Yes-associated Protein Activation Research Articles

Evidence that CRISPR-Cas9 Y537S-mutant expressing breast cancer cells activate Yes-associated protein 1 to driving the conversion of normal fibroblasts into cancer-associated fibroblasts.

Endocrine therapy (ET) has improved the clinical outcomes of Estrogen receptor alpha-positive (ERɑ +) breast cancer (BC) patients, even though resistance to ET remains a clinical issue. Mutations in the hormone-binding domain of ERɑ represent an acquired intrinsic mechanism of ET resistance. However, the latter also depends on the multiple functional interactions between BC cells and the tumor microenvironment (TME). Here, we investigated how the most common Y537S-ERɑ mutation may influence the behavior of fibroblasts, the most prominent component of the TME. We conducted coculture experiments with normal human foreskin fibroblasts BJ1-hTERT (NFs), cancer-associated fibroblasts (CAFs), isolated from human BC specimens, and Y537S CRISPR-expressing MCF-7 BC cells (MCF-7YS). Mass spectrometry (MS) and Metacore analyses were performed to investigate how the functional interactions between BC cells/fibroblasts may affect their proteomic profile. The impact of fibroblasts on BC tumor growth and metastatic potential was evaluated in nude mice. Mutant BC conditioned medium (CM) affected the morphology/proliferation/migration of both NFs and CAFs. 198 deregulated proteins signed the proteomic similarity profile of NFs exposed to the YS-CM and CAFs. Among the upregulated proteins, Yes-associated protein 1 (YAP1) was the main central hub in the direct interaction network. Increased YAP1 protein expression and activity were confirmed in NFs treated with MCF-7YS-CM. However, YAP1 activation appears to crosstalk with the insulin growth factor-1 receptor (IGF-1R). Higher amount of IGF-1 were noticed in the MCF-7YS-CM cells compared to the MCF-7P, and IGF-1 immunodepletion reversed the enhanced YAP1 expression and activity. Mutant cells upon exposure to the NF- and CAF-CM exhibited an enhanced proliferation/growth/migration/invasion compared to the MCF-7P. MCF-7YS cells when implanted with CAFs showed an early relative increased tumor volume compared to YS alone. No changes were observed when MCF-7P cells were co-implanted with CAFs. Compared with that in MCF-7P cells, the metastatic burden of MCF-7YS cells was intrinsically greater, and this effect was augmented upon treatment with NF-CM and further increased with CAF-CM. YS mutant BC cells induced the conversion of fibroblasts into CAFs, via YAP, which represent a potential therapeutic target which interrupt the functional interactions between mutant cells/TME and to be implemented in the novel therapeutic strategy of a subset of metastatic BC patients carrying the frequent Y537S mutations.

Hypoxia-reducedYAP phosphorylation enhances expression of Mucin5AC in nasal epithelial cells of chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is an upper respiratory disease characterized by persistent inflammation of the nasal mucosa. However, the mechanism of abnormal Mucin5AC expression by CRSwNP epithelial cells is not fully understood. We investigated the potential role of yes-associated protein (YAP) underlying the mechanism of excessive epithelial Mucin5AC expression in CRSwNP in a hypoxic model. Tissue biopsies of CRSwNP (n = 60), chronic rhinosinusitis without nasal polyps (CRSsNP) (n = 9) and healthy controls (n = 30) were investigated together with a well-established hypoxic model of primary human nasal epithelial cells (hNECs). The expression levels of hypoxia inducible factor (HIF)-1α and YAP, and the effect of the signaling axis on mucus secretion in hNECs were analyzed. We observed a significant elevated expression levels of YAP in patients with CRSwNP and CRSsNP compared to controls. In addition, HIF-1α expression of CRSwNP was higher than that of control group. Under hypoxic conditions, HIF-1α was found to regulate the upregulation of YAP in hNECs. Further investigations revealed that HIF-1α facilitated the activation and nuclear localization of active-YAP by reducing the phosphorylation of YAP. This mechanism appeared to be linked to HIF-1α-mediated inhibition of LATS 1 phosphorylation and subsequent YAP degradation. HIF-1α was shown to promote the expression of P63 and the levels of Mucin5AC in hNECs by enhancing YAP activation. Our findings indicated that hypoxia enhances YAP activation by decreasing p-LATS 1 and YAP phosphorylation. This has the potential to impact on the proliferation of basal cells and the differentiation of goblet cells in CRSwNP, ultimately leading to a pathological condition characterized by excessive Mucin5AC expression.

