YAP Signaling Research Articles

High Glucose Activates YAP Signaling to Promote Vascular Inflammation.

Background and AimsThe YAP/TAZ signaling is known to regulate endothelial activation and vascular inflammation in response to shear stress. Moreover, YAP/TAZ signaling plays a role in the progression of cancers and renal damage associated with diabetes. However, whether YAP/TAZ signaling is also implicated in diabetes-associated vascular complications is not known.MethodsThe effect of high glucose on YAP/TAZ signaling was firstly evaluated in vitro on endothelial cells cultured under static conditions or subjected to shear stress (either laminar or oscillatory flow). The impact of diabetes on YAP/TAZ signaling was additionally assessed in vivo in db/db mice.ResultsIn vitro, we found that YAP was dephosphorylated/activated by high glucose in endothelial cells, thus leading to increased endothelial inflammation and monocyte attachment. Moreover, YAP was further activated when high glucose was combined to laminar flow conditions. YAP was also activated by oscillatory flow conditions but, in contrast, high glucose did not exert any additional effect. Interestingly, inhibition of YAP reduced endothelial inflammation and monocyte attachment. Finally, we found that YAP is also activated in the vascular wall of diabetic mice, where inflammatory markers are also increased.ConclusionWith the current study we demonstrated that YAP signaling is activated by high glucose in endothelial cells in vitro and in the vasculature of diabetic mice, and we pinpointed YAP as a regulator of high glucose-mediated endothelial inflammation and monocyte attachment. YAP inhibition may represent a potential therapeutic opportunity to improve diabetes-associated vascular complications.

VGLL4 inhibits YAP1/TEAD signaling to suppress the epidermal squamous cell carcinoma cancer phenotype.

Epidermal squamous cell carcinoma (SCC) develops in response to ultraviolet light exposure and is among the most common cancers. The transglutaminase 2 cancer cell survival protein stimulates the activity of the YAP1/TEAD transcription complex to drive the expression of genes that promote aggressive epidermal SCC cell invasion, migration, and tumor formation. Therefore, we are interested in mechanisms that may inhibit these events. Vestigial-like protein-4 (VGLL4) is a transcription cofactor/tumor suppressor that inhibits several pro-cancer pathways including YAP1 signaling. Our present studies show that VGLL4 inhibits YAP1/TEAD-dependent transcription to reduce the expression of YAP1 target genes (CCND1, CYR61, and CTGF) and pro-cancer collagen genes (COL1A2and COL3A1). We further show that loss of these YAP1 regulated genes is required for VGLL4 suppression of the cancer cell phenotype, as forced CCND1 or COL1A2 expression partially restores the aggressive cancer phenotype in VGLL4 expressing cells. Consistent with these findings, VGLL4 expression reduces tumor formation, and this is associated with reduced CCND1, CYR61, CTGF, COL1A2, and COL1A3 mRNA and protein levels, and reduced EMT marker expression. These findings indicate that VGLL4 suppresses the malignant epidermal SCC cancer phenotype by inhibiting YAP1/TEAD-dependent pro-cancer signaling.

Gout-induced endothelial impairment: The role of SREBP2 transactivation of YAP.

Gout is a multifaceted inflammatory disease involving vascular impairments induced by hyperuricemia. Experiments using human umbilical vein endothelial cells treated with uric acid (UA), monosodium urate (MSU), or serum from gout patients showed increased expression of pro-inflammatory genes (ie, VCAM1, ICAM1, CYR61, CCNA1, and E2F1) with attendant increase in monocyte adhesion. Mechanistically, UA- or MSU-induced SREBP2 expression and its transcriptional activity. RNA sequencing analysis and real-time PCR showed the induction of YAP signaling and pro-inflammatory pathways in HUVECs transfected with adenovirus-SREBP2. The SREBP2 knockdown by siRNA partially abolished UA- or MSU-induced YAP activity, pro-inflammatory gene expression, and monocytes adhesion. Vascular intima from transgenic mice overexpressing SREBP2 in endothelium or mice with hyperuricemia exhibited activated YAP signaling and increased expression of pro-inflammatory genes. Betulin, an SREBP pharmacological inhibitor, attenuated the UA-, MSU-, or gout serum-induced endothelial cell inflammation and dysfunction. In the human study, endothelial cell function, assessed by EndoPAT, was negatively correlated with serum UA level among gouty patients and healthy controls. Collectively, UA or MSU causes endothelial dysfunction via SREBP2 transactivation of YAP. Betulin inhibition of SREBP2 may restrain gout-induced endothelial dysfunction.

