[Mechanism of resveratrol in protecting ovary in poor ovarian response mice by regulating Hippo signaling pathway].

This study observed the protective effect of resveratrol(Res) on ovarian function in poor ovarian response(POR) mice by regulating the Hippo signaling pathway and explored the potential mechanism of Res in inhibiting ovarian cell apoptosis. Female mice with regular estrous cycles were randomly divided into a blank group, a model group, and low-and high-dose Res groups(20 and 40 mg·kg~(-1)), with 20 mice in each group. The blank group received an equal volume of 0.9% saline solution by gavage, while the model group and Res groups received suspension of glycosides of Triptergium wilfordii(GTW) at 50 mg·kg~(-1) by gavage for two weeks to induce the model. After modeling, the low-and high-dose Res groups were continuously treated with drugs by gavage for two weeks, while the blank group and the model group received an equal volume of 0.9% saline solution by gavage. Ovulation was induced in all groups on the day following the end of treatment. Finally, 12 female mice were randomly selected from each group, and the remaining eight female mice were co-housed with male mice at a ratio of 1∶1. Changes in the estrous cycle of mice were observed using vaginal cytology smears. The number of ovulated eggs, ovarian wet weight, ovarian index, and pregnancy rate of mice were measured. The le-vels of anti-Mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in serum were determined using enzyme-linked immunosorbent assay(ELISA). Ovarian tissue morphology and ovarian cell apoptosis were observed using hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining, respectively. The protein expression levels of yes-associated protein(YAP) 1 and transcriptional coactivator with PDZ-binding motif(TAZ) were detected by immunohistochemistry(IHC), while the changes in protein expression levels of mammalian sterile 20-like kinase(MST) 1/2, large tumor suppressor(LATS) 1/2, YAP1, TAZ, B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were determined by Western blot. The results showed that compared with the blank group, the model group had an increased rate of estrous cycle disruption in mice, a decreased number of normally developing ovarian follicles, an increased number of blocked ovarian follicles, increased ovarian granulosa cell apoptosis, decreased ovulation, reduced ovarian wet weight and ovarian index, increased serum FSH and LH levels, decreased AMH and E_2 levels, decreased protein expression levels of YAP1 and TAZ in ovarian tissues, increased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and decreased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Additionally, the number of embryos per litter significantly decreased after co-housing. Compared with the model group, the low-and high-dose Res groups exhibited reduced estrous cycle disruption rates in mice, varying degrees of improvement in the number and morphology of ovarian follicles, reduced numbers of blocked ovarian follicles, improved ovarian granulosa cell apoptosis, increased ovulation, elevated ovarian wet weight and ovarian index, decreased serum FSH and LH levels, increased AMH and E_2 levels, elevated protein expression levels of YAP1 and TAZ in ovarian tissues, decreased relative expression levels of MST1/2, LATS1/2, and Bax proteins, and increased relative expression levels of YAP1, TAZ, and Bcl-2 proteins. Furthermore, the number of embryos per litter increased to varying degrees after co-housing. In conclusion, Res effectively inhibits ovarian cell apoptosis in mice and improves ovarian responsiveness. Its mechanism may be related to the regulation of key molecules in the Hippo pathway.

Trans-3, 5, 4'-trimethoxystilbene restrains non-small-cell lung carcinoma progression via suppressing M2 polarization through inhibition of m6A modified circPACRGL-mediated Hippo signaling.