Early anti-inflammatory polarization of macrophages ameliorates post-surgical inflammation and osseointegration around titanium implants in mice

Dental implants are considered a superior option for the replacement of missing teeth. However, the invasive nature of the surgical procedure often results in significant postoperative inflammation, and the prolonged healing period of 3 to 6 months presents a notable disadvantage. High mobility group box 1 (HMGB1) is a critical mediator of acute inflammation following surgical injury, which can hinder the onset of osseointegration. This study aims to examine whether the inhibition of HMGB1 can mitigate acute inflammation and subsequently enhance osseointegration. The findings indicate that HMGB1 inhibition markedly reduces inflammation and promotes bone repair in murine models. Further in vitro investigations into the regulatory mechanisms of HMGB1 in macrophages reveal its role in increasing Yes-associated protein (YAP) activity, which contributes to pro-inflammatory polarization. Additionally, conditioned media derived from macrophages influenced by HMGB1 significantly impair the migratory and osteogenic capabilities of bone marrow-derived mesenchymal stem cells, which are essential for bone regeneration. In vivo experiments further validate that the administration of exogenous HMGB1 exacerbates postoperative acute inflammation and obstructs osseointegration. The study concludes that inhibiting HMGB1 fosters an anti-inflammatory polarization of macrophages, leading to diminished postoperative acute inflammation and expedited osseointegration around dental implants in mice.

Piezo1-Mediated Mechanotransduction Contributes to Disturbed Flow-Induced Atherosclerotic Endothelial Inflammation.

Disturbed flow generates oscillatory shear stress (OSS), which in turn leads to endothelial inflammation and atherosclerosis. Piezo1, a biomechanical force sensor, plays a crucial role in the cardiovascular system. However, the specific role of Piezo1 in atherosclerosis remains to be fully elucidated. We detected the expression of Piezo1 in atherosclerotic mice and endothelial cells from regions with disturbed blood flow. The pharmacological inhibitor Piezo1 inhibitor (GsMTx4) was used to evaluate the impact of Piezo1 on plaque progression and endothelial inflammation. We examined Piezo1's direct response to OSS invitro and its effects on endothelial inflammation. Furthermore, mechanistic studies were conducted to explore the potential molecular cascade through which Piezo1 mediates endothelial inflammation in response to OSS. Our findings revealed the upregulation of Piezo1 in apoE-/- (apolipoprotein E) atherosclerotic mice, which is associated with disturbed flow. Treatment with GsMTx4 not only delayed plaque progression but also mitigated endothelial inflammation in both chronic and disturbed flow-induced atherosclerosis. Piezo1 was shown to facilitate calcium ions (Ca2+)influx in response to OSS, thereby activating endothelial inflammation. This inflammatory response was attenuated in the absence of Piezo1. Additionally, we identified that under OSS, Piezo1 activates the Ca2+/CaM/CaMKII (calmodulin/calmodulin-dependent protein kinases Ⅱ) pathways, which subsequently stimulate downstream kinases FAK (focal adhesion kinase) and Src. This leads to the activation of the OSS-sensitive YAP (yes-associated protein), ultimately triggering endothelial inflammation. Our study highlights the key role of Piezo1 in atherosclerotic endothelial inflammation, proposing the Piezo1-Ca2+/CaM/CaMKII-FAK/Src-YAP axis as a previously unknown endothelial mechanotransduction pathway. Piezo1 is expected to become a potential therapeutic target for atherosclerosis and cardiovascular diseases.

