Balance of bone and marrow fat formation is critical for bone homeostasis. The imbalance of bone homeostasis will cause various bone diseases, such as osteoporosis. However, the precise mechanisms governing osteoporotic bone loss and marrow adipose tissue (MAT) accumulation remain poorly understood. By analysis of publicly available databases from bone samples of osteoporosis patients, we found that the expression of intraflagellar transport 20 (IFT20) and WW domain containing transcription regulator 1 (WWTR1) were significantly downregulated in osteoblast lineage cells. Additionally, we found that double deletions of IFT20 and WWTR1 in osteoblasts resulted in a significant accumulation of MAT and bone loss. Moreover, IFT20 and WWTR1 deficiency in osteoblasts exacerbated bone-fat imbalance in ovariectomy (OVX)- and high-fat-diet (HFD)-induced osteoporosis mouse models. Mechanistically, we found that deletions of IFT20 and WWTR1 in osteoblasts synergistically inhibited osteogenesis and promoted adipogenesis and osteoclastogenesis. We also found that IFT20 interacted with TGF-β receptor type II (TβRII) to enhance TβRII stability by blocking c-Cbl-mediated ubiquitination and degradation of TβRII. WWTR1 transcriptionally upregulated TβRII expression by directly binding its promoter. These findings indicate that targeting IFT20/WWTR1 may be a potential therapeutic strategy for the treatment of osteoporosis.

Targeted therapies are effective cancer treatments when accompanied by accurate diagnostic tests that can help identify patients that will respond to those therapies. The YAP/TAZ-TEAD axis is activated and plays a causal role in several cancer types, and TEAD inhibitors are currently in early-phase clinical trials in cancer patients. However, a lack of a reliable way to identify tumors with YAP/TAZ-TEAD activation for most cancer types makes it difficult to determine which tumors will be susceptible to TEAD inhibitors. Here, we used a combination of RNA-seq and bioinformatic analysis of metastatic melanoma cells to develop a YAP/TAZ gene signature. We found that the genes in this signature are TEAD-dependent in several melanoma cell lines, and that their expression strongly correlates with YAP/TAZ activation in human melanomas. Using DepMap dependency data, we found that this YAP/TAZ signature was predictive of melanoma cell dependence upon YAP/TAZ or TEADs. Importantly, this was not limited to melanoma because this signature was also predictive when tested on a panel of over 1000 cancer cell lines representing numerous distinct cancer types. Our results suggest that YAP/TAZ gene signatures like ours may be effective tools to predict tumor cell dependence upon YAP/TAZ-TEAD, and thus potentially provide a means to identify patients likely to benefit from TEAD inhibitors.

Nonalcoholic steatohepatitis (NASH) is emerging as the most common cause of liver disease. Although many studies in mouse NASH models have suggested therapies, translation to humans is poor, with no approved drugs for NASH. One explanation may lie in differences between mouse and human hepatocytes. We used NASH diet-fed chimeric mice reconstituted with human hepatocytes (hu-liver mice) to test a mechanism-based hepatocyte-targeted small interfering RNA (siRNA), GalNAc-siTaz, shown previously to block the progression to fibrotic NASH in mice. Following ablation of endogenous hepatocytes, male mice were reconstituted with human hepatocytes from a single donor with the rs738409-C/G PNPLA3 risk variant, resulting in ∼95% human hepatocyte reconstitution. The mice were then fed a high-fat choline-deficient l-amino acid-defined diet for 6 weeks to induce NASH, followed by six weekly injections of GalNAc-siTAZ to silence hepatocyte-TAZ or control GalNAc-siRNA (GalNAc-control) while still on the NASH diet. GalNAc-siTAZ lowered human hepatic TAZ and IHH, a TAZ target that promotes NASH fibrosis. Most important, GalNAc-siTAZ decreased liver inflammation, hepatocellular injury, hepatic fibrosis, and profibrogenic mediator expression versus GalNAc-control, indicating that GalNAc-siTAZ decreased the progression of NASH in mice reconstituted with human hepatocytes. In conclusion, silencing TAZ in human hepatocytes suppresses liver fibrosis in a hu-liver model of NASH.