Regulatory effect of microRNA-223-3p on breast cancer cell processes via the Hippo/Yap signaling pathway.

According to the 2018 global cancer statistics, the incidence and mortality rates of breast cancer are increasing gradually, which seriously threatens the health of women. MicroRNA-223-3p (miR-223-3p) can promote the proliferation and invasion of breast cancer cells. Hippo/Yes-related protein (Yap) signaling pathway activation has been found in a variety of tumors. The present study aimed to investigate the potential mechanism of miR-223-3p in breast cancer. The Cell Counting Kit-8 assay was used to detect cell viability and flow cytometry was used to detect apoptosis. The abilities of cell migration and invasion were detected using scratch and Transwell assays, as well as reverse transcription-quantitative PCR and western blotting to detect gene and protein expression, respectively. The current results demonstrated that miR-223-3p transcription levels were increased in breast cancer cells, and inhibition of miR-223-3p gene expression decreased cell proliferation, migration and invasion. Additionally, inhibition of miR-223-3p expression inhibited epithelial-mesenchymal transition (EMT) in breast cancer cells. miR-223-3p promoted cell proliferation, migration, invasion and EMT, and the western blotting results demonstrated that miR-223-3p inhibition increased the phosphorylation of Yap1 and the protein expression levels of large tumor suppressor kinase 1. In conclusion, results from the present results suggested that miR-223-3p may promote cell proliferation, migration, invasion and EMT through the Hippo/Yap signaling pathway. Therefore, miR-223-3p may be a potential biomarker for breast cancer.

CCL2‐ Dependent Macrophage Recruitment by Glioblastoma Stem Cells: The Role of Gα12 and YAP Signaling

Glioblastoma multiforme (GBM), the most malignant form of brain tumor, has an average survival rate of less than two years, associated with recurrence despite conventional interventions. GBM tumors contain myeloid cells, recruited in response to inflammatory cytokines which play a role in the tumor progression and may also contribute to immune suppression. Tumor associated macrophages interact with tumor cells and conversely tumor cells contribute to generation of the inflammatory immune cell environment. Gα12 protein-coupled receptors are regulated by inflammatory mediators often generated in the tumor cell environment. Stimulation of these receptors activates the transcriptional co-activator YAP (Yes-associated protein 1) which can in turn induce inflammatory gene expression. Our previous studies showed that knockdown of YAP in GSC-23 PDX cells significantly attenuated in vitro stemness, proliferation, and migration. In vivo experiments showed that YAP knockdown PDX cells formed significantly smaller tumors and had lower lethality than wild-type cells. Our more recent in vivo studies with GSC-23 cells suggested that there is a significant inflammatory response in the tumor cell environment. In order to determine if Gα12/YAP signaling in this glioma stem cell tumor line contributed to macrophage infiltration we utilized an in vitro. Transwell migration assay with human THP-1 macrophages on the top and GSC-23 cells on the bottom. Remarkably, the presence of GSC-23 cells strongly enhanced THP-1 cell migration and this could be mimicked almost fully by replacing the cells with medium in which they were cultured for 24 hours. One of the most efficacious mediators of macrophage migration is CCL2 (MCP-1). The involvement of CCL2 and its receptor CCR2 on THP-1 cell migration was demonstrated by the ability of the CCR2 blocker RS102895 to significantly inhibit the migratory response elicited by GSC-23 cells or cell medium. To understand whether the generation of CCL2 and thus the effect of the tumor cells on macrophage recruitment was specifically regulated, we examined GSC-23 cells in which YAP or Gα12 were deleted. YAP knockout prevented GSC-23 cell mediated migration suggesting that YAP regulates production of CCL2 by the GSC-23 cells. We observed that CCL2 mRNA levels were in fact significantly lower in YAP knockdown compared to WT cells. Notably this appeared to depend on the presence of the THP-1 cells suggesting that macrophage crosstalk to the GSC-23 tumor cells controls CCL2 generation. IL-6 mRNA levels were similarly regulated by THP-1 signaling to GSC-23 cells through YAP. Using a Gliovis tool to analyze data sets of GBM tumors, we determined that there is a highly significant Pearson's correlation between YAP1 and CCL2 gene expression as well as a correlation between YAP1 and IL-6 expression. Further of studies are underway to explore mechanisms responsible for this glioma stem cell /macrophages crosstalk in co-culture experiments. We suggest that YAP mediated cytokine generation by glioma stem cells represents a critical point for therapeutic intervention to disrupt recruitment of macrophages and interfere with their potential role in creating an immunosuppressive myeloid cell tumor microenvironment.