Advancements of anticancer agents by targeting the Hippo signalling pathway: biological activity, selectivity, docking analysis, and structure-activity relationship.

The Hippo signalling pathway is prominent andgoverns cell proliferation and stem cell activity, acting as a growth regulator and tumour suppressor. Defects in Hippo signalling and hyperactivation of its downstream effector's Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) play roles in cancer development, implying that pharmacological inhibition of YAP and TAZ activity could be an effective cancer treatment strategy. Conversely, YAP and TAZ can also have beneficial effects in promoting tissue repair and regeneration following damage, therefore their activation may be therapeutically effective in certain instances. Recently, a complex network of intracellular and extracellular signalling mechanisms that affect YAP and TAZ activity has been uncovered. The YAP/TAZ-TEAD interaction leads to tumour development and the protein structure of YAP/TAZ-TEAD includes three interfaces and one hydrophobic pocket. There are clinical and preclinical trial drugs available to inhibit the hippo signalling pathway, but these drugs have moderate to severe side effects, so researchers are in search of novel, potent, and selective hippo signalling pathway inhibitors. In this review, we have discussed the hippo pathway in detail, including its structure, activation, and role in cancer. We have also provided the various inhibitors under clinical and preclinical trials, and advancement of small molecules their detailed docking analysis, structure-activity relationship, and biological activity. We anticipate that the current study will be a helpful resource for researchers.

Inhibiting Yes-associated protein alleviates CCl4 liver fibrosis in mice by reducing epithelial mesenchymal transition

To explore whether Yes-associated protein (YAP) affects occurrence and progression of liver fibrosis by regulating epithelial-mesenchymal transition (EMT). In a 8-week-old C57BL/6 mouse model of CCl4-induced liver fibrosis, the effect of verteporfin (a YAP inhibitor) intervention was assessed with HE staining and by detecting liver biochemistry and expressions of YAP and EMT-related genes using immunohistochemistry and Western blotting. Transcriptome and proteomic sequencing and informatics analysis were used to investigate the main downstream pathways of YAP in liver fibrosis. Serum levels of YAP, N-cadherin, vimentin and Twist were examined in 60 healthy individuals, 60 patients with chronic hepatitis B (CHB), and 60 patients with HBV-related liver cirrhosis. In another 24 C57BL/6 mice, the effects of Twist inhibitor alone or in combination with harmine (a YAP activator) on CCl4-induced liver fibrosis were evaluated by histopathological examination and Western blotting. The mouse models of liver fibrosis showed obvious structural damages of the liver lobes with formation of pseudolobules, and verteporfin treatment significantly improved these pathologies and lowered plasma ALT and AST levels of the mice. Transcriptome and proteomic sequencing and informatics analysis suggested that N-cadherin and Twist were differentially expressed in liver fibrosis in close correlation with YAP. Inhibition of YAP obviously downregulated hepatic N-cadherin and Twist protein expressions in the mice with liver fibrosis. In patients with CHB and liver cirrhosis, serum levels of YAP elevated obviously with the severity of liver fibrosis and were significantly correlated with N-cadherin, vimentin and Twist levels. In mice with liver fibrosis, inhibiting Twist effectively improved liver inflammation and fibrosis, while the combined treatment with YAP activator worsened hepatic collagen fiber deposition and increased hepatic YAP and α-SMA expressions. EMT is an important pathogenic mechanism of liver fibrosis, and inhibiting YAP can alleviate liver fibrosis by reducing EMT.

BRAF Modulates the Interplay Between Cell-Cell and Cell-Extracellular Matrix Adhesions in PECAM-1-Mediated Mechanotransduction.