BACKGROUND: Non-small-cell lung carcinoma (NSCLC) accounts for ∼85% of all lung carcinomas. Trans-3,5,4'-trimethoxystilbene (TMS) shows strong anti-tumor activity and induces tumor cell apoptosis. However, its function and mechanism in NSCLC still require investigation. METHODS: PMA was used to treated THP-1 cells for macrophage differentiation. The abundance and m6A modification of circPACRGL were examined with qRT-PCR and MeRIP. Colony forming, transwell, wound healing, and Western blotting assays were applied to analyze proliferation, invasion, migration, and EMT. Macrophage polarization was determined through flow cytometry analysis of M1 and M2 markers. The interplay between circPACRGL, IGF2BP2 and YAP1 was validated by RNA pull-down and RIP assays. Mice received subcutaneous injection of NSCLC cells as a mouse model of subcutaneous tumor. RESULTS: CircPACRGL was upregulated in NSCLC cells, but it was reduced by TMS treatment. CircPACRGL depletion blocked proliferation, migration, and invasion in H1299 and H1975 cells. TMS suppressed these malignant behaviors, but it was abolished by circPACRGL overexpression. In addition, NSCLC-derived exosomes delivered circPACRGL into THP-1 cells to promote its M2 polarization, but TMS inhibited these effects by downregulating exosomal circPACRGL. Mechanically, exosomal circPACRGL bound to IGF2BP2 to improve the stability of YAP1 mRNA and regulate Hippo signaling in polarized THP-1 cells. TMS inhibited NSCLC growth via suppressing Hippo signaling and M2 polarization in vivo. CONCLUSION: TMS restrains M2 polarization and NSCLC progression by reducing circPACRGL and inhibiting exosomal circPACRGL-mediated Hippo signaling. Thus, these findings provide a novel mechanism underlying NSCLC progression and potential therapeutic targets.

Natural compound library screening to identify berberine as a treatment for rheumatoid arthritis.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) play a critical role on the exacerbation and deterioration of rheumatoid arthritis (RA). Aberrant activation of FLS pyroptosis signaling is responsible for the hyperplasia of synovium and destruction of cartilage of RA. This study investigated the screened traditional Chinese medicine berberine (BBR), an active alkaloid extracted from the Coptis chinensis plant, that regulates the pyroptosis of FLS and secretion of inflammatory factors in rheumatoid arthritis. METHODS: First, BBR was screened using a high-throughput drug screening strategy, and its inhibitory effect on RA-FLS was verified by in vivo and in vitro experiments. Second, BBR was intraperitoneally administrated into the collagen-induced arthritis rat model, and the clinical scores, arthritis index, and joint HE staining were evaluated. Third, synovial tissues of CIA mice were collected, and the expression of NLRP3, cleaved-caspase-1, GSDMD-N, Mst1, and YAP was detected by Western blot. RESULTS: The administration of BBR dramatically alleviated the severity of collagen-induced arthritis rat model with a decreased clinical score and inflammation reduction. In addition, BBR intervention significantly attenuates several pro-inflammatory cytokines (interleukin-1ß, interleukin-6, interleukin-17, and interleukin-18). Moreover, BBR can reduce the pyroptosis response (caspase-1, NLR family pyrin domain containing 3, and gasdermin D) of the RA-FLS in vitro, activating the Hippo signaling pathway (Mammalian sterile 20-like kinase 1, yes-associated protein, and transcriptional enhanced associate domains) so as to inhibit the pro-inflammatory effect of RA-FLS. CONCLUSION: These results support the role of BBR in RA and may have therapeutic implications by directly repressing the activation, migration of RA-FLS, which contributing to the attenuation of the progress of CIA. Therefore, targeting PU.1 might be a potential therapeutic approach for RA. Besides, BBR inhibited RA-FLS pyroptosis by downregulating of NLRP3 inflammasomes (NLRP3, caspase-1) and eased the pro-inflammatory activities via activating the Hippo signaling pathway, thereby improving the symptom of CIA.

Impact of De Novo Cholesterol Biosynthesis on the Initiation and Progression of Breast Cancer.

Cholesterol (CHOL) is a multifaceted lipid molecule. It is an essential structural component of cell membranes, where it cooperates in regulating the intracellular trafficking and signaling pathways. Additionally, it serves as a precursor for vital biomolecules, including steroid hormones, isoprenoids, vitamin D, and bile acids. Although CHOL is normally uptaken from the bloodstream, cells can synthesize it de novo in response to an increased requirement due to physiological tissue remodeling or abnormal proliferation, such as in cancer. Cumulating evidence indicated that increased CHOL biosynthesis is a common feature of breast cancer and is associated with the neoplastic transformation of normal mammary epithelial cells. After an overview of the multiple biological activities of CHOL and its derivatives, this review will address the impact of de novo CHOL production on the promotion of breast cancer with a focus on mammary stem cells. The review will also discuss the effect of de novo CHOL production on in situ and invasive carcinoma and its impact on the response to adjuvant treatment. Finally, the review will discuss the present and future therapeutic strategies to normalize CHOL biosynthesis.

