, the activation of Hippo/MST is regulated by its dimerization and autophosphorylation^{49,50,51,52,53}. Although Acan-Hippo can form as a dimer (Fig. 3H), its intrinsic kinase activity is relatively low as compared with human MST1 (Fig. 3I,J). Moreover, Acan-Hippo failed to form a complex with human adaptor protein SAV1 (Fig. 3K). These functional deficiencies could explain why Acan-Hippo has limited ability to rescue YAP’s cytoplasmic localization in the MST/MAP4K-8KO cells (Fig. 2D). These data indicated that both Mats/MOB1 and Hippo/MST may gradually acquire the abilities to regulate Warts/LATS from their unicellular ancestors in evolution.

Collectively, our findings suggested opposite evolutionary directions for the three Hippo pathway ancestor components. As for both Tetra-Mats and Acan-Hippo, their intrinsic activities/functions were further acquired or strengthened when evolved to their human orthologs (Fig. 3L); while the intrinsic activity of Fonti-Warts was diluted in this evolutionary process (Fig. 3L). Given the crucial role of Warts/LATS in relaying the upstream signaling to downstream gene transcription in the Hippo pathway, these opposite evolutionary trends may pioneer the fine-tuned regulation of LATS kinase in human.

Exploring the oncogenic alteration of the Hippo pathway in evolution

Accumulating evidences suggest that dysregulation of the Hippo pathway and hyperactivation of YAP have been frequently associated with various types of human cancers including breast, liver, lung, ovary, colon, kidney, brain and others^8,9,54. Moreover, recent human cancer genome studies also connected the genetic alteration of the Hippo pathway components with cancer development^55,56. Given the evolutionarily conserved roles for the Hippo pathway components, it will be interesting to characterize the oncogenic alteration of the Hippo pathway in evolution.

To achieve this, we profiled the protein sequences of the Hippo pathway core components from unicellular species to human, assigned each amino acid residue in these components with a conservation score, and analyzed the conservative distribution of the Hippo pathway missense mutations derived from two cancer genome databases TCGA and COSMIC (Fig. 4A). Actually, these cancer-derived missense mutations were widely distributed through the whole protein sequences, but not significantly correlated with the conservation of the mutated residues (Fig. 4A,B). These results suggested that the Hippo pathway missense mutations could be randomly occurred in tumorigenesis (i.e., “passenger” mutations). Interestingly, when all the TCGA missense mutations were experimentally characterized using a series of Hippo pathway component KO cells⁵⁷, a group of missense mutations that can functionally inhibit the Hippo signaling activity were found highly enriched in the residues with high conservation scores (Fig. 4B). These findings highlighted the significant role of evolutionarily conserved residues in maintaining a normal tumor suppressive function of the Hippo pathway.

Next, we subjected the Hippo-associated conserved missense mutations to 32 types of human cancers and found that they were largely enriched in several types of cancers, such as mesothelioma (MESO) and uterine corpus endometrial carcinoma (UCEC) (Fig. 4C). MESO is known to contain genetic alteration in multiple Hippo pathway components such as NF2 and LATS2⁵⁸. Our comparative analysis further suggested MST1, MAP4K4 and LATS1 as another group of Hippo pathway components, whose conserved missense mutations may also involve in MESO development (Fig. 4C). Unexpectedly, UCEC was found significantly enriched with conserved missense mutations in multiple Hippo pathway components including KIBRA, MST1, MST2, MAP4K3, MAP4K6, MAP4K7, LATS1 and LATS2 (Fig. 4C). Since Hippo signaling dysregulation has not been well characterized in UCEC, next, we experimentally tested some of the conserved missense mutations for several frequently-mutated Hippo pathway components in UCEC. These missense mutations were chosen from our unpublished Hippo pathway somatic mutation screen and were at least reported in different patient samples based on public cancer databases (i.e., TCGA, COSMICS). As shown in Fig. 4D,E, these tested missense mutations all failed the Hippo pathway components LATS1, LATS2, MST2 and MAP4K3 to rescue YAP’s cytoplasmic localization in their corresponding KO cells, suggesting that alteration of the Hippo pathway components at their evolutionarily conserved residues could play an important role in UCEC development.

Taken together, our study not only provided a novel insight into the cancer-related Hippo signaling dysregulation from an evolutionary perspective, but also indicated a new pathological relevance for the Hippo pathway in UCEC.

