Blastocyst-like Structures in the Peripheral Retina of Young Adult Beagles.

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

YAP1/TAZ activity maintains vascular integrity and organismal survival.

Radiation therapy is one of the major treatment modalities for patients with cancers. However, ionizing radiation (IR) damages not only cancer cells but also the surrounding vascular endothelial cells (ECs). Hippo pathway effector genes Yap1 and Taz are the two transcriptional coactivators that have crucial roles in tissue homeostasis and vascular integrity in various organs. However, their function in adult ECs at the steady state and after IR is poorly understood. Here, we report sex- and context-dependent roles of endothelial YAP1/TAZ in maintaining vascular integrity and organismal survival. EC-specific Yap1/Taz deletion compromised systemic vascular integrity, resulting in lethal circulation failure preferentially in male mice. Furthermore, EC-specific Yap1/Taz deletion induced acute lethality upon sublethal IR that was closely associated with exacerbated systemic vascular dysfunction and circulation failure. Consistent with these findings, RNA-seq analysis revealed downregulation of tight junction genes in Yap1/Taz-deleted ECs. Collectively, our findings highlight the importance of endothelial YAP1/TAZ for maintaining adult vascular function, which may provide clinical implications for preventing organ injury after radiation therapy.

Yes-associated protein 1 translocation through actin cytoskeleton organization in trophectoderm cells.

A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells.

Identification of YAP1 as a novel downstream effector of the FGF2/STAT3 pathway in the pathogenesis of renal tubulointerstitial fibrosis.

Chronic kidney disease is a global health problem and eventually develops into an end-stage renal disease (ESRD). It is now widely believed that renal tubulointerstitial fibrosis (TIF) plays an important role in the progression of ESRD. Renal tubular epithelial-mesenchymal transition (EMT) is an important cause of TIF. Studies have shown that FGF2 is highly expressed in fibrotic renal tissue, although the mechanism remains unclear. We found that FGF2 can activate STAT3 and induce EMT in renal tubular epithelial cells. STAT3, an important transcription factor, was predicted by the JASPAR biological database to bind to the promoter region of YAP1. In this study, STAT3 was shown to promote the expression of the downstream target gene YAP1 through transcription, promote EMT of renal tubular epithelial cells, and mediate the occurrence of renal TIF. This study provides a theoretical basis for the involvement of the FGF2/STAT3/YAP1 signaling pathway in the process of renal interstitial fibrosis and provides a potential target for the treatment of renal fibrosis.

The novel long non-coding RNA LATS2-AS1-001 inhibits gastric cancer progression by regulating the LATS2/YAP1 signaling pathway via binding to EZH2.

BACKGROUND: To explore the expression pattern and role of the novel long non-coding RNA LATS2 antisense transcript 1 (LATS2-AS1-001) in gastric cancer (GC). METHODS: qRT-PCR was applied to evaluate LATS2-AS1-001 expression and correlation with LATS2 in GC. In vitro experiments were performed to investigate the role of LATS2-AS1-001 in GC cells. RNA immunoprecipitation (RIP) was performed to assess the interaction between EZH2 and LATS2-AS1-001. LATS2/YAP1 signaling pathway proteins were detected by immunoblot. Oncomine and KMPLOT data analysis was conducted to assess the prognostic value of YAP1 in GC. RESULTS: Decreased expression levels of LATS2-AS1-001 and LATS2 were confirmed in 357 GC tissues compared with the normal mucosa. A strong positive correlation between LATS2-AS1-001 and LATS mRNA expression was found in Pearson Correlation analysis (r = 0.719, P < 0.001). Furthermore, ROC curve analysis revealed areas under the curves for LATS2-AS1-001 and LATS2 of 0.7274 and 0.6865, respectively (P < 0.001), which indicated that LATS2-AS1-001 and LATS could be used as diagnostic indicators in GC. Moreover, ectopic expression of LATS2-AS1-001 decreased cell viability, induced G0/G1 phase arrest, and inhibited cell migration and invasion in GC cells. Mechanistically, overexpressing LATS2-AS1-001 upregulated LATS2 and induced YAP1 phosphorylation via binding to EZH2. Oncomine and KMPLOT database analysis demonstrated YAP1 was highly expressed in human GC samples, and high YAP1 expression predicted poor patient prognosis in GC. CONCLUSION: This study revealed that lncRNA LATS2-AS1-001 might serve as a potential diagnostic index in GC and act as a suppressor of GC progression.