TAZ (WWTR1) is a transcriptional co-activator regulated by Hippo signaling, mechano-transduction, and G-protein couple receptors. Once activated, TAZ and its paralogue, YAP1, regulate gene expression programs promoting cell proliferation, survival, and differentiation, thus controlling embryonic development, tissue regeneration, and aging. YAP and TAZ are also frequently activated in tumors, particularly in poorly differentiated and highly aggressive malignancies. Yet, mutations of YAP/TAZ or of their upstream regulators do not fully account for their activation in cancer, raising the possibility that other upstream regulatory pathways, still to be defined, are altered in tumors. In this work, we set out to identify novel regulators of TAZ by means of a siRNA-based screen. We identified 200 genes able to modulate the transcriptional activity of TAZ, with prominence for genes implicated in cell-cell contact, cytoskeletal tension, cell migration, WNT signaling, chromatin remodeling, and interleukins and NF-kappaB signaling. Among these genes we identified was BRCC3, a component of the BRCA1 complex that guards genome integrity and exerts tumor suppressive activity during cancer development. The loss of BRCC3 or BRCA1 leads to an increased level and activity of TAZ. Follow-up studies indicated that the cytoplasmic BRCA1 complex controls the ubiquitination and stability of TAZ. This may suggest that, in tumors, inactivating mutations of BRCA1 may unleash cell transformation by activating the TAZ oncogene.

UNASSIGNED: While Yap and Wwtr1 regulate resident cardiac fibroblast to myofibroblast differentiation following cardiac injury, their role specifically in activated myofibroblasts remains unexplored.
UNASSIGNED: We assessed the pathophysiological and cellular consequence of genetic depletion of Yap alone (Yap fl/fl ;Postn MCM ) or Yap and Wwtr1 (Yap fl/fl ;Wwtr1 fl/+ ;Postn MCM ) in adult mouse myofibroblasts following myocardial infarction and identify and validate novel downstream factors specifically in cardiac myofibroblasts that mediate pathological remodeling.
UNASSIGNED: Following myocardial infarction, depletion of Yap in myofibroblasts had minimal effect on heart function while depletion of Yap/Wwtr1 resulted in smaller scars, reduced interstitial fibrosis, and improved ejection fraction and fractional shortening. Single cell RNA sequencing of interstitial cardiac cells 7 days post infarction showed suppression of pro-fibrotic genes in fibroblasts derived from Yap fl/fl ,Wwtr1 fl/+ ;Postn MCM hearts. In vivo myofibroblast depletion of Yap/Wwtr1 as well in vitro knockdown of Yap/Wwtr1 dramatically decreased RNA and protein expression of the matricellular factor Ccn3. Administration of recombinant CCN3 to adult mice following myocardial infarction remarkably aggravated cardiac function and scarring. CCN3 administration drove myocardial gene expression of pro-fibrotic genes in infarcted left ventricles implicating CCN3 as a novel driver of cardiac fibrotic processes following myocardial infarction.
UNASSIGNED: Yap/Wwtr1 depletion in myofibroblasts attenuates fibrosis and significantly improves cardiac outcomes after myocardial infarction and we identify Ccn3 as a factor downstream of Yap/Wwtr1 that contributes to adverse cardiac remodeling post MI. Myofibroblast expression of Yap, Wwtr1, and Ccn3 could be further explored as potential therapeutic targets for modulating adverse cardiac remodeling post injury.

Healthy progression of human pregnancy relies on cytotrophoblast (CTB) progenitor self-renewal and its differentiation toward multinucleated syncytiotrophoblasts (STBs) and invasive extravillous trophoblasts (EVTs). However, the underlying molecular mechanisms that fine-tune CTB self-renewal or direct its differentiation toward STBs or EVTs during human placentation are poorly defined. Here, we show that Hippo signaling cofactor WW domain containing transcription regulator 1 (WWTR1) is a master regulator of trophoblast fate choice during human placentation. Using human trophoblast stem cells (human TSCs), primary CTBs, and human placental explants, we demonstrate that WWTR1 promotes self-renewal in human CTBs and is essential for their differentiation to EVTs. In contrast, WWTR1 prevents induction of the STB fate in undifferentiated CTBs. Our single-cell RNA sequencing analyses in first-trimester human placenta, along with mechanistic analyses in human TSCs revealed that WWTR1 fine-tunes trophoblast fate by directly regulating WNT signaling components. Importantly, our analyses of placentae from pathological pregnancies show that extreme preterm births (gestational time ≤28 wk) are often associated with loss of WWTR1 expression in CTBs. In summary, our findings establish the critical importance of WWTR1 at the crossroads of human trophoblast progenitor self-renewal versus differentiation. It plays positive instructive roles in promoting CTB self-renewal and EVT differentiation and safeguards undifferentiated CTBs from attaining the STB fate.