Discussion

In this study, we focused on the evolutionary history of the Hippo pathway by addressing three questions. First, why is the paralogous gene number for the Hippo pathway components different between Drosophila and human? Second, although an intact and conserved Hippo pathway can be traced to unicellular species, how was this pathway evolved to be a functional one from its ancestor components? Third, what is the connection between the Hippo pathway evolution and its oncogenic alteration in cancer development?

As for the current Hippo signaling research, most of our studies are being carried out in the species/model systems ranging from Drosophila to mammals. As compared with Drosophila, one of the major differences in the mammalian Hippo pathway is the development of paralogous genes for many Hippo pathway components. Our current study suggested that the turning point of this paralogous gene number change happened in the process of whole genome duplication in fish (Fig. 1A). In addition to the Hippo pathway, our unpublished data also showed that the increase of paralogous gene number in other key signaling pathways including PI3K-AKT, WNT, NOTCH, TGF-β, was also initiated in fish. Through it, the complexity and stability of these key signaling pathways were further developed, which maximized their abilities to control tissue/organism homeostasis. In addition, we found that TAZ was originated from Yorkie during the whole genome duplication in fish (Figs. 1D and S1B), providing an evolutionary insight into their redundant roles in the mammalian Hippo pathway.

To trace the origin of the Hippo pathway, we majorly focused on the firstly emerged Hippo ancestor components Mats, Hippo and Warts in the unicellular species Tetrahymena thermophile, Acanthamoeba castellanii and Fonticula alba, respectively (Fig. 2A). In contrast to Capsapsora owczarzaki that has already developed an intact Hippo pathway⁴⁰, these unicellular species only have one or two Hippo pathway components (Fig. 2A), allowing us to characterize their intrinsic activities at the “true origin” of the Hippo pathway. Interestingly, these three Hippo pathway ancestor components all showed conserved functions even in human cells (Fig. 2B) and shared key domains and regulatory resides with their orthologs in Capsapsora owczarzaki, Drosophila and human (Fig. S2 and S3). These data suggest that the conserved role of the Hippo pathway could be originated from its ancestor components.

As compared to their human orthologs, both Mats and Hippo unicellular ancestors were evolved in a “forward” direction where their activities in the Hippo pathway were turned to be strengthened in evolution (Fig. 3L). Surprisingly, the evolution of Warts unicellular ancestor seems to be occurred in a “backward” manner (Fig. 3L), since its intrinsic kinase activity was much higher than that of its human ortholog LATS (Fig. 3A–F). These findings implicated that regulation of the Warts activity could be a central event driving the Hippo pathway evolution. During the course that Warts activity was decreased from its ancestor, the adaptor function of Mats and kinase activity of Hippo were both strengthened, a process accompanied by the emergence of additional upstream regulators and downstream effectors in the Hippo pathway (Fig. 2A). Based on this hypothesis, this activity adjustment process from the three Hippo ancestor components could be gradually compromised in the species where most of the Hippo pathway components have been emerged, such as Capsapsora owczarzaki. Indeed, Capsapsora owczarzaki Hippo protein has shown a similar level of activity to its Drosophila ortholog in regulating Drosophila Yorkie⁴⁰. Taken together, our findings provide a novel insight into the Hippo pathway evolution from its ancestor components to an intact pathway in unicellular organisms.

Cancer can be considered as part of an evolution that is now taking place in human, where its associated mutations may result in novel functions and selective advantages^59,60. As a well-established tumor suppressor pathway in control of tissue homeostasis, the Hippo pathway kindly plays a negative role in this evolutionary progress. Although the functionally validated missense mutations in the Hippo pathway were highly enriched in the conserved sites (Fig. 4B), the ratio is relatively low (Fig. 4B) and the associated cancer type number is also limited (Fig. 4C). In contrast, a large portion of the cancer-derived missense mutations were found at the sites with low conservation scores (less than 7), but they did not affect the Hippo pathway activity (Fig. 4A,B). These findings suggest that mutations at the sites with low conservation may result in novel oncogenic functions for the Hippo pathway components to bypass an active Hippo signaling pathway in tumorigenesis.