IL-6/YAP1/ß-catenin signaling is involved in intervertebral disc degeneration.

Yes-associated protein 1 (YAP1) is a transcriptional coactivator and negative regulator of the Hippo pathway. It regulates diverse cellular processes, such as cell proliferation, contact inhibition, and tissue size. However, the role of YAP1 in intervertebral disc degeneration (IDD) remains elusive. Here, we demonstrated that YAP1 was activated by Interleukin 6 (IL-6) through tyrosine phosphorylation in nucleus pulposus cells (NP cells). Overexpression of YAP1 decreased Sox-9, Col-II, aggrecan expression, whereas increased matrix metalloproteinases 13 level. In contrast, knockdown of YAP1 by small interfering RNA (siRNA) showed opposite effects and rescued IL-6 induced NP cells degeneration. In addition, western blot showed that IL-6 treatment increased YAP1 and ß-catenin protein level; co-immunoprecipitation (Co-IP) and immunofluorescence analysis showed that IL-6 enhanced YAP1 and ß-catenin interaction and nuclear accumulation. Knockdown of ß-catenin by siRNA blocked IL-6 treatment or YAP1 overexpression induced degeneration. Moreover, we found that verteporfin, a specific inhibitor of YAP1, effectively alleviated IDD development in rat disks. Taken together, our findings indicated that YAP1 plays an important role in IDD, and ß-catenin is essential for IL-6/YAP1 signaling.

Long noncoding RNA APTR contributes to osteosarcoma progression through repression of miR-132-3p and upregulation of yes-associated protein 1.

A growing amount of evidence has shown that long noncoding RNAs (lncRNAs) play crucial roles in osteosarcoma (OS). However, little knowledge is available about the functional roles and molecular mechanisms of lncRNA Alu-mediated p21 transcriptional regulator (APTR) in OS. Herein, APTR expression was demonstrated to be significantly upregulated in OS tumor tissues and four OS cell lines (including MG63, 143B, Saos-2, and HOS) compared with the adjacent tissues and human osteoblast cell line hFOB1.19, respectively. We confirmed miR-132-3p to be a target for APTR, and its expression was demonstrated to be inhibited by APTR. In functional terms, knockdown of APTR and overexpression of miR-132-3p both, remarkably repressed human OS cell proliferation, invasion and migration, and induced apoptosis. Also, Yes-associated protein 1 (YAP1) was determined as an inhibitory target of miR-132-3p. Moreover, our findings demonstrated that the repression of YAP1 protein expression and the suppression of Ki-67, MMP9, and Bcl2 expression induced by APTR knockdown required increased miR-132-3p. Thus, APTR contributed to OS progression through repression of miR-132-3p and upregulation of YAP1 expression. Therefore, we have uncovered a novel regulatory mechanism by which the APTR/miR-132-3p/YAP1 axis can regulate OS progression.

HNF4α and CDX2 Regulate Intestinal YAP1 Promoter Activity.

The Hippo pathway is important for tissue homeostasis, regulation of organ size andgrowth in most tissues. The co-transcription factor yes-associated protein 1 (YAP1) serves as a maindownstream effector of the Hippo pathway and its dysregulation increases cancer development andblocks colonic tissue repair. Nevertheless, little is known about the transcriptional regulation ofYAP1 in intestinal cells. The aim of this study to identify gene control regions in the YAP1 gene andtranscription factors important for intestinal expression. Bioinformatic analysis of caudal typehomeobox 2 (CDX2) and hepatocyte nuclear factor 4 alpha (HNF4α) chromatin immunoprecipitatedDNA from differentiated Caco-2 cells revealed potential intragenic enhancers in the YAP1 gene.Transfection of luciferase-expressing YAP1 promoter-reporter constructs containing the potentialenhancer regions validated one potent enhancer of the YAP1 promoter activity in Caco-2 and T84cells. Two potential CDX2 and one HNF4α binding sites were identified in the enhancer by in silicotranscription factor binding site analysis and protein-DNA binding was confirmed in vitro usingelectrophoretic mobility shift assay. It was found by chromatin immunoprecipitation experimentsthat CDX2 and HNF4α bind to the YAP1 enhancer in Caco-2 cells. These results reveal a previouslyunknown enhancer of the YAP1 promoter activity in the YAP1 gene, with importance for highexpression levels in intestinal epithelial cells. Additionally, CDX2 and HNF4α binding areimportant for the YAP1 enhancer activity in intestinal epithelial cells.