Vemurafenib induces senescence in acute myeloid leukemia and myelodysplastic syndrome by activating the HIPPO signaling pathway: implications for potential targeted therapy.

BACKGROUND: The outcome of Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remain dismal despite the development of treatment. Targeted therapy is gaining more and more attention in improving prognosis. METHODS: Expression of BRAF was analyzed by RT-qPCR in AML and MDS patients. Cells viability treated by drugs was measured by CCK-8 assay. Network pharmacology and RNA-sequence were used to analyze the mechanism of drugs and verified in vitro and xenograft tumor model. RESULTS: Here we showed that BRAF was overexpressed in AML and MDS patients, and correlated with poor prognosis. The BRAF inhibitor-Vemurafenib (VEM) could significantly induce senescence, proliferation inhibition and apoptosis in AML cells, which can be enhanced by Bortezomib (BOR). This inhibitory effect was also verified in CD34 + cells derived from AML patients. Mechanistically, we showed that VEM combined with BOR could turn on HIPPO signaling pathway, thereby inducing cellular senescence in AML cells and xenograft mouse. CONCLUSIONS: Taken together, our findings demonstrate a significant upregulation of BRAF expression in AML and MDS patients, which is associated with unfavorable clinical outcomes. We also discovered that the BRAF inhibitor Vemurafenib induces cellular senescence through activation of the HIPPO signaling pathway. Analysis of BRAF expression holds promise as a prognostic indicator and potential therapeutic target for individuals with AML and MDS.

Breast Cancer Therapeutics and Hippo Signaling Pathway: Novel MicroRNA-Gene-Protein Interaction Networks.

The Hippo signaling pathway is a master regulator of development, cell proliferation, and apoptosis in particular, and it plays an important role in tissue regeneration, controlling organ size, and cancer suppression. Dysregulation of the Hippo signaling pathway has been implicated in breast cancer, a highly prevalent cancer affecting 1 out of every 15 women worldwide. While the Hippo signaling pathway inhibitors are available, they are suboptimal, for example, due to chemoresistance, mutation, and signal leakage. Inadequate knowledge about the Hippo pathway connections and their regulators limits our ability to uncover novel molecular targets for drug development. We report here novel microRNA (miRNA)-gene and protein-protein interaction networks in the Hippo signaling pathway. We employed the GSE miRNA dataset for the present study. The GSE57897 dataset was normalized and searched for differentially expressed miRNAs, and their targets were searched using the miRWalk2.0 tool. From the upregulated miRNAs, we observed that the hsa-miR-205-5p forms the biggest cluster and targets four genes involved in the Hippo signaling pathway. Interestingly, we found a novel connection between two Hippo signaling pathway proteins, angiomotin (AMOT) and mothers against decapentaplegic homolog 4 (SMAD4). From the downregulated miRNAs, hsa-miR-16-5p, hsa-miR-7g-5p, hsa-miR-141-3p, hsa-miR-103a-3p, hsa-miR-21-5p, and hsa-miR-200c-3p, target genes were present in the pathway. We found that PTEN, EP300, and BTRC were important cancer-inhibiting proteins, form hubs, and their genes interact with downregulating miRNAs. We suggest that targeting proteins from these newly unraveled networks in the Hippo signaling pathway and further research on the interaction of hub-forming cancer-inhibiting proteins can open up new avenues for next-generation breast cancer therapeutics.

The Ethyl acetate extract from Celastrus orbiculatus suppresses non-small-cell lung cancer by activating Hippo signaling and inhibiting YAP nuclear translocation.