TNIK, a novel activator of FAK and YAP signaling, is a therapeutic target in Lung Squamous Cell Carcinoma

Distal chromosome 3q amplification (3q26-29, also known as the 3q amplicon) is the most frequent genomic alteration in lung squamous cell carcinoma (LSCC). Analysis of LSCC samples from the TCGA reveals that nearly 50% of LSCC patients harbor the 3q amplicon that includes the resident protein kinase gene TNIK. Recent studies have pinpointed TNIK as a potential oncogenic driver in cancer cells with distal 3q amplification; however, the therapeutic potential of TNIK remains unexplored. We found that TNIK was highly expressed in LSCC cells with the 3q amplicon, while its expression was modest in cells that lacked the 3q amplicon, consistent with data from the TCGA. To evaluate TNIK as a target in LSCC, we generated stable, doxycycline-inducible cells expressing shRNA to deplete TNIK from LSCC cells and conducted functional assays to measure cell proliferation and survival. TNIK knockdown or inhibition of its kinase activity with a small molecule inhibitor significantly diminished the viability of LSCC cells with 3q amplification in vitro and in cell line-derived xenograft mouse models. We also observed that TNIK inhibitors significantly abrogated the growth of LSCC patient-derived xenografts and showed that TNIK inhibition induced apoptotic cell death in LSCC cells that harbor the 3q amplicon. Importantly, TNIK depletion or catalytic inhibition in LSCC cells that lack the 3q amplicon had no significant effect on cell survival. Finally, we used a combination of bioinformatics and proteomic analysis (RPPA, peptide mapping, and mass spectrometry) to define the underlying mechanisms driving TNIK mediated cancer cell survival. We identified the tumor suppressor MERLIN as a novel TNIK substrate and determined that TNIK phosphorylates MERLIN at serine 13 and 315. We also show that TNIK is required to maintain FAK activation and stabilize the YAP transcription factor, two oncogenic pathways inhibited by MERLIN. In conclusion, our results demonstrate that TNIK maintains survival of LSCC cells through modulation of a novel TNIK-MERLIN-YAP/FAK signaling pathway and validate TNIK inhibitors in pre-clinical models of LSCC, including patient-derived xenografts. In summary, we have pinpointed the protein kinase TNIK as a promising therapeutic target for the treatment of LSCC patients with distal chromosome 3q amplification.

Genetic and acquired heterotopic ossification are driven by a self‐amplifying positive feedback loop of Hedgehog signaling

Heterotopic ossification (HO), which refers to extra-skeletal pathological bone formation, occurs as a common complication after injury or in genetic disorders. However, limited by poor understanding of the underlying cellular and molecular mechanisms, there is currently no effective treatment and surgical eviction often results in recurrence. We have found that in both genetic and injury-induced HO, activated ectopicexpression of a hedgehog ligand drives HO progression by forming a positive feedback loop with Yap activation that results in non-cell-autonomous and self-propagation of osteoblast differentiation of wild type cells. In mouse models of progressive osseous heteroplasia (POH), a human disease caused by null mutations in GNASthat encodes Gαs, we found previously that Hedgehog signaling was activated, which is both necessary and sufficient to induce HO. We recently further found that progressively expanded ectopic bone was formed by progressively recruited wild type cells. Mechanistically, the Gnas-/-mesenchymal cells differentiate into osteoblasts and recruit wild type cells to form bone by activatingexpression of Hedgehog ligands. Secreted Hedgehog further induces Yap activation that directly activating more Hedgehogligand expression in surrounding wild type cells. Activated Yap and Hedgehog signaling induces osteoblast differentiation and this self-propagating positive feedback loop is both necessary and sufficient for ectopic bone formation and expansion and can act independently of Gαs. Importantly, Hedgehog activation was also found in fibrodysplasia ossificans progressive (FOP), Achilles tendon puncture (ATP)-induced HO mouse models and in human HO samples. Genetic removal of Hedgehog and Yap abolished HO not only in POH, but also in FOP and acquired HO mouse models. We therefore identify a Hedgehog and Yap-driven, self- amplifying and self-propagating osteoblast differentiation as a common cellular and molecular mechanism underlying HO initiation and expansion. Our work highlights the importance of rare genetic disease studies in identifying a shared core pathological mechanism underlying a class of diseases. Our results further suggest that the Hedgehog pathway is a promising new therapeutic target for HO without affecting the normal bone.