Pseudo-3D Topological Alignments Regulate Mechanotransduction and Maturation of Smooth Muscle Cells.

Smooth muscle cells (SMCs) sense and respond to mechanical stimuli in their extracellular microenvironments (ECMs), playing a crucial role in muscle tissue engineering. Increasing evidence from topological cues-mediated mechanotransduction of SMCs in ECMs has suggested some potential underlying mechanisms of how SMC functions and maturation are regulated by their mechanosensing leading to transduction. However, how the expression of yes-associated protein 1 (YAP) influences the phenotypic shift from synthetic to contractile is still controversial. Here, pseudo-3D topological alignments mimicking native muscle tissues are generated using laser-cutter engraving to explore the influence of topological cues on SMC mechanotransduction and maturation. The analysis of topological cue-mediated mechanotransduction and maturation marker expression revealed YAP is involved in mechanotransduction for SMCs cultured on cross-patterned substrates in the presence of cell-cell interactions. Moreover, these SMCs with YAP-linked mechanosensing showed higher expression of calponin, indicating a shift toward contractile phenotypes in vitro and in vivo. Furthermore, it showed skeletal muscle cells has different mechanosensing and maturation mechanisms compared to SMCs, revealing muscle type-dependent different sensing of topological cues, and converting into maturation-associated signaling cascades. This study provides insights into the regulation of SMC mechanotransduction and maturation by topological cues, suggesting the involvement of YAP-linked signaling pathways in this process.

Establishment of Induced Pancreatic Stem Cells by Yes-Associated Protein 1.

Recently, we and others generated induced tissue-specific stem/progenitor (iTS/iTP) cells. The advantages of iTS/iTP cells compared with induced pluripotent stem (iPS) cells are (1) easier generation, (2) efficient differentiation, and (3) no teratomas formation. In this study, we generated mouse induced pancreatic stem cells (iTS-P cells) by the plasmid vector expressing Yes-associated protein 1 (YAP). The iTS-P YAP9 cells expressed Foxa2 (endoderm marker) and Pdx1 (pancreatic marker) while the expressions of Oct3/4 and Nanog (marker of embryonic stem [ES] cells) in iTS-P YAP9 cells was significantly lower compared with those in ES cells. The iTS-P YAP9 cells efficiently differentiated into insulin-expressing cells compared with ES cells. The ability to generate autologous iTS cells may be applied to diverse applications of regenerative medicine.

Yes-associated protein-1 overexpression in ocular surface squamous neoplasia; a potential diagnostic marker and therapeutic target.

Yes-associated protein-1 (YAP-1) is a Hippo system transcription factor, which serves as an oncogene in squamous cell carcinoma, and several solid tumors when the Hippo pathway is dysregulated. Yet, the activity of YAP-1 in ocular surface squamous neoplasia (OSSN) has not been determined. Here, we investigate the relationship between YAP-1 overexpression and OSSN. Using a cross-sectional study design, we recruited 227 OSSN patients from the University Teaching Hospitals in Lusaka, Zambia. Immunohistochemistry was used to assess YAP-1 protein overexpression in tumor tissue relative to surrounding benign squamous epithelium. OSSN patient samples (preinvasive, n = 62, 27% and invasive, n = 165, 73%) were studied. One hundred forty-nine invasive tumors contained adjacent preinvasive tissue, bringing the total number of preinvasive lesions examined to 211 (62 + 149). There was adjacent benign squamous epithelium in 50.2% (114/227) of OSSN samples. Nuclear YAP- 1 was significantly overexpressed in preinvasive (Fisher's (F): p <.0001, Monte Carlo (MC): p <.0001) and invasive (F: p <.0001, MC: p <.0001) OSSN in comparison to adjacent benign squamous epithelium when analyzed for basal keratinocyte positive count, staining intensity, expression pattern, and Immunostaining intensity-distribution index. YAP-1 expression did not differ between preinvasive and invasive OSSN (p >.05), keratinizing and non- keratinizing cancer (p >.05), or between T1/T2 and T3/T4 stages in invasive tumors (p >.05). However, grade 2 and 3 tumors had significantly stronger nucleus YAP-1 overexpression intensity than grade 1 tumors (F: p = .0078, MC: p = .0489). By immunohistochemistry, we identified significant overexpression (upregulation of YAP-1 protein expression) in preinvasive and invasive OSSN lesions compared to neighboring benign squamous epithelium. YAP-1 expression was significantly higher in poorly and moderately differentiated invasive squamous cancer than in well-differentiated carcinomas. Overexpression of YAP-1 within the margin of preinvasive and invasive OSSN, but not in the neighboring normal epithelium, indicates that it plays a role in the development and progression of OSSN.