BACKGROUND: Celastrus orbiculatus Thunb. is a medicinal plant that has been widely used for thousands of years in China, and the ethyl acetate extract (Celastrus orbiculatus Thunb. Extract, COE) from its stem was reported to exert antitumor and anti-inflammatory effects in various preclinical studies. However, the anti-non-small-cell lung cancer activity of COE and its potential mechanism are not yet fully understood. PURPOSE: To investigate the antitumor effects of COE on non-small-cell lung cancer (NSCLC) cells and explore its molecular mechanism from the perspective of Hippo signaling, YAP nuclear translocation, and reactive oxygen species (ROS) generation. METHODS: The effects of COE on proliferation, cell cycle arrest, apoptosis, stemness, and senescence in NSCLC cell lines were determined by CCK-8, clone formation, flow cytometry, and ß-galactosidase staining assays. The effects of COE on Hippo signaling were investigated by Western blotting. The intracellular expression and distribution of YAP were analyzed by immunofluorescence assay. DCFH-DA probe combined with flow cytometry was used to detect intracellular total ROS levels in NSCLC cells after COE treatment. Xenograft tumor model was established, and the animal living image system was employed to analyze the effects of COE on the Hippo-YAP signaling in vivo. RESULT: COE significantly inhibited NSCLC activity in vitro and in vivo, mainly by proliferation inhibition, cycle arrest, apoptosis promotion, senescence promotion, and stemness downregulation. COE strongly activated Hippo signaling and inhibited YAP expression and nuclear retention. Activation of Hippo signaling induced by COE was associated with ROS-mediated phosphorylation of MOB1. CONCLUSION: This study demonstrated that COE inhibited NSCLC through activating Hippo signaling and suppressing YAP nuclear translocation, in which ROS may play a role in the phosphorylation of the MOB1 protein.

Therapeutic targeting of TEAD transcription factors in cancer.

The Hippo signaling pathway inhibits the activity of the oncogenic YAP (Yes-associated protein)/TAZ (transcriptional co-activator with PDZ-binding motif)-TEAD (TEA/ATTS domain) transcriptional complex. In cancers, inactivating mutations in upstream Hippo components and/or enhanced activity of YAP/TAZ and TEAD have been observed. The activity of this transcriptional complex can be effectively inhibited by targeting the TEAD family of transcription factors. The development of TEAD inhibitors has been driven by the discovery that TEAD has druggable hydrophobic pockets, and is currently at the clinical development stage. Three small molecule TEAD inhibitors are currently being tested in Phase I clinical trials. In this review, we highlight the role of TEADs in cancer, discuss various avenues through which TEAD activity can be inhibited, and outline the opportunities for the administration of TEAD inhibitors.

Circadian signatures of anterior hypothalamus in time-restricted feeding.

Background: Meal timing resets circadian clocks in peripheral tissues, such as the liver, in seven days without affecting the phase of the central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Anterior hypothalamus plays an essential role in energy metabolism, circadian rhythm, and stress response. However, it remains to be elucidated whether and how anterior hypothalamus adapts its circadian rhythms to meal timing. Methods: Here, we applied transcriptomics to profile rhythmic transcripts in the anterior hypothalamus of nocturnal female mice subjected to day- (DRF) or night (NRF)-time restricted feeding for seven days. Results: This global profiling identified 128 and 3,518 rhythmic transcripts in DRF and NRF, respectively. NRF entrained diurnal rhythms among 990 biological processes, including 'Electron transport chain' and 'Hippo signaling' that reached peak time in the late sleep and late active phase, respectively. By contrast, DRF entrained only 20 rhythmic pathways, including 'Cellular amino acid catabolic process', all of which were restricted to the late active phase. The rhythmic transcripts found in both DRF and NRF tissues were largely resistant to phase entrainment by meal timing, which were matched to the action of the circadian clock. Remarkably, DRF for 36 days partially reversed the circadian clock compared to NRF. Conclusions: Collectively, our work generates a useful dataset to explore anterior hypothalamic circadian biology and sheds light on potential rhythmic processes influenced by meal timing in the brain (www.circametdb.org.cn).

YAP1 activation and Hippo pathway signaling in the pathogenesis and treatment of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA), the second most common primary liver cancer, is a highly lethal epithelial cell malignancy exhibiting features of cholangiocyte differentiation. iCCAs can potentially develop from multiple cell types of origin within liver, including immature or mature cholangiocytes, hepatic stem cells/progenitor cells, and from transdifferentiation of hepatocytes. Understanding the molecular mechanisms and genetic drivers that diversely drive specific cell lineage pathways leading to iCCA has important biological and clinical implications. In this context, activation of the YAP1-TEAD dependent transcription, driven by Hippo-dependent or -independent diverse mechanisms that lead to the stabilization of YAP1 is crucially important to biliary fate commitment in hepatobiliary cancer. In preclinical models, YAP1 activation in hepatocytes or cholangiocytes is sufficient to drive their malignant transformation into iCCA. Moreover, nuclear YAP1/TAZ is highly prevalent in human iCCA irrespective of the varied etiology, and significantly correlates with poor prognosis in iCCA patients. Based on the ubiquitous expression and diverse physiologic roles for YAP1/TAZ in the liver, recent studies have further revealed distinct functions of active YAP1/TAZ in regulating tumor metabolism, as well as the tumor immune microenvironment. In the current review, we discuss our current understanding of the various roles of the Hippo-YAP1 signaling in iCCA pathogenesis, with a specific focus on the roles played by the Hippo-YAP1 pathway in modulating biliary commitment and oncogenicity, iCCA metabolism, and immune microenvironment. We also discuss the therapeutic potential of targeting the YAP1/TAZ-TEAD transcriptional machinery in iCCA, its current limitations, and what future studies are needed to facilitate clinical translation.