Here, we systematically analyzed the Hippo pathway in evolution by elucidating its paralogous gene history, tracing its conserved functions to its unicellular ancestor components and evaluating its cancer-related dysregulation from an evolutionary perspective. However, exactly how the Hippo pathway is able to largely reserve its conserved functions from its unicellular ancestor species to human is still a mystery. In addition, as a key signaling pathway in maintaining tissue homeostasis and preventing cancer development, it is possible that the Hippo pathway could also play a role in regulating an organism’s evolution, which deserves further investigation.

Materials and Methods

Antibodies and chemicals

For Western blotting, anti-α-tubulin (T6199, 1:5000 dilution) and anti-Flag (M2) (F3165-5MG, 1:5000 dilution) antibodies were obtained from Sigma-Aldrich. Anti-Myc (sc-40, 1:500 dilution) and anti-GST antibodies (sc-138, 1:1000 dilution) were purchased from Santa Cruz Biotechnology. Anti-phospho-YAP (S127) (4911S, 1:1000 dilution) and anti-phospho-LATS1 (Thr1079) (8654S, 1:1000 dilution) antibodies were purchased from Cell Signaling Technology. Anti-hemagglutinin (HA) monoclonal antibody (901514, 1:2000 dilution) was obtained from BioLegend. Anti-Thiophosphate ester antibody (ab92670, 1:1000 dilution) was purchased from Abcam. The YAP and MBP polyclonal antibodies were generated as previously described^57,61. ATP-γ-S kinase substrate (ab138911) and p-Nitrobenzyl mesylate (ab138910) were obtained from Abcam. For immunostaining, an anti-YAP (sc-101199, 1:200 dilution) monoclonal antibody was purchased from Santa Cruz Biotechnology. Anti-Flag polyclonal antibody (F7425, 1:5000 dilution) was obtained from Sigma-Aldrich.

Constructs and viruses

Plasmids encoding the indicated genes were obtained from the Human ORFeome V5.1 library or purchased from Harvard Plasmid DNA Resource Core. For Tetrahymena thermophila Mats, Acanthamoeba castellanii Hippo and Fonticula alba Warts, their sequences were optimized for expression in Homo sapiens, synthesized and cloned into a mammalian gene expression vector. All constructs were generated via polymerase chain reaction (PCR) and subcloned into a pDONOR201 vector using Gateway Technology (Invitrogen) as the entry clones. Gateway-compatible destination vectors with the indicated SFB tag, HA tag, Myc tag, GST tag or MBP tag were used to express various fusion proteins. PCR-mediated mutagenesis was used to generate all the indicated site mutations.

Cell culture and transfection

HEK293T cells were purchased from ATCC and kindly provided by Dr. Junjie Chen (MD Anderson Cancer Center). HEK293A cells were purchased from ThermoFisher and kindly provided by Dr. Jae-Il Park (MD Anderson Cancer Center). MDA-MB-231 cells were purchased from ATCC and kindly provided by Dr. Mien-Chie Hung (MD Anderson Cancer Center). HEK293T, HEK293A and MDA-MB-231 cells were maintained in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in 5% CO₂ (v/v) and 1% penicillin and streptomycin.

Plasmid transfection was performed using polyethylenimine and Lipofectamine 3000 (ThermoFisher).

Searches of the Hippo pathway genes in different species

The genome data of unicellular eukaryotes were downloaded from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) genome database. For each main invertebrate phylum and vertebrate class, two species were chosen as representative species from unicellular organisms to multicellular organism. Genes encoding the indicated Hippo pathway core components were isolated from genome sequences of the selected species. The protein sequences of the human and mouse Hippo pathway components were used as queries for the TBLASTN search. The blast results were filtered with e-value <10⁻¹⁰. The introns of the predicted sequences were removed by Genewise (https://www.ebi.ac.uk/Tools/psa/genewise/) by taking their human orthologous genes as queries⁶². The following unique domains that were confirmed by PFAM and SMART databases^63,64 were subjected to the gene searches: the TED domain was used to identify Scalloped/TEAD; the Mob1 phocein region was used to identify Mats/MOB1; both the catalytic domain of the serine/threonine kinase and the MOB1-binding domain were used to identify Warts/LATS; both the catalytic domain of the serine/threonine kinases and C-terminal SARAH domain were used to identify Hippo/MST; both the WW domain and C2 domain were used to identify Kibra/WWC; both the catalytic domain of the serine/threonine kinase and CNH domain were used to identify Misshapen or Happyhour/MAP4K; the B41 and ERM domains were used to identify Merlin/NF2; two WW domains and coiled-coil domain were used to identify Yorkie/YAP; one WW domain and coiled-coil domain were used to identify TAZ (WWTR1); two WW domains were used to identify Salvador/SAV1. All the Hippo pathway candidate genes were further verified via the phylogenetic reconstruction by taking their human genes as controls. Phylogenetic reconstruction for each dataset was conducted as Maximum Likelihood trees using MEGA X with a Tamura-Nei model and 1000 times bootstrap consensus⁶⁵.