Mechanotransduction, the process of how cells sense and convert mechanical stimuli into biochemical response, is crucial in the migration of leukocytes or cancer cells through the endothelium during inflammation or metastasis. Migrating cells exert forces on the endothelium through cell surface adhesion molecules, such as platelet endothelial adhesion molecule PECAM-1, and this is essential for a successful transmigration. To study PECAM-1-mediated mechanotransduction, we applied PECAM-1-antibody-coated magnetic beads and exerted about 40 pN force on the endothelial monolayer. We show that force increases cell-ECM adhesion in the cell center and is accompanied by the opening of cell-cell junctions. Upon depletion of the MEK/ERK kinase, BRAF force increases cell-ECM adhesion both at the cell periphery and in the cell center, but this does not result in the opening of cell-cell junctions. Decreasing cell-ECM adhesion in BRAF-depleted cells through FAK inhibition results in the remodeling of cell-cell junctions. Force-induced increase in cell-ECM adhesion in the cell center correlates with the activation of the transcriptional cofactor Yes-associated protein (YAP). Furthermore, the induced activation of YAP through LATS inhibition prevents junctional remodeling in control cells. Thus, the activation of YAP might determine the strength of cell-cell junctions during PECAM-1-mediated mechanotransduction.

Wedelolactone Attenuates Liver Fibrosis and Hepatic Stellate Cell Activation by Suppressing the Hippo Pathway.

Liver fibrosis is a commonly observed pathological phenomenon that occurs during the progression of various types of chronic liver diseases. The Hippo pathway is closely associated with the pathogenesis of liver fibrosis. Previous studies have shown that wedelolactone (WED) has a significant antihepatic fibrosis effect, whereas the target and mechanism underlying WED remain elusive. In this study, we found that WED significantly alleviated liver fibrosis and injury by inhibiting the expression of Yes-associated protein (YAP) and tafazzin (TAZ). In an in vitro model, WED suppressed the activation of hepatic stellate cells (HSCs) induced by transforming growth factor (TGF-β1), as well as the mRNA and protein expression of α-smooth muscle actin (α-SMA), YAP, and TAZ. The allosteric regulation of YAP by WED was confirmed using MD and cellular thermal shift assay. Moreover, specific knockdown or inhibition of YAP did not enhance the suppressive effect of WED on HSC activation or protein expression associated with fibrosis. These findings demonstrated that the administration of WED effectively alleviated liver fibrosis by suppressing the Hippo/YAP/TAZ pathways. In addition, YAP activity may be regulated by WED via allosteric regulation.

7290 Hippo Signaling Regulates Zona Glomerulosa Cell Fate In The Adrenal Cortex Of Mice

Abstract Disclosure: N. Abou Nader: None. N. Jakuc: None. M. Meinsohn: None. L. Charrier: None. M. Paquet: None. G. St-Jean: None. J. Brind’Amour: None. D. Boerboom: None. J. Mao: None. D. Pepin: None. G. Zamberlam: None. D.T. Breault: None. A. Boyer: None. The Hippo signaling is a major signaling pathway that regulates processes such as cell proliferation and differentiation during both embryogenesis and postnatal homeostatic maintenance in several tissues. Our recent findings, using the Nr5a1-cre model, have highlighted the essential role of Lats1 and Lats2 (large tumor suppressor homolog kinases 1/2), the main kinases of the Hippo signaling cascade, in maintaining proper differentiation of mouse developing adrenocortical cells by suppressing their differentiation into myofibroblast-like cells. However, since the abnormal transdifferentiation of Lats1/2-negative adrenocortical cells occurs before the establishment of zonation when using the Nr5a1-cre model, the role of Hippo signaling in zona glomerulosa (zG) cells could not be assessed with this model. To overcome this limitation, we inactivated Lats1/2 in aldosterone-producing zG cells using the zG cell-specific AS-cre strain (Lats1flox/flox; Lats2flox/flox; AScre/+). We report here that specific loss of Lats1/2 in steroidogenic zG cells results in the accumulation of an important extracellular matrix (ECM) in the zG an outer zona fasciculata (zF) by 4 weeks of age; a phenotype that does not seem to progress with aging. In addition, tracing experiments demonstrate that the inactivation of Lats1/2 leads to the ectopic transdifferentiation of the zG cells into the ECM-producing cells rather than into zF cells. We then performed RNAseq experiments on whole adrenals which further suggested that the ECM producing cells derived from zG cells share characteristics with chondroblast/osteoblast mesenchymal progenitors among other cell lineages and, that the ectopic transdifferentiation of zG cells triggers a significant inflammation/immune response in the adrenal cortex. Our study also demonstrates that the increased activity of the Hippo pathway effectors YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) is responsible for the ectopic transdifferentiation of the zG cells as their concomitant inactivation with Lats1/2 (Yapflox/flox; Tazflox/flox; Lats1flox/flox; Lats2flox/flox; AScre/+) rescues the observed phenotype. Finally, we also demonstrate that the expression of a constitutively active form of YAP (YAP5SA) in zG cells (Rosa26YAP5SA/+; AScre/+) does not alter their identity as severely as the loss of Lats1/2 but leads to adrenal hyperplasia. Together, these novel findings underscore the crucial role of Hippo signaling in maintaining the identity of zG cells in the adrenal cortex. Presentation: 6/2/2024