Asperosaponin VI protects mice from sepsis by regulating Hippo and Rho signaling pathway.

OBJECTIVE: To explore the novel protective effect of Asperosaponin VI (AVI) on sepsis and its potential mechanism. METHODS: In in vitro experiments, bone marrow mononuclear cells and THP-1-derived cells were used to evaluate the viability of AVI treatment. Besides, the quantitative real-time PCR and Western blot were adopted to explore the protective effect of AVI on LPS-induced inflammation. For in vivo work, the effect of AVI on mice was evaluated by using both CLP-induced and the LPS-induced sepsis mice model. The fluctuation of anal temperature and the behavior of mice were recorded after surgery. Further, the content of bacteria in peritoneal lavage fluid was detected, as well as the levels of ALT, AST, LD and LDH in serum with ELISA. H&E staining and real-time PCR were used to evaluate the histopathology of liver, spleen and lung. Finally, relevant signaling pathways were detected by Western blot, real-time PCR and immunohistochemistry. RESULTS: AVI inhibited the expression of inflammatory factors in both CLP-induced and LPS-induced sepsis mice models, and reduced the number of bacteria in abdominal lavage fluid. The preventive treatment with AVI alleviated sepsis-induced organ injuries, reduced inflammatory responses, which was through inhibiting Hippo and Rho signaling pathway. CONCLUSIONS: This study indicated that AVI effectively protected mice from sepsis by down-regulating the activation of Hippo signaling and Rho family, and reducing inflammation and organ damage. However, conventional treatment was using antibiotics, and its mechanism was different with AVI.

Alpha-Ketoglutarate Attenuates Colitis in Mice by Increasing Lactobacillus Abundance and Regulating Stem Cell Proliferation via Wnt-Hippo Signaling.

SCOPE: Inflammatory bowel disease is an inflammatory gastrointestinal disorder associated with intestinal barrier damage, cell proliferation disorder, and dysbiosis of the intestinal microbiota. It remains unknown whether alpha-ketoglutarate (α-KG) can alleviate colitis in mice. METHODS AND RESULTS: Six-week-old male C57BL/6 mice supplemented with or without 0.5% α-KG (delivered in the form of sodium salt) are subjected to drinking water or 2.5% DSS to induce colitis. The results show that α-KG administration is attenuated the severity of colitis, as is indicated by reduced body-weight loss, colon shortening and colonic hyperplasia, and repressed proinflammatory cytokine secretion in DSS-challenged mice. Additionally, DSS-induced increases in malondialdehyde (MDA) and hydrogen peroxide (H2 O2 ), and decreases in glutathione (GSH) levels are attenuated by α-KG administration. Further study shows that the protective effect of α-KG is associated with restoring gut barrier integrity by enhancing the expression of tight junction proteins, increasing Lactobacillus levels, and regulating gut hyperplasia by the Wnt-Hippo signaling pathway in DSS-induced colitis. CONCLUSION: Collectively, the data provided herein demonstrate that α-KG administration is attenuated mucosal inflammation, barrier dysfunction, and gut microflora dysbiosis. This beneficial effect is associated with increased Lactobacillus levels and regulated colon hyperplasia by the Wnt-Hippo signaling pathway.

Targeting Hippo signaling pathway by phytochemicals in cancer therapy.