Sequence alignment and phylogenetic analysis

Sequence alignments were performed by ClustalW with default parameters and visualized by BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Homologous domains, motifs and important residues were checked by alignment. The nucleotide sequences were generated based on the aligned protein sequences and used to reconstruct phylogenetic trees. Specifically, the gaps and amino acids with missing information were deleted, allowing all amino acid sequences in a good alignment for phylogenetic reconstruction.

The conservation of the Hippo pathway components was estimated by the maximum likelihood paradigm in ConSurf server⁶⁶ using the multiple sequence alignment (MSA) data from all the selected species. The evolutionary tree for each dataset was generated based on the species’ evolutionary relationship as estimated by Timetree (http://www.timetree.org)⁶⁷. The conservation score is a relative value and calculated by ConSurf. Based on the relative degree of conservation, each amino acid site was assigned with a value from 1 to 9. Score one indicated the most variable sites while score 9 represented the most conserved sites. Functional divergence between YAP and TAZ was estimated by the program DIVERGE 3.0⁴⁴.

Characterization of the genes saved after whole genome duplication

We used the Ohnologs⁶⁸, a database of paralogous genes that were remained from whole genome duplication, to calculate the q score between the Hippo pathway genes and their homologous genes, and quantitatively assess the statistical significance of each paralogous pair. To identify syntenic clusters among the YAP and TAZ genomic regions in the indicated species, we used BioMart⁶⁹ to get the information of around 1000 genes surrounding the genes of interest and matched them based on the homolog gene information obtained from BioMart. The syntenic clusters were visualized using R packages genoPlotR and OmicCircos^70,71.

Coevolution analysis

Genetic distance from the multiple sequence alignment data was generated by Mega X with default parameters⁶⁵. To examine the co-evolution of the Hippo pathway core components, we used linear regression analysis to determine the correlation between pairwise evolutionary distances among the Hippo pathway components. Pearson’s correlation coefficient (r) was generated as the output of linear regression analysis to assess the overall evolutionary correlation between any two-gene pairs of interest.

Immunofluorescent staining

Immunofluorescent staining was performed as described previously⁷². Briefly, cells cultured on coverslips were fixed with 4% paraformaldehyde for 10 minutes at room temperature and then extracted with 0.5% Triton X-100 solution for 5 minutes. After blocking with Tris-buffered saline with Tween 20 containing 1% bovine serum albumin, the cells were incubated with primary antibodies for 1 hour at room temperature. After that, the cells were washed and incubated with fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies for 1 hour. To visualize nuclear DNA, cells were counterstained with 100 ng/mL 4′,6-diamidino-2-phenylindole (DAPI) for 2 minutes. The coverslips were mounted onto glass slides with an anti-fade solution and visualized under a Nikon Ti2-E inverted microscope.

Gene inactivation by CRISPR/Cas9 system

The indicated MOB1A/B and LATS1/2 double knockout HEK293A cells were generated through the CRISPR/Cas9 system as described previously⁵⁷. Briefly, five distinct single-guide RNAs (sgRNA) were designed by CHOPCHOP website (https://chopchop.rc.fas.harvard.edu), cloned into lentiGuide-Puro vector (Addgene plasmid # 52963), and transfected into HEK293A cells with lentiCas9-Blast construct (Addgene plasmid # 52962). The next day, cells were selected with puromycin (2 μg/ml) for two days and subcloned to form single colonies. Knockout cell clones were screened by Western blotting to verify the loss of targeted protein expression. The MST/MAP4K-8KO HEK293A cells were kindly provided by Dr. Kun-Liang Guan (University of California at San Diego).

RNA extraction, reverse transcription and real-time PCR

RNA samples were extracted with TRIzol reagent (Invitrogen). Reverse transcription assay was performed using the Script Reverse Transcription Supermix Kit (Bio-Rad) according to the manufacturer’s instructions. Real-time PCR was performed using Power SYBR Green PCR master mix (Applied Biosystems). For quantification of gene expression, the 2^−ΔΔCt method was used. GAPDH expression was used for normalization.