Pressure loading regulates the stemness of liver cancer stem cells via YAP/BMF signaling axis.

Cancer stem cells (CSCs) are considered the major cause of the occurrence, progression, chemoresistance/radioresistance, recurrence, and metastasis of cancer. Increased interstitial fluid pressure (IFP) is a key feature of solid tumors. Our previous study showed that the distribution of liver cancer stem cells (LCSCs) correlated with the mechanical heterogeneity within liver cancer tissues. However, the regulation of liver cancer's mechanical microenvironment on the LCSC stemness is not fully understood. Here, we employed a cellular pressure-loading device to investigate the effects of normal IFP (5 mmHg), as well as increased IFP (40 and 200 mmHg) on the stemness of LCSCs. Compared to the control LCSCs (exposure to 5 mmHg pressure loading), the LCSCs exposed to 40 mmHg pressure loading exhibited significantly upregulated expression of CSC markers (CD44, EpCAM, Nanog), enhanced sphere and colony formation capacities, and tumorigenic potential, whereas continuously increased pressure to 200 mmHg suppressed the LCSC characteristics. Mechanistically, pressure loading regulated Yes-associated protein (YAP) activity and Bcl-2 modifying factor (BMF) expression. YAP transcriptionally regulated BMF expression to affect the stemness of LCSCs. Knockdown of YAP and overexpression of BMF attenuated pressure-mediated stemness and tumorgenicity, while YAP-deficient and BMF-deletion recused pressure-dependent stemness on LCSCs, suggesting the involvement of YAP/BMF signaling axis in this process. Together, our findings provide a potential target for overcoming the stemness of CSCs and elucidate the significance of increased IFP in cancer progression.

YAP promotes global mRNA translation to fuel oncogenic growth despite starvation.

Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) play fundamental roles in stem/progenitor cell expansion during homeostasis, and their dysregulation often leads to tissue overgrowth. Here, we show that YAP activation is sufficient to overcome the restriction of global protein synthesis induced by serum starvation, enabling cells to sustain proliferation and survival despite an unfavorable environment. Mechanistically, YAP/TAZ selectively promoted the mTORC1-dependent translation of mRNAs containing 5' terminal oligopyrimidine (5'TOP) motifs, ultimately increasing the cellular polysome content. Interestingly, DNA damage-inducible transcript 4 (DDIT4), a negative regulator of mTORC1, was upregulated by serum starvation but repressed by YAP/TAZ. DDIT4 was sufficient to suppress the translation and transformative potential of uveal melanoma cells, which are often serum unresponsive due to G protein mutations. Our findings reveal a vital role for protein synthesis as a key modality of YAP/TAZ-induced oncogenic transformation and indicate the potential for targeting mTORC1 or translation to treat YAP/TAZ-driven malignancies.