Application of molecular testing in clinical practice has led to significant advances in the classification of soft tissue sarcomas. Despite remarkable progress, there are still challenging cases that remain unclassified. In this study, we present an unusual spindle cell sarcoma arising in the abdominal cavity of a 37-year-old female. An extensive panel of immunostains was nonspecific for a line of differentiation and the tumor was subjected to targeted RNA sequencing for further classification. The findings showed a novel WWTR1::AFF2 fusion, which was further confirmed by break-apart FISH analysis for WWTR1 gene rearrangement. The tumor was attached to the wall of sigmoid colon and showed a highly cellular proliferation of plump spindle to epithelioid cells arranged in intersecting fascicles. Areas of extensive endometriosis were identified adjacent to the tumor. The immunoprofile was significant for reactivity with desmin, calponin, WT-1, ER, and PR, while negative for CD10, SMA, caldesmon, pan-keratin, ALK, CD117, and S100. The patient is alive and well after 11 months of follow-up. The exact histogenesis of this sarcoma remains unclear, however, the presence of adjacent endometriosis and coexpression of WT1/ER/PR raises the possibility of an unusual endometrioid stromal sarcoma, occurring outside the GYN tract. Additional cases are needed to establish the recurrent potential of this fusion event and to better define its pathogenesis and clinical behavior.

Clear cell carcinoma (CCC) of the cervix (cCCC) is a rare and aggressive type of human papillomavirus (HPV)-negative cervical cancer with limited effective treatment options for recurrent or metastatic disease. Historically, CCCs of the lower genital tract were associated with in utero diethylstilbestrol exposure; however, the genetic landscape of sporadic cCCCs remains unknown. Here we sought to define the molecular underpinning of cCCCs. Using a combination of whole-exome, targeted capture, and RNA-sequencing, we identified pathogenic genetic alterations in the Hippo signaling pathway in 50% (10/20) of cCCCs, including recurrent WWTR1 S89W somatic mutations in 40% (4/10) of the cases harboring mutations in the Hippo pathway. Irrespective of the presence or absence of Hippo pathway genetic alterations, however, all primary cCCCs analyzed in this study (n = 20) harbored features of Hippo pathway deregulation at the transcriptomic and protein levels. In vitro functional analysis revealed that expression of the WWTR1 S89W mutation leads to reduced binding of TAZ to 14-3-3, promoting constitutive nuclear translocation of TAZ and Hippo pathway repression. WWTR1 S89W expression was found to lead to acquisition of oncogenic behavior, including increased proliferation, migration, and colony formation in vitro as well as increased tumorigenesis in vivo, which could be reversed by targeted inhibition of the TAZ/YAP1 complex with verteporfin. Finally, xenografts expressing WWTR1 S89W displayed a shift in tumor phenotype, becoming more infiltrative as well as less differentiated, and were found to be composed of cells with conspicuous cytoplasmic clearing as compared to controls. Our results demonstrate that Hippo pathway alterations are likely drivers of cCCCs and likely contribute to the clear cell phenotype. Therapies targeting this pathway may constitute a new class of treatment for these rare, aggressive tumors. © 2022 The Pathological Society of Great Britain and Ireland.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.

Yes-associated protein (YAP) and TAZ are transcriptional cofactors that sit at the crossroad of several signaling pathways involved in cell growth and differentiation. As such, they play essential functions during embryonic development, regeneration, and, once deregulated, in cancer progression. In this review, we will revise the current literature and provide an overview of how YAP/TAZ control transcription. We will focus on data concerning the modulation of the basal transcriptional machinery, their ability to epigenetically remodel the enhancer-promoter landscape, and the mechanisms used to integrate transcriptional cues from multiple pathways. This reveals how YAP/TAZ activation in cancer cells leads to extensive transcriptional control that spans several hallmarks of cancer. The definition of the molecular mechanism of transcriptional control and the identification of the pathways regulated by YAP/TAZ may provide therapeutic opportunities for the effective treatment of YAP/TAZ-driven tumors.