The current era of cancer research has been continuously advancing upon identifying novel aspects of tumorigenesis and the principal mechanisms behind the unleashed proliferation, invasion, drug resistance and immortality of cancer cells in hopes of exploiting these findings to achieve a more effective treatment for cancer. In pursuit of this goal, the identification of the first components of an extremely important regulatory pathway in Drosophila melanogaster that largely determines cell fate during the developmental stages, ended up in the discovery of the highly sophisticated Hippo signaling cascade. Soon after, it was revealed that deregulation of the components of this pathway either via mutations or through epigenetic alterations can be observed in a vast variety of tumors and these alterations greatly contribute to the neoplastic transformation of cells, their survival, growth and resistance to therapy. As more hidden aspects of this pathway such as its widespread entanglement with other major cellular signaling pathways are continuously being uncovered, many researchers have sought over the past decade to find ways of therapeutic interventions targeting the major components of the Hippo cascade. To date, various approaches such as the use of exogenous targeting miRNAs and different molecular inhibitors have been recruited herein, among which naturally occurring compounds have shown a great promise. On such a basis, in the present work we review the current understanding of Hippo pathway and the most recent evidence on targeting its components using natural plant-derived phytochemicals.

Honokiol Affects Stem Cell Viability by Suppressing Oncogenic YAP1 Function to Inhibit Colon Tumorigenesis.

Honokiol (HNK) is a biphenolic compound that has been used in traditional medicine for treating various ailments, including cancers. In this study, we determined the effect of HNK on colon cancer cells in culture and in a colitis-associated cancer model. HNK treatment inhibited proliferation and colony formation while inducing apoptosis. In addition, HNK suppressed colonosphere formation. Molecular docking suggests that HNK interacts with reserve stem cell marker protein DCLK1, with a binding energy of -7.0 Kcal/mol. In vitro kinase assays demonstrated that HNK suppressed the DCLK1 kinase activity. HNK also suppressed the expression of additional cancer stem cell marker proteins LGR5 and CD44. The Hippo signaling pathway is active in intestinal stem cells. In the canonical pathway, YAP1 is phosphorylated at Ser127 by upstream Mst1/2 and Lats1/2. This results in the sequestration of YAP1 in the cytoplasm, thereby not allowing YAP1 to translocate to the nucleus and interact with TEAD1-4 transcription factors to induce gene expression. However, HNK suppressed Ser127 phosphorylation in YAP1, but the protein remains sequestered in the cytoplasm. We further determined that this occurs by YAP1 interacting with PUMA. To determine if this also occurs in vivo, we performed studies in an AOM/DSS induced colitis-associated cancer model. HNK administered by oral gavage at a dose of 5mg/kg bw for 24 weeks demonstrated a significant reduction in the expression of YAP1 and TEAD1 and in the stem marker proteins. Together, these data suggest that HNK prevents colon tumorigenesis in part by inducing PUMA-YAP1 interaction and cytoplasmic sequestration, thereby suppressing the oncogenic YAP1 activity.

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Hepatocellular carcinoma (HCC) is one of the most common malignant tumor worldwide, with high incidence and mortality. However, the exact mechanisms leading to HCC development remain unclear. The cores of the Hippo signaling pathway consist of a kinase cascade to transmit signals, which inhibits the transcriptional coactivator translocate into the nucleus and reduces the transcription of downstream proliferation-related genes. Hippo signaling pathway regulates liver development and regeneration after liver resection, and it is also related to the occurrence of HCC. The Hippo pathway regulates proliferation, apoptosis, metastasis, autophagy, metabolic reprogramming of HCC cells, affects the tumor immune microenvironment, and participates multiple-drug resistance. Further study on the role of Hippo signaling pathway in HCC is important to develop new therapeutic targets.

Extracellular matrix and Hippo signaling as therapeutic targets of antifibrotic compounds for uterine fibroids.