Yes-Associated Protein Is Crucial for Constitutive Androstane Receptor-Driven Hepatocyte Proliferation But Not for Induction of Drug Metabolism Genes in Mice.

Background and AimsConstitutive androstane receptor (CAR) agonists, such as 1,4‐bis [2‐(3,5‐dichloropyridyloxy)] benzene (TCPOBOP), are known to cause robust hepatocyte proliferation and hepatomegaly in mice along with induction of drug metabolism genes without any associated liver injury. Yes‐associated protein (Yap) is a key transcription regulator that tightly controls organ size, including that of liver. Our and other previous studies suggested increased nuclear localization and activation of Yap after TCPOBOP treatment in mice and the potential role of Yap in CAR‐driven proliferative response. Here, we investigated a direct role of Yap in CAR‐driven hepatomegaly and hepatocyte proliferation using hepatocyte‐specific Yap‐knockout (KO) mice.Approach and ResultsAdeno‐associated virus 8‐thyroxine binding globulin promoter‐Cre recombinase vector was injected to Yap‐floxed mice for achieving hepatocyte‐specific Yap deletion followed by TCPOBOP treatment. Yap deletion did not decrease protein expression of CAR or CAR‐driven induction of drug metabolism genes (including cytochrome P450 [Cyp] 2b10, Cyp2c55, and UDP‐glucuronosyltransferase 1a1 [Ugt1a1]). However, Yap deletion substantially reduced TCPOBOP‐induced hepatocyte proliferation. TCPOBOP‐driven cell cycle activation was disrupted in Yap‐KO mice because of delayed (and decreased) induction of cyclin D1 and higher expression of p21, resulting in decreased phosphorylation of retinoblastoma protein. Furthermore, the induction of other cyclins, which are sequentially involved in progression through cell cycle (including cyclin E1, A2, and B1), and important mitotic regulators (such as Aurora B kinase and polo‐like kinase 1) was remarkably reduced in Yap‐KO mice. Microarray analysis revealed that 26% of TCPOBOP‐responsive genes that were mainly related to proliferation, but not to drug metabolism, were altered by Yap deletion. Yap regulated these proliferation genes through alerting expression of Myc and forkhead box protein M1, two critical transcriptional regulators of CAR‐mediated hepatocyte proliferation.ConclusionsOur study revealed an important role of Yap signaling in CAR‐driven hepatocyte proliferation; however, CAR‐driven induction of drug metabolism genes was independent of Yap.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Cholesterol metabolic enzyme Ggpps regulates epicardium development and ventricular wall architecture integrity in mice

During embryonic heart development, the progenitor cells in the epicardium would migrate and differentiate into noncardiomyocytes in myocardium and affect the integrity of ventricular wall, but the underlying mechanism has not been well studied. We have found that myocardium geranylgeranyl diphosphate synthase (Ggpps), a metabolic enzyme for cholesterol biosynthesis, is critical for cardiac cytoarchitecture remodelling during heart development. Here, we further reveal that epicardial Ggpps could also regulate ventricular wall architecture integrity. Epicardium-specific deletion of Ggpps before embryonic day 10.5 (E10.5) is embryonic lethal, whereas after E13.5 is survival but with defects in the epicardium and ventricular wall structure. Ggpps deficiency in the epicardium enhances the proliferation of epicardial cells and disrupts cell‒cell contact, which makes epicardial cells easier to invade into ventricular wall. Thus, the fibroblast proliferation and coronary formation in myocardium were found enhanced that might disturb the coronary vasculature remodelling and ventricular wall integrity. These processes might be associated with the activation of YAP signalling, whose nuclear distribution is blocked by Ggpps deletion. In conclusion, our findings reveal a potential link between the cholesterol metabolism and heart epicardium and myocardium development in mammals, which might provide a new view of the cause for congenital heart diseases and potential therapeutic target in pathological cardiac conditions.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Injury-mediated stiffening persistently activates muscle stem cells through YAP and TAZ mechanotransduction.