An involvement of Hippo-yes-associated protein pathway in biliary epithelial senescence in primary biliary cholangitis.

BACKGROUND & AIMS: Accumulating evidence suggest that Hippo-yes-associated protein (YAP) pathway plays important roles in development and repair after injuries in biliary system. We disclosed that senescent biliary epithelial cells (BECs) participate in the pathogenesis of primary biliary cholangitis (PBC). We hypothesized that dysregulation of Hippo-YAP pathway may be associated with biliary epithelial senescence in pathogenesis of PBC. APPROACH & RESULTS: Cellular senescence was induced in cultured BECs by treatment with serum depletion or glycochenodeoxycholic acid. The expression and activity of YAP1 were significantly decreased in senescent BECs (p<0.01). Cellular senescence and apoptosis were significantly increased (p<0.01) and a proliferation activity and a 3D-cyst formation activity were significantly decreased (p<0.01) by a knockdown of YAP1 in BECs. The expression of YAP1 were immunohistochemically determined in livers taken from the patients with PBC (n = 79) and 79 control diseased and normal livers and its association with senescent markers p16INK4a and p21WAF1/Cip1 was analyzed. The nuclear expression of YAP1, which indicates activation of YAP1, was significantly decreased in BECs in small bile ducts involved in cholangitis and ductular reactions in PBC, compared to control livers (p<0.01). The decreased expression of YAP1 was seen in senescent BECs showing expression of p16INK4a and p21WAF1/Cip1 in bile duct lesions. CONCLUSION: Dysregulation of Hippo-YAP1 pathway may be involved in the pathogenesis of PBC in association with biliary epithelial senescence.

Retinal Src homology region 2-containing protein tyrosine phosphatase 2 silencing alleviates diabetic retinopathy via suppressing inflammatory response and oxidative stress by regulating Yes-associated protein 1 activity.

Diabetic retinopathy (DR) is the prevalent microvascular complication of diabetes mellitus (DM), and it may lead to permanent blindness. The previous publication has indicated that both inflammatory response and oxidative stress are critical factors involved in DR progression, however, the accurate regulatory mechanism remains to be revealed. Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), a member of the protein tyrosine phosphatase family, was reported to play a role in diabetic nephropathy, whereas its function in DR was unknown and required further exploration. The level of phosphorylated, not the total, SHP2 increased in the retinas of rats with streptozotocin injection-induced DM. Further, the intravitreal injection of SHP2 shRNA lentivirus alleviated retinal pathological changes, and inhibited inflammatory response and oxidative stress, which were accompanied with Yes-associated protein 1 (YAP1) deactivation in DR rats. Additional co-immunoprecipitation results confirmed the interaction of SHP2 and YAP1. Collectively, our data preliminarily show that DR amelioration-induced by SHP2 inhibition in rats may attribute to the deactivation of YAP1 pathway.

The prognostic effect of HER2 heterogeneity and YAP1 expression in HER2 positive breast cancer patients: a retrospective study.