The sequence information for each primer used for gene expression analysis is as follows:

CTGF-Forward: 5′-CCAATGACAACGCCTCCTG-3′;

CTGF-Reverse: 5′-GAGCTTTCTGGCTGCACCA-3′;

CYR61-Forward: 5′-AGCCTCGCATCCTATACAACC-3′;

CYR61-Reverse: 5′-GAGTGCCGCCTTGTGAAAGAA-3′;

GAPDH-Forward: 5′-ATGGGGAAGGTGAAGGTCG-3′;

GAPDH-Reverse: 5′-GGGGTCATTGATGGCAACAATA-3′.

In vitro kinase assay

SFB-LATS1 or its kinase-dead mutant (K734R), SFB-MST1 or its kinase-dead mutant (K59R), SFB-Acanthamoeba castellanii Hippo and SFB-Fonticula alba Warts were expressed in HEK293T cells for 48 hours. The indicated kinases were then pulled down by S protein beads, washed three times in washing buffer (40 mM HEPES, 250 mM NaCl), once in kinase buffer (30 mM HEPES, 50 mM potassium acetate, 5 mM MgCl2), and subjected to the kinase assay in the presence of cold ATP (500 μM) and 2 μg bacterially-purified GST- YAP or MBP-LATS1-C3 (a region containing LATS1 hydrophobic motif)⁵⁷. The reaction mixture was incubated at 30 °C for 30 min, terminated with 2x SDS loading buffer and subjected to SDS–PAGE. Phosphorylation of YAP and LATS1-HM were determined by YAP S127 and LATS1 T1079 phospho-antibodies, respectively. As for ATP-γ-S-based kinase assay, the purified SFB-tagged kinases were subjected to kinase assay in the presence of 500 μM ATP-γ-S. The reaction mixture was incubated at 30 °C for 30 min, supplemented with 2.5 mM p-Nitrobenzyl mesylate (PNBM) at room temperature for 1 h, terminated with 2x SDS loading buffer and subjected to SDS–PAGE. The autophosphorylation of the indicated kinases was detected by an anti-Thiophosphate-ester antibody.

Human cancer genome analyses

The missense mutation information of the Hippo pathway genes was downloaded from cancer genome databases TCGA (via cBioportal) and COSMIC^73,74, and compared with the gene conservation data for the Hippo pathway components and our unpublished experimental screen data.

Quantification and statistical analysis

Each experiment was repeated twice or more unless otherwise noted. There were no samples excluded for the analyses in this study. Data were analyzed by Student’s t-test. SD was used for error estimation. A p value < 0.05 was considered statistically significant.

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Acknowledgements

We thank Drs. Feng Qiao (University of California, Irvine) and Bao-Liang Song (Wuhan University) for coordinating the Wuhan University visiting student program. This work was supported by a NIH grant (GM126048) and an American Cancer Society Research Scholar Grant (RSG-18-009-01-CCG) to WW.

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These authors contributed equally: Yuxuan Chen and Han Han.

Authors and Affiliations

Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, 92697, USA
Yuxuan Chen, Han Han, Gayoung Seo, Rebecca Elizabeth Vargas, Bing Yang, Kimberly Chuc & Wenqi Wang
Department of Ecology, College of Life Sciences, Wuhan University, Wuhan, Hubei, 430072, China
Yuxuan Chen & Huabin Zhao

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Yuxuan Chen
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Han Han
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Gayoung Seo
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Huabin Zhao
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Contributions

W.W. conceived and designed the study. Y.C. performed the evolutionary analyses. H.H. and W.W. performed the experiments with assistance from G.S., R.E.V., B.Y. and K.C. H.Z. analyzed the data and revised the manuscript. Y.C. and W.W. wrote the manuscript.

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Correspondence to Wenqi Wang.

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Chen, Y., Han, H., Seo, G. et al. Systematic analysis of the Hippo pathway organization and oncogenic alteration in evolution. Sci Rep 10, 3173 (2020). https://doi.org/10.1038/s41598-020-60120-4

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Received: 29 December 2019
Accepted: 06 February 2020
Published: 21 February 2020
DOI: https://doi.org/10.1038/s41598-020-60120-4

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