The skeletal muscle microenvironment transiently remodels and stiffens after exercise and injury, as muscle ages, and in myopathic muscle; however, how these changes in stiffness affect resident muscle stem cells (MuSCs) remains understudied. Following muscle injury, muscle stiffness remained elevated after morphological regeneration was complete, accompanied by activated and proliferative MuSCs. To isolate the role of stiffness on MuSC behavior and determine the underlying mechanotransduction pathways, we cultured MuSCs on strain-promoted azide-alkyne cycloaddition hydrogels capable of in situ stiffening by secondary photocrosslinking of excess cyclooctynes. Using pre- to post-injury stiffness hydrogels, we found that elevated stiffness enhances migration and MuSC proliferation by localizing yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (WWTR1; TAZ) to the nucleus. Ablating YAP and TAZ in vivo promotes MuSC quiescence in postinjury muscle and prevents myofiber hypertrophy, demonstrating that persistent exposure to elevated stiffness activates mechanotransduction signaling maintaining activated and proliferating MuSCs.

The Dynamic Counterbalance of RAC1-YAP/OB-Cadherin Coordinates Tissue Spreading with Stem Cell Fate Patterning.

Tissue spreading represents a key morphogenetic feature of embryonic development and regenerative medicine. However, how molecular signaling orchestrates the spreading dynamics and cell fate commitment of multicellular tissue remains poorly understood. Here, it is demonstrated that the dynamic counterbalance between RAC1–YAP and OB‐cadherin plays a key role in coordinating heterogeneous spreading dynamics with distinct cell fate patterning during collective spreading. The spatiotemporal evolution of individual stem cells in spheroids during collective spreading is mapped. Time‐lapse cell migratory trajectory analysis combined with in situ cellular biomechanics detection reveal heterogeneous patterns of collective spreading characteristics, where the cells at the periphery are faster, stiffer, and directional compared to those in the center of the spheroid. Single‐cell sequencing shows that the divergent spreading result in distinct cell fate patterning, where differentiation, proliferation, and metabolism are enhanced in peripheral cells. Molecular analysis demonstrates that the increased expression of RAC1–YAP rather than OB‐cadherin facilitated cell spreading and induced differentiation, and vice versa. The in vivo wound healing experiment confirms the functional role of RAC1–YAP signaling in tissue spreading. These findings shed light on the mechanism of tissue morphogenesis in the progression of development and provide a practical strategy for desirable regenerative therapies.

Lymph node metastasis-derived gastric cancer cells educate bone marrow-derived mesenchymal stem cells via YAP signaling activation by exosomal Wnt5a

Lymph node metastasis (LNM), a common metastatic gastric-cancer (GC) route, is closely related to poor prognosis in GC patients. Bone marrow-derived mesenchymal stem cells (BM-MSCs) preferentially engraft at metastatic lesions. Whether BM-MSCs are specifically reprogrammed by LNM-derived GC cells (LNM-GCs) and incorporated into metastatic LN microenvironment to prompt GC malignant progression remains unknown. Herein, we found that LNM-GCs specifically educated BM-MSCs via secretory exosomes. Exosomal Wnt5a was identified as key protein mediating LNM-GCs education of BM-MSCs, which was verified by analysis of serum exosomes collected from GC patients with LNM. Wnt5a-enriched exosomes induced YAP dephosphorylation in BM-MSCs, whereas Wnt5a-deficient exosomes exerted the opposite effect. Inhibition of YAP signaling by verteporfin blocked LNM-GC exosome- and serum exosome-mediated reprogramming in BM-MSCs. Analysis of MSC-like cells obtained from metastatic LN tissues of GC patients (GLN-MSCs) confirmed that BM-MSCs incorporated into metastatic LN microenvironment, and that YAP activation participated in maintaining their tumor-promoting phenotype and function. Collectively, our results show that LNM-GCs specifically educated BM-MSCs via exosomal Wnt5a-elicited activation of YAP signaling. This study provides new insights into the mechanisms of LNM in GC and BM-MSC reprogramming, and will provide potential therapeutic targets and detection indicators for GC patients with LNM.