Mechanotransduction pathways regulating YAP nuclear translocation under Yoda1 and vibration in osteocytes

Yes-associated protein (YAP) is a mechanosensitive protein crucial for bone remodeling. Although research has identified pathways and components involved in YAP regulation, the precise mechanisms of its localization during Piezo1 activation or vibration remain unclear. Piezo1, a mechanosensitive ion channel, allows calcium ions to flow into cells upon activation. Recent studies suggest that combining Yoda1, a Piezo1 activator, with low-magnitude high-frequency (LMHF) vibration (>30 Hz, <1 g acceleration) enhances YAP nuclear translocation. This combination potentially improves the mechanoresponse and therapeutic efficacy of LMHF vibration in bone cells. This study aims to elucidate how Yoda1 and LMHF vibration regulate mechanosensitive structures and pathways, leading to YAP nuclear translocation in MLO-Y4 osteocyte like cells. We investigated the roles of the cytoskeleton and nuclear envelope (NE) in YAP activation under combined LMHF vibration and Yoda1 treatments. Additionally, we analyzed differentially expressed genes (DEGs) in MLO-Y4 cells subjected to these treatments and in Piezo1 knockdown MLO-Y4 cells exposed to vibration. Our findings indicated that increased YAP nuclear translocation with combined treatment may result from the distinct effects of Yoda1 and vibration. Specifically, Yoda1 influenced YAP through mechanisms involving actin and NE dynamics, while LMHF vibration may modulate YAP via the interleukin 6 (IL6)/signal transducer and activator of transcription 3 (STAT3) axis. This study provides new insights and potential therapeutic targets for osteocyte-related pathologies.

EDA Fibronectin Microarchitecture and YAP Translocation During Wound Closure.

Fibronectin (Fn) is an extracellular matrix glycoprotein with mechanosensitive structure-function. EDA Fn, a Fn isoform, is not present in adult tissue but is required for tissue repair. Curiously, EDA Fn is linked to both regenerative and fibrotic tissue repair. Given that Fn mechanoregulates cell behavior, Fn EDA organization during wound closure might play a role in mediating these differing responses. One mechanism by which cells sense and respond to their microenvironment is by activating a transcriptional co-activator, Yes-associated protein (YAP). Interestingly, YAP activity is not only required for wound closure, but similarly linked to both regenerative and fibrotic repair. Therefore, this study aims to evaluate how, during normal and fibrotic wound closure, EDA Fn organization might modulate YAP translocation by culturing human dermal fibroblasts on polydimethylsiloxane (PDMS) substrates mimicking normal (soft: 18 kPa) and fibrotic (stiff: 146 kPa) wounded skin. On stiffer substrates mimicking fibrotic wounds, fibroblasts assembled an aligned EDA Fn matrix comprising thinner fibers, suggesting increased microenvironmental tension. To evaluate if cell binding to the EDA domain of Fn was essential to overall matrix organization, fibroblasts were treated with Irigenin, which inhibits binding to the EDA domain within Fn. Blocking adhesion to EDA led to randomly organized EDA Fn matrices with thicker fibers, suggesting reduced microenvironmental tension even during fibrotic wound closure. To evaluate if YAP signaling plays a role in EDA Fn organization, fibroblasts were treated with CA3, which suppresses YAP activity in a dose-dependent manner. Treatment with CA3 also led to randomly organized EDA Fn matrices with thicker fibers, suggesting a potential connected mechanism of reducing tension during fibrotic wound closure. Next, YAP activity was assessed to evaluate the impact of EDA Fn organization. Interestingly, fibroblasts migrating on softer substrates mimicking normal wounds increased YAP activity but on stiffer substrates, decreased YAP activity. When fibroblasts on stiffer substrates were treated with Irigenin or CA3, fibroblasts increased YAP activity. These results suggest there may be disrupted signaling between EDA Fn organization and YAP translocation during fibrotic wound closure that could be restored when reestablishing normal EDA Fn matrix organization to instead drive regenerative wound repair.

Identifying adeno-associated virus (AAV) vectors that efficiently target high grade glioma cells, for in vitro monitoring of temporal cell responses.