Background: This study sought to estimate the prognostic effect of intratumoral heterogeneity (ITH) and Yes-associated protein 1 (YAP1) intensity in human epidermal growth factor receptor 2 (HER2) positive breast cancer patients. We also investigated individualized adjuvant therapy for YAP1-sufficient patients and HER2 heterogeneous patients. Methods: The relationship between prognostic outcomes and clinicopathological variables in 1,650 retrieved breast cancer patients was evaluated. The HER2 intensity and YAP1 expression in HER2-ITH and non-ITH (NITH) patients were also estimated. All patients were followed-up, regardless of whether or not they received intensive treatment, to explore individualized adjuvant therapy for YAP1-sufficient patients and HER2 heterogeneous patients. Results: Over-expression and nuclear localization of YAP1 were significant in HER2-ITH patients. The over-expression of YAP1 and the presence of ITH affected the prognosis of HER2 positive patients. YAP1 intensity and lymph nodes metastases was more obviously affected the survival of HER2-ITH patients, while the prognosis of NITH patients were correlated with clinical Tumor-Node-Metastasis (cTNM) stage and lymph-vascular space invasion (LVSI) status. HER2-NITH patients and YAP1-insufficient patients benefited from a combination of Trastuzumab and Pertuzumab/Lapatinib, while Capecitabine significantly decreased the relapse risk of ITH patients and YAP1-sufficient patients. Conclusions: YAP1 overexpression and nuclear localization was usually observed in HER2-ITH patients. For HER2-NITH patients, an advanced stage of cTNM and LVSI status increased the recurrent risk, and intensified Pertuzumab or Lapatinib treatment (combination with Trastuzumab) improved their survival. For HER2-ITH patients, the overexpression of YAP1 and pathological lymph nodes (pLN) metastases increased recurrent risk, and intensified Capecitabine treatment improved their survival. YAP1 overexpression contributed to a poor prognostic outcome, especially when HER2 signal intensity was insufficient.

Exosomal PTEN as a Predictive Marker of Aggressive Gliomas.

Background: Liquid biopsies have emerged as convenient alternative diagnostic methods to invasive biopsies, by evaluating disease-specific biomarkers and monitoring the disease risk noninvasively. Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) is a potent tumor suppressor, and its deletion/mutations are common in gliomas. Objective: Evaluate the feasibility of non-invasive detection of PTEN and its downstream genes in serum exosomes of glioma patients. Materials and methods: PTEN, Yes-associated-protein 1 (YAP1), and lysyl oxidase (LOX) transcript expression were monitored through polymerase chain reaction (PCR) in serum exosomes and their paired tumor tissues. The impact of PTEN and its axis genes expression on the overall survival (OS) was monitored. Results: Out of the 106 glioma serum samples evaluated, PTEN was retained/lost in 65.4%/34.6% of the tumor samples while it was retained/lost in 67.1%/32.9% of their paired exosomal fractions. PTEN expression in both tissue and paired exosomal fractions was observed in 48.11% of the samples. Sanger sequencing detected three mutations (Chr10: 89720791(A>G), Chr10:89720749(C>T), and Chr10:89720850(A>G). Both PTEN-responsive downstream genes (YAP1) and LOX axis were upregulated in the PTEN-deficient samples. PTEN loss was associated with poor survival in the glioma patients (hazard ratio (HR) 0.68, confidence interval (CI): 0.35-1.31, P = 0.28). The OS of the exosomal PTEN cohort coincided with the tumor-tissue PTEN devoid group (HR 1.08, CI: 0.49-2.36, P = 0.85). While, old age yielded the worst prognosis; gender, location, and grade were not prognostic of OS in the multivariate analysis. Conclusions: PTEN and its responsive genes YAP1 and LOX can be detected in serum exosomes and can serve as essential tools for the non-invasive evaluation/identification of aggressive gliomas.

YAP1 protein expression has variant prognostic significance in small cell lung cancer (SCLC) stratified by histological subtypes.

BACKGROUND: Recently, expression of YAP1, a nuclear effector of an inactivated HIPPO pathway, has been identified as one of four molecular subtypes of SCLC. However, the clinicopathological relevance and prognostic significance of YAP1 expression in SCLC stratified by histological subtypes has not been systematically reported to date. METHODS: Tumor sections and corresponding formalin-fixed paraffin-embedded (FFPE) samples of 297 SCLC patients were retrieved from the pathological specimen repository and were subsequently reviewed by pathologists. Forty-six C-SCLCs (combined SCLCs) (15.5%) and 251P-SCLCs (pure SCLCs) (84.5%) were identified respectively. YAP1 expression was examined by immunohistochemistry (IHC) and assessed semi-quantitatively on tumor tissue array (TMA). Propensity score was used to match C-SCLCs and P-SCLCs in a ratio of 1 to 2 to balance age, gender, tumor stage and treatment methods. Finally, 46C-SCLCs and 92P-SCLCs were included for prognostic analysis. RESULTS: The positive rate of YAP1 expression was significantly higher in C-SCLCs than P-SCLCs before matching (52.2% vs 29.1%, P = 0.004). After matching by propensity score, the prescribed clinical parameters were well balanced between P-SCLCs and C-SCLCs. Expression of YAP1 was associated worse overall survival (OS) (5- year OS%, 39.0% vs. 74.9%, P = 0.013) and was an independent risk factor for OS (HR = 2.93, 95% CI: 1.01-8.51; P = 0.048) exclusively in C-SCLC. Univariate survival analysis in subgroups of different clinical variables also confirmed the prognostic impact of YAP1 was most significant in C-SCLC. But for P-SCLCs, expression of YAP1 showed no prognostic impact. CONCLUSIONS: Expression of YAP1 in small cell components of C-SCLC was significantly higher than that in P-SCLC. Besides, it served as an unfavorable predictor for OS in C-SCLC but not in P-SCLC, which suggested different entities of small cell components with variant YAP1 expression and potential different targetable oncogenic pathway between C-SCLC and P-SCLC.