BACKGROUND: Uterine fibroids are highly prevalent, collagen-rich, mechanically stiff, fibrotic tumors for which new therapeutic options are needed. Increased extracellular matrix (ECM) stiffness activates mechanical signaling and Hippo/YAP promoting fibroid growth, but no prior studies have tested either as a therapeutic target. We tested the hypothesis that injection of a purified form of collagenase Clostridium histolyticum (CCH) that selectively digests type I and type III collagens would alter ECM stiffness, Hippo signaling, and selectively reduce fibroid cell growth. We also used two FDA-approved drugs, verteporfin and nintedanib, to elucidate the role of Hippo/YAP signaling in uterine fibroid and myometrial cells. METHODS: The clinical trial was registered (NCT02889848). Stiffness of samples was measured by rheometry. Protein expression in surgical samples was analyzed via immunofluorescence. Protein and gene expression in uterine fibroid or myometrial cell lines were measured by real time PCR and western blot, and immunofluorescence. RESULTS: Injection of CCH at high doses (0.1-0.2 mg/cm3 ) into fibroids resulted in a 46% reduction in stiffness in injected fibroids compared to controls after 60 days. Levels of the cell proliferation marker proliferative cell nuclear antigen (PCNA) were decreased in fibroids 60 days after injection at high doses of CCH. Key Hippo signaling factors, specifically the transcriptionally inactive phosphorylated YAP (p-YAP), was increased at high CCH doses, supporting the role of YAP in fibroid growth. Furthermore, inhibition of YAP via verteporfin (YAP inhibitor) decreased cell proliferation, gene and protein expression of key factors promoting fibrosis and mechanotransduction in fibroid cells. Additionally, the anti-fibrotic drug, nintedanib, inhibited YAP and showed anti-fibrotic effects. CONCLUSIONS: This is the first report that in vivo injection of collagenase into uterine fibroids led to a reduction in Hippo/YAP signaling and crucial genes and pathways involved in fibroid growth. These results indicate that targeting ECM stiffness and Hippo signaling might be an effective strategy for uterine fibroids.

New insights into the core Hippo signaling and biological macromolecules interactions in the biology of solid tumors.

As an evolutionarily conserved pathway, Hippo signaling pathway impacts different pathology and physiology processes such as wound healing, tissue repair/size and regeneration. When some components of Hippo signaling dysregulated, it affects cancer cells proliferation. Moreover, the relation Hippo pathway with other signaling including Wnt, TGFß, Notch, and EGFR signaling leaves effect on the proliferation of cancer cells. Utilizing a number of therapeutic approaches, such as siRNAs and long noncoding RNA (lncRNA) to prevent cancer cells through the targeting of Hippo pathways, can provide new insights into cancer target therapy. The purpose of present review, first of all, is to demonstrate the importance of Hippo signaling and its relation with other signaling pathways in cancer. It also tries to demonstrate targeting Hippo signaling progress in cancer therapy.

LINC00673 is activated by YY1 and promotes the proliferation of breast cancer cells via the miR-515-5p/MARK4/Hippo signaling pathway.

BACKGROUND: An increasing number of studies have shown that long noncoding RNAs (lncRNAs) play essential roles in tumor initiation and progression. LncRNAs act as tumor promoters or suppressors by targeting specific genes via epigenetic modifications and competing endogenous RNA (ceRNA) mechanisms. In this study, we explored the function and detailed mechanisms of long intergenic nonprotein coding RNA 673 (LINC00673) in breast cancer progression. METHODS: Quantitative real-time PCR (qRT-PCR) was used to examine the expression of LINC00673 in breast cancer tissues and in adjacent normal tissues. Gain-of-function and loss-of function experiments were conducted to investigate the biological functions of LINC00673 in vitro and in vivo. We also explored the potential role of LINC00673 as a therapeutic target using antisense oligonucleotide (ASO) in vivo. RNA sequencing (RNA-seq), dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP) assay, and rescue experiments were performed to uncover the detailed mechanism of LINC00673 in promoting breast cancer progression. RESULTS: In the present study, LINC00673 displayed a trend of remarkably increased expression in breast cancer tissues and was associated with poor prognosis in breast cancer patients. Importantly, LINC00673 depletion inhibited breast cancer cell proliferation by inhibiting the cell cycle and increasing apoptosis. Furthermore, ASO therapy targeting LINC00673 substantially suppressed breast cancer cell proliferation in vivo. Mechanistically, LINC00673 was found to act as a ceRNA by sponging miR-515-5p to regulate MARK4 expression, thus inhibiting the Hippo signaling pathway. Finally, ChIP assay showed that the transcription factor Yin Yang 1 (YY1) could bind to the LINC00673 promoter and increase its transcription in cis. CONCLUSIONS: YY1-activated LINC00673 may exert an oncogenic function by acting as a sponge for miR-515-5p to upregulate the MARK4 and then inhibit Hippo signaling pathway, and may serve as a potential therapeutic target.