To improve the translation of preclinical cancer research data to successful clinical effect, there is an increasing focus on the use of primary patient-derived cancer cells with limited growth in culture to reduce geneticand phenotype drift. However, these primary lines are less amenable to standardly used methods of exogenous DNA introduction. Adeno-associated viral (AAV) vectors display tropism for a wide range of human tissues, avidly infect primary cells and have a good safety profile. In the present study, we therefore used a next-generation sequencing (NGS) barcoded AAV screening method to assess transduction capability of a panel of 36 AAVs in primary cell lines representing high-grade glioma (HGG) brain tumours including glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). As proof of principle, we created a reporter construct to analyse activity of the transcriptional co-activators yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ). Transcriptional activation was monitored by promoter-driven expression of the Timer fluorescent tag, a protein that fluoresces green immediately after transcription and transitions to red fluorescence over time. As expected, attempts to express the reporter in primary HGG cells from plasmid expression vectors were unsuccessful. Using the top candidate from the AAV screen, we demonstrate successful AAV-mediated transduction of HGG cells with the YAP/TAZ dynamic activity reporter. In summary, the NGS-screening approach facilitated screening of many potential AAVs, identifying vectors that can be used to study the biology of primary HGG cells.

Activation of Yes-Associated Protein Is Indispensable for Transformation of Kidney Fibroblasts into Myofibroblasts during Repeated Administration of Cisplatin.

Cisplatin is a potent chemotherapy medication that is used to treat various types of cancer. However, it can cause nephrotoxic side effects, which lead to acute kidney injury (AKI) and subsequent chronic kidney disease (CKD). Although a clinically relevant in vitro model of CKD induced by repeated administration of low-dose cisplatin (RAC) has been established, its underlying mechanisms remain poorly understood. Here, we compared single administration of high-dose cisplatin (SAC) to repeated administration of low-dose cisplatin (RAC) in myofibroblast transformation and cellular morphology in a normal rat kidney fibroblast NRK-49F cell line. RAC instead of SAC transformed the fibroblasts into myofibroblasts as determined by α-smooth muscle actin, enlarged cell size as represented by F-actin staining, and increased cell flattening as expressed by the semidiameter ratio of attached cells to floated cells. Those phenomena, as well as cellular senescence, were significantly detected from the time right before the second administration of cisplatin. Interestingly, inhibition of the interaction between Yes-associated protein (YAP) and the transcriptional enhanced associated domain (TEAD) using Verteporfin remarkedly reduced cell size, cellular senescence, and myofibroblast transformation during RAC. These findings collectively suggest that YAP activation is indispensable for cellular hypertrophy, senescence, and myofibroblast transformation during RAC in kidney fibroblasts.

A non-canonical repressor function of JUN restrains YAP activity and liver cancer growth

Yes-associated protein (YAP) and its homolog, transcriptional coactivator with PDZ-binding motif (TAZ), are the main transcriptional downstream effectors of the Hippo pathway. Decreased Hippo pathway activity leads to nuclear translocation of YAP/TAZ where they interact with TEAD transcription factors to induce target gene expression. Unrestrained YAP/TAZ activity can lead to excessive growth and tumor formation in a short time, underscoring the evolutionary need for tight control of these two transcriptional coactivators. Here, we report that the AP-1 component JUN acts as specific repressor of YAP/TAZ at joint target sites to decrease YAP/TAZ activity. This function of JUN is independent of its heterodimeric AP-1 partner FOS and the canonical AP-1 function. Since expression of JUN is itself induced by YAP/TAZ, our work identifies a JUN-dependent negative feedback loop that buffers YAP/TAZ activity at joint genomic sites. This negative feedback loop gets disrupted in liver cancer to unlock the full oncogenic potential of YAP/TAZ. Our results thus demonstrate an additional layer of control for the interplay of YAP/TAZ and AP-1.