Regulation of ferroptosis in cancer cells by YAP/TAZ and Hippo pathways: The therapeutic implications.

Ferroptosis is a novel form of iron-dependent cell death characterized by lipid peroxidation. While the importance and disease relevance of ferroptosis is gaining recognition, much remains unknown about various genetic and non-genetic determinants of ferroptosis. Hippo signaling pathway is an evolutionarily conserved pathway that responds to various environmental cues and controls organ size, cell proliferation, death, and self-renewal capacity. In cancer biology, Hippo pathway is a potent tumor suppressing mechanism and its dysregulation contributes to apoptosis evasion, cancer development, metastasis, and treatment resistance. Hippo dysregulation leads to aberrant activation of YAP and TAZ, the two major transcription co-activators of TEADs, that induce the expression of genes triggering tumor-promoting phenotypes, including enhanced cell proliferation, self-renewal and apoptosis inhibition. The Hippo pathway is regulated by the cell-cell contact and cellular density/confluence. Recently, ferroptosis has also been found being regulated by the cellular contact and density. The YAP/TAZ activation under low density, while confers apoptosis resistance, renders cancer cells sensitivity to ferroptosis. These findings establish YAP/TAZ and Hippo pathways as novel determinants of ferroptosis. Therefore, inducing ferroptosis may have therapeutic potential for YAP/TAZ-activated chemo-resistant and metastatic tumor cells. Reciprocally, various YAP/TAZ-targeting treatments under clinical development may confer ferroptosis resistance, limiting the therapeutic efficacy.

Effects of YAP1 and SFRP2 overexpression on the biological behavior of colorectal cancer cells and their molecular mechanisms.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies worldwide and has a high mortality rate. With the development of tumor molecular biology, more and more attention is being paid to the mechanisms of cell pathways in colorectal carcinogenesis, such as the Hippo/Yes-associated protein 1 (YAP1) and Wnt/ß-catenin signaling pathways. The abnormal expression of YAP1 and ß-catenin have been reported in CRC, and can lead to excessive cell proliferation, and eventually, tumor formation. Secreted frizzled-related protein 2 (SFRP2) levels have been found to be decreased in a variety of cancers, and SFRP2 is an antagonist that binds directly to Wnt signal. At present, the molecular basis of colorectal tumors is still not fully understood. In the present study, we sought to identify the molecular mechanisms underlying YAP1 and SFRP2 in the development of CRC. METHODS: We constructed CRC cell lines that stably overexpressed YAP1 and SFRP2 using lentivirus packaging and cell infection. The levels of expression of the proteins were evaluated by western blot and immunofluorescence assays. Protein complex immunoprecipitation (Co-IP) was used to detect the interaction between YAP1, SFRP2, and ß-catenin. The functional roles of YAP1 and SFRP2 in CRC was determined by a Cell Counting Kit-8 (CCK8) proliferation assay and flow cytometric apoptosis assay. RESULTS: The data of the present study showed that the overexpression of SFRP2 promoted the expression of YAP1 and ß-catenin protein, and the overexpression of YAP1 promoted the expression of ß-catenin protein. YAP1 overexpression promoted cell proliferation, while SFRP2 overexpression inhibited cell proliferation and promoted cell apoptosis. CONCLUSIONS: Our findings showed that the expression of YAP1, SFRP2, and ß-catenin is correlated in CRC cells. The Hippo pathway and Wnt pathway interact with each other in the pathogenesis of CRC, and YAP1 and SFRP2 are involved in the formation and development of CRC.