Claudin-2 inhibits renal clear cell carcinoma progression by inhibiting YAP-activation

BackgroundClaudin-2 expression is upregulated in multiple cancers and promotes cancer malignancy. Remarkably, the regulation of claudin-2 expression in kidney cell lines contrasts its reported regulation in other organs. However, claudin-2 role in renal clear cell carcinoma (RCC) remains unknown despite its predominant expression in the proximal tubular epithelium (PTE), the site of RCC origin.MethodsPublicly available and independent patient databases were examined for claudin-2 association with RCC. The novel protein function was validated in vitro and in vivo by gain or loss of function assays. Mechanistic results were concluded by Mass spectroscopy, immunoprecipitation and mutational studies, and functional evaluations.ResultsWe show that the significant decrease in claudin-2 expression characterized PTE cells and Ex-vivo cultured mouse kidney subjected to dedifferentiation. Inhibition of claudin-2 was enough to induce mesenchymal plasticity and invasive mobility in these models. Further, a progressive loss of claudin-2 expression associated with the RCC progression and poor patient survival. Overexpression of claudin-2 in RCC-derived cancer cells inhibited tumorigenic abilities and xenograft tumor growth. These data supported a novel tumor-suppressive role of claudin-2 in RCC. Mechanistic insights further revealed that claudin-2 associates with YAP-protein and modulates its phosphorylation (S127) and nuclear expression. The tumor suppressive effects of claudin-2 expression were lost upon deletion of its PDZ-binding motif emphasizing the critical role of the PDZ-domain in claudin-2 interaction with YAP in regulating RCC malignancy.ConclusionsOur results demonstrate a novel kidney specific tumor suppressive role for claudin-2 protein and further demonstrate that claudin-2 co-operates with the YAP signaling in regulating the RCC malignancy.

A TNFR2-hnRNPK Axis Promotes Primary Liver Cancer Development via Activation of YAP Signaling in Hepatic Progenitor Cells.

Most primary liver cancer (PLC) cases progress mainly due to underlying chronic liver inflammation, yet the underlying mechanisms of inflammation-mediated PLC remain unclear. Here we uncover a TNF receptor II (TNFR2)-hnRNPK-YAP signaling axis in hepatic progenitor cells (HPC) essential for PLC development. TNFR2, but not TNF receptor I (TNFR1), was required for TNFα-induced activation of YAP during malignant transformation of HPCs and liver tumorigenesis. Mechanistically, heterogeneous nuclear ribonuclear protein K (hnRNPK) acted downstream of TNFα-TNFR2 signaling to directly interact with and stabilize YAP on target gene promoters genome-wide, therefore coregulating the expression of YAP target genes. Single-cell RNA sequencing confirmed the association of TNFR2-hnRNPK with YAP expression and the pathologic importance of HPC. Accordingly, expressions of TNFR2, hnRNPK, and YAP were all upregulated in PLC tissues and were strongly associated with poor prognosis of PLC including patient survival. Collectively, this study clarifies the differential roles of TNFRs in HPC-mediated tumorigenesis, uncovering a TNFR2-hnRNPK-centered mechanistic link between the TNFα-mediated inflammatory milieu and YAP activation in HPCs during PLC development. SIGNIFICANCE: This work defines how hnRNPK links TNFα signaling and Hippo pathway transcription coactivator YAP in hepatic progenitor cells during primary liver tumorigenesis.

Piezo1 impairs hepatocellular tumor growth via deregulation of the MAPK-mediated YAP signaling pathway

Accumulating evidence has revealed the mechanosensitive ion channel protein Piezo1 is contributing to tumorigenesis. However, its role in hepatocellular carcinoma (HCC) remains unexplored. In this study, we demonstrated that Piezo1 was expressed in the HepG2 cell line and depletion of Piezo1 impaired proliferation and migration, as well as increased apoptosis in these cells. Using a Piezo1-specific activator, Yoda1, we identified that calcium entry induced by Yoda1 resulted in phosphorylation of JNK, p38, and ERK, thereby activating the mitogen-activated protein kinase (MAPK) pathway, in a dose- and time-dependent manner. More strikingly, Piezo1 activation integrated with YAP signaling to control the nuclear translocation of YAP and regulation of its target genes. JNK, p38, and ERK (MAPK signaling) regulated Yoda1-induced YAP activation. Consistent with the association of calpain with Piezo1, we also found that calpain activity was decreased by siRNA-mediated knockdown of Piezo1. In addition, the growth of HCC tumors was inhibited in Piezo1 haploinsufficient mice. Together, our findings establish that the Piezo1/MAPK/YAP signaling cascade is essential for HepG2 cell function. These results highlight the importance of Piezo1 in HCC and the potential utility of Piezo1 as a biomarker and therapeutic target.