Angiotensin II depends on hippo/YAP signaling to reprogram angiogenesis and promote liver fibrosis

Liver fibrosis is a chronic pathological process in which the abnormal proliferation of connective tissue is induced by various pathogenic factors. During the process of fibrosis, excessive angiogenesis is observed. Physiological angiogenesis has the potential to impede the progression of liver fibrosis through augmenting matrix metalloenzyme activity; however, pathological angiogenesis can exacerbate liver fibrosis by promoting collagen accumulation. Therefore, a key scientific research focus in the treatment of liver diseases is to search for the “on-off” mechanism that regulates angiogenesis from normal proliferation to pathological proliferation. In this study, we found that excessive angiogenesis appeared during the initial phase of hepatic fibrosis without mesenchymal characteristics. In addition, angiogenesis accompanied by significant endothelial-to-mesenchymal transition (EndMT) was observed in mice after the intraperitoneal injection of angiotensin II (Ang II). Interestingly, the changes in Yes-associated protein (YAP) activity in endothelial cells (ECs) can affect the regulation of angiogenesis by Ang II. The results of in vitro experiments revealed that the regulatory influence of Ang II on ECs was significantly attenuated upon suppression of YAP activity. Furthermore, the function of Ang II in regulating angiogenesis during fibrosis was investigated in liver-specific transgenic mice. The results revealed that Ang II gene deletion could restrain liver fibrosis and EndMT. Meanwhile, Ang II deletion downregulated the profibrotic YAP signaling pathway in ECs. The small molecule AT1R agonist olmesartan targeting Ang II-YAP signaling could also alleviate liver fibrosis. In conclusion, this study identified Ang II as a pivotal regulator of EndMT during the progression of liver fibrosis and evaluated the therapeutic effect of the Ang II-targeted drug olmesartan on liver fibrosis.

Retinoic acid-induced protein 14 links mechanical forces to Hippo signaling

Cells sense and respond to various mechanical forces from the extracellular matrix primarily by modulating the actin cytoskeleton. Mechanical forces can be translated into biochemical signals in a process called mechanotransduction. Yes-associated protein (YAP) is an effector of Hippo signaling and a mediator of mechanotransduction, but how mechanical forces regulate Hippo signaling is still an open question. We propose that retinoic acid-induced protein 14 (RAI14) responds to mechanical forces and regulates Hippo signaling. RAI14 positively regulates the activity of YAP. RAI14 interacts with NF2, a key component of the Hippo pathway, and the interaction occurs on filamentous actin. When mechanical forces are kept low in cells, NF2 dissociates from RAI14 and filamentous actin, resulting in increased interactions with LATS1 and activation of the Hippo pathway. Clinical data show that tissue stiffness and expression of RAI14 and YAP are upregulated in tumor tissues and that RAI14 is strongly associated with adverse outcome in patients with gastric cancer. Our data suggest that RAI14 links mechanotransduction with Hippo signaling and mediates Hippo-related biological functions such as cancer progression.

A highly sensitive reporter system to monitor endogenous YAP1/TAZ activity and its application in various human cells.

The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity invitro and Venus fluorescence invivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/β-catenin/TCF- and IL-1β/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and β-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts.

Neuro-mesenchymal interaction mediated by a β2 adrenergic-nerve growth factor feedforward loop promotes colorectal cancer progression.

Cancer-associated fibroblasts (CAFs) and nerves, components of the tumor microenvironment, have each been shown to directly promote gastrointestinal cancers. However, it remains unknown whether these cells interact with each other to regulate cancer progression. We found that in colorectal cancer (CRC) norepinephrine induces ADRB2-dependent nerve growth factor (NGF) secretion from CAFs, which in turn increases intra-tumor sympathetic innervation and norepinephrine accumulation. Adrenergic stimulation accelerates CRC growth through ADRA2A/Gi-mediated activation of Yes-Associated Protein (YAP). NGF from CAFs directly enhances CRC cell growth via the PI3K/AKT pathway. Treatment with a tropomyosin receptor kinase (Trk) inhibitor decreased YAP and AKT activation and CRC progression in mice. In human CRC, high NGF expression is associated with the mesenchymal-like tumor subtype and poor patient survival. These findings suggest a central role for reciprocal CAF-nerve crosstalk in promoting CRC progression. Blocking this feedforward loop with a Trk inhibitor may represent a potential therapeutic approach for CRC.