YAP1/MMP7/CXCL16 axis affects efficacy of neoadjuvant chemotherapy via tumor environment immunosuppression in triple-negative breast cancer.

BACKGROUND: To evaluate the association of potential YAP1/MMP7/CXCL16 axis and tumor infiltrating lymphocytes (TILs) related chemo-response in triple-negative breast cancer (TNBC) patients. METHODS: We estimated the messenger RNA (mRNA) expression levels of Yes-associated protein 1 (YAP1), MMP7, and CXCL16 in paired TNBC tumor/para-tumor tissues by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and performed statistical analysis according to neoadjuvant chemotherapy (NAC) response. Based on The Cancer Genome Atlas (TCGA) data, we noticed outstanding expression of MMP7/CXCL16 in TNBC cases, as well as associations between MMP7/CXCL16 and HIPPO-YAP1-relevant kinases. We also performed gene set enrichment analysis (GSEA) between MMP7/CXCL16 and YAP1-associated pathways. Western blotting assay was employed to evaluate YAP1/MMP7/CXCL16 expression in vitro and their modulation sequence. Logistic model stepwise regression analysis was used to assess YAP1, MMP7, CXCL16, and TILs as therapeutic predictors. Residual cancer burden (RCB) score was calculated and statistically analyzed according to intensity of these variables, and receiver operating characteristic (ROC) curve also showed their predictive value in NAC response. Recruitment efficacy for CD4+/CD8+ TIL cells (TCGA data) as well as quantified TIL cells density were both explored according to YAP1, MMP7, and CXCL16 expression level. RESULTS: Up-regulation of YAP1/MMP7 and down-regulation of CXCL16 were both significant in TNBC cases with poor NAC response. Inhibition of YAP1 induced down-regulation of MMP7 and up-regulation of CXCL16, whereas inhibition of MMP7 also induced up-regulation of CXCL16. It was also shown that MMP7/CXCL16 was enriched in the YAP1-related pathway. Activation of the YAP1/MMP7/CXCL16 axis obviously affected RCB of TNBC cases. The ROC curve also supported the predictive value of YAP1/MMP7/CXCL16 axis and TILs density in NAC response prospect. The density of TILs, meanwhile, demonstrated a strong link with the YAP1/MMP7/CXCL16 axis. Over expression of YAP1/MMP7 significantly suppressed recruitment of CD4+/CD8+ TILs, while CXCL16 over expression had a beneficial impact on anti-tumor immune. CONCLUSIONS: Over expression of causes up-regulation of MMP7 and down-regulation of CXCL16, which suppressed CD4+/CD8+ TILs recruitment and indirectly affected NAC response of TNBC patients.

NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output.

Most prostate cancer (PCa) deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa (mCRPC); however, the mechanism and key players leading to this are not fully understood. While studying the role of tousled-like kinase 1 (TLK1) and never in mitosis gene A (NIMA)-related kinase 1 (NEK1) in a DNA damage response (DDR)-mediated cell cycle arrest in LNCaP cells treated with bicalutamide, we uncovered that overexpression of wt-NEK1 resulted in a rapid conversion to androgen-independent (AI) growth, analogous to what has been observed when YAP1 is overexpressed. We now report that overexpression of wt-NEK1 results in accumulation of YAP1, suggesting the existence of a TLK1>NEK1>YAP1 axis that leads to adaptation to AI growth. Further, YAP1 is co-immunoprecipitated with NEK1. Importantly, NEK1 was able to phosphorylate YAP1 on six residues in vitro, which we believe are important for stabilization of the protein, possibly by increasing its interaction with transcriptional partners. In fact, knockout (KO) of NEK1 in NT1 PCa cells resulted in a parallel decrease of YAP1 level and reduced expression of typical YAP-regulated target genes. In terms of cancer potential implications, the expression of NEK1 and YAP1 proteins was found to be increased and correlated in several cancers. These include PCa stages according to Gleason score, head and neck squamous cell carcinoma, and glioblastoma, suggesting that this co-regulation is imparted by increased YAP1 stability when NEK1 is overexpressed or activated by TLK1, and not through transcriptional co-expression. We propose that the TLK1>NEK1>YAP1 axis is a key determinant for cancer progression, particularly during the process of androgen-sensitive to -independent conversion during progression to mCRPC.