YAP and β-Catenin Cooperate to Drive Oncogenesis in Basal Breast Cancer.

Targeting cancer stem cells (CSC) can serve as an effective approach toward limiting resistance to therapies. While basal-like (triple-negative) breast cancers encompass cells with CSC features, rational therapies remain poorly established. We show here that the receptor tyrosine kinase Met promotes YAP activity in basal-like breast cancer and find enhanced YAP activity within the CSC population. Interfering with YAP activity delayed basal-like cancer formation, prevented luminal to basal transdifferentiation, and reduced CSC. YAP knockout mammary glands revealed a decrease in β-catenin target genes, suggesting that YAP is required for nuclear β-catenin activity. Mechanistically, nuclear YAP interacted with β-catenin and TEAD4 at gene regulatory elements. Proteomic patient data revealed an upregulation of the YAP signature in basal-like breast cancers. Our findings demonstrate that in basal-like breast cancers, β-catenin activity is dependent on YAP signaling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/β-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal-like breast cancers. SIGNIFICANCE: These findings show that YAP cooperates with β-catenin in basal-like breast cancer to regulate CSCs and that targeting this interaction may be a novel CSC therapy for patients with basal-like breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2116/F1.large.jpg.

OLIG2 maintenance is not essential for diffuse intrinsic pontine glioma cell line growth but regulates tumor phenotypes.

Diffuse intrinsic pontine glioma (DIPG) is a pediatric lethal high-grade brainstem glioma with no effective therapies. OLIG2 (oligodendrocyte transcription factor 2) was reported to be critical for the growth of a DIPG cell line CCHMC-DIPG-1. Surprisingly, we found that the CCHMC-DIPG-1 cells express little OLIG2 and exhibit a mesenchymal phenotype, which raised a question regarding the role of OLIG2 in the growth of DIPG cells. We evaluated the function of OLIG2 in different DIPG cell lines through molecular and genetic approaches and performed transcriptomic and genomic landscape profiling including whole-genome bisulfite sequencing, RNA-seq, ATAC-seq, and ChIP-seq. shRNA-mediated knockdown and CRISPR-Cas9-mediated knockout approaches were utilized to assess OLIG2 functions in DIPG cell growth. We found that DIPG cells are phenotypically heterogeneous and exhibit the characteristics of distinct malignant gliomas including proneural, classical, and mesenchymal subtypes. OLIG2 knockdown did not impact the growth of CCHMC-DIPG-1 cells, wherein OLIG2 is epigenetically silenced. Moreover, OLIG2 deletion did not substantially impair OLIG2-expressing proneural-like DIPG growth but led to an upregulation of HIPPO-YAP1 and epidermal growth factor receptor (EGFR) signaling and a tumor phenotype shift. Targeting HIPPO-YAP1 and EGFR signaling in OLIG2-deficient DIPG cells inhibited tumor cell growth. Our data indicate that OLIG2 is dispensable for DIPG growth but regulates the phenotypic switch of DIPG tumor cells. OLIG2 downregulation leads to deregulation of adaptive YAP1 and EGFR signaling. Targeting YAP1 and EGFR pathways inhibits the growth of OLIG2-deficient DIPG cells, pointing to a therapeutic potential by targeting adaptive signaling to treat DIPG tumors with nominal OLIG2 expression.

The migration of metastatic breast cancer cells is regulated by matrix stiffness via YAP signalling

Matrix stiffness is a driver of breast cancer progression and mechanosensitive transcriptional activator YAP plays an important role in this process. However, the interplay between breast cancer and matrix stiffness, and the significance of this interplay remained largely unknown. Here, we showed an increase in YAP nuclear localization and a higher proliferation rate in both highly metastatic MDA-MB-231 cells and the non-metastatic counterpart MCF-7 cells when they were exposed to the stiff matrix. However, in response to the stiff matrix highly metastatic MDA-MB-231 cells instead of MCF-7 cells exhibited upregulated mobility, which was shown to be YAP-dependent. Consistently, MDA-MB-231 cells exhibited different focal adhesion dynamics from MCF-7 cells in response to matrix stiffness. These results suggested a YAP-dependent mechanism through which matrix stiffness regulates the migratory potential of metastatic breast cancer cells.