The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells.

Cancer stem cells (CSCs) are proposed to drive tumor initiation and progression. Yet, our understanding of the cellular and molecular mechanisms that underlie CSC properties is limited. Here we show that the activity of TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in breast CSCs. TAZ protein levels and activity are elevated in prospective CSCs and in poorly differentiated human tumors and have prognostic value. Gain of TAZ endows self-renewal capacity to non-CSCs. In epithelial cells, TAZ forms a complex with the cell-polarity determinant Scribble, and loss of Scribble--or induction of the epithelial-mesenchymal transition (EMT)--disrupts the inhibitory association of TAZ with the core Hippo kinases MST and LATS. This study links the CSC concept to the Hippo pathway in breast cancer and reveals a mechanistic basis of the control of Hippo kinases by cell polarity.

FAM/USP9x, a deubiquitinating enzyme essential for TGFbeta signaling, controls Smad4 monoubiquitination.

The assembly of the Smad complex is critical for TGFbeta signaling, yet the mechanisms that inactivate or empower nuclear Smad complexes are less understood. By means of siRNA screen we identified FAM (USP9x), a deubiquitinase acting as essential and evolutionarily conserved component in TGFbeta and bone morphogenetic protein signaling. Smad4 is monoubiquitinated in lysine 519 in vivo, a modification that inhibits Smad4 by impeding association with phospho-Smad2. FAM reverts this negative modification, re-empowering Smad4 function. FAM opposes the activity of Ectodermin/Tif1gamma (Ecto), a nuclear factor for which we now clarify a prominent role as Smad4 monoubiquitin ligase. Our study points to Smad4 monoubiquitination and deubiquitination as a way for cells to set their TGFbeta responsiveness: loss of FAM disables Smad4-dependent responses in several model systems, with Ecto being epistatic to FAM. This defines a regulative ubiquitination step controlling Smads that is parallel to those impinging on R-Smad phosphorylation.

A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis.

TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.

An optogenetic method for interrogating YAP1 and TAZ nuclear-cytoplasmic shuttling.

The shuttling of transcription factors and transcriptional regulators into and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive LOV (light-oxygen-voltage) domain from Avena sativa is used to sequester fluorescently labelled transcriptional regulators YAP1 and TAZ (also known as WWTR1) on the surface of mitochondria and to reversibly release them upon blue light illumination. After dissociation, fluorescent signals from the mitochondria, cytoplasm and nucleus are extracted by a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidence that, despite high intercellular variability, YAP1 import and export rates correlate within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration, we show that their rates of entry and exit are correlated. Furthermore, combining the optogenetic release of YAP1 with lattice light-sheet microscopy reveals high heterogeneity of YAP1 dynamics within different cytoplasmic regions, demonstrating the utility and versatility of our tool to study protein dynamics. This article has an associated First Person interview with Anna M. Dowbaj, joint first author of the paper.

F-actin dynamics regulates mammalian organ growth and cell fate maintenance.

BACKGROUND & AIMS: In vitro, cell function can be potently regulated by the mechanical properties of cells and of their microenvironment. Cells measure these features by developing forces via their actomyosin cytoskeleton, and respond accordingly by regulating intracellular pathways, including the transcriptional coactivators YAP/TAZ. Whether mechanical cues are relevant for in vivo regulation of adult organ homeostasis, and whether this occurs through YAP/TAZ, remains largely unaddressed. METHODS: We developed Capzb conditional knockout mice and obtained primary fibroblasts to characterize the role of CAPZ in vitro. In vivo functional analyses were carried out by inducing Capzb inactivation in adult hepatocytes, manipulating YAP/Hippo activity by hydrodynamic tail vein injections, and treating mice with the ROCK inhibitor, fasudil. RESULTS: We found that the F-actin capping protein CAPZ restrains actomyosin contractility: Capzb inactivation alters stress fiber and focal adhesion dynamics leading to enhanced myosin activity, increased traction forces, and increased liver stiffness. In vitro, this rescues YAP from inhibition by a small cellular geometry; in vivo, it induces YAP activation in parallel to the Hippo pathway, causing extensive hepatocyte proliferation and leading to striking organ overgrowth. Moreover, Capzb is required for the maintenance of the differentiated hepatocyte state, for metabolic zonation, and for gluconeogenesis. In keeping with changes in tissue mechanics, inhibition of the contractility regulator ROCK, or deletion of the Yap1 mechanotransducer, reverse the phenotypes emerging in Capzb-null livers. CONCLUSIONS: These results indicate a previously unsuspected role for CAPZ in tuning the mechanical properties of cells and tissues, which is required in hepatocytes for the maintenance of the differentiated state and to regulate organ size. More generally, it indicates for the first time that mechanotransduction has a physiological role in maintaining liver homeostasis in mammals. LAY SUMMARY: The mechanical properties of cells and tissues (i.e. whether they are soft or stiff) are thought to be important regulators of cell behavior. Herein, we found that inactivation of the protein CAPZ alters the mechanical properties of cells and liver tissues, leading to YAP hyperactivation. In turn, this profoundly alters liver physiology, causing organ overgrowth, defects in liver cell differentiation and metabolism. These results reveal a previously uncharacterized role for mechanical signals in the maintenance of adult liver homeostasis.

SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors.

The molecular determinants of malignant cell behaviours in breast cancer remain only partially understood. Here we show that SHARP1 (also known as BHLHE41 or DEC2) is a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer. SHARP1 is regulated by the p63 metastasis suppressor and inhibits TNBC aggressiveness through inhibition of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α (HIFs). SHARP1 opposes HIF-dependent TNBC cell migration in vitro, and invasive or metastatic behaviours in vivo. SHARP1 is required, and sufficient, to limit expression of HIF-target genes. In primary TNBC, endogenous SHARP1 levels are inversely correlated with those of HIF targets. Mechanistically, SHARP1 binds to HIFs and promotes HIF proteasomal degradation by serving as the HIF-presenting factor to the proteasome. This process is independent of pVHL (von Hippel-Lindau tumour suppressor), hypoxia and the ubiquitination machinery. SHARP1 therefore determines the intrinsic instability of HIF proteins to act in parallel to, and cooperate with, oxygen levels. This work sheds light on the mechanisms and pathways by which TNBC acquires invasiveness and metastatic propensity.

Self-regulation of the head-inducing properties of the Spemann organizer.

The Spemann organizer stands out from other signaling centers of the embryo because of its broad patterning effects. It defines development along the anteroposterior and dorsoventral axes of the vertebrate body, mainly by secreting antagonists of growth factors. Qualitative models proposed more than a decade ago explain the organizer's region-specific inductions (i.e., head and trunk) as the result of different combinations of antagonists. For example, head induction is mediated by extracellular inhibition of Wnt, BMP, and Nodal ligands. However, little is known about how the levels of these antagonists become harmonized with those of their targets and with the factors initially responsible for germ layers and organizer formation, including Nodal itself. Here we show that key ingredients of the head-organizer development, namely Nodal ligands, Nodal antagonists, and ADMP ligands reciprocally adjust each other's strength and range of activity by a self-regulating network of interlocked feedback and feedforward loops. A key element in this cross-talk is the limited availability of ACVR2a, for which Nodal and ADMP must compete. By trapping Nodal extracellularly, the Nodal antagonists Cerberus and Lefty are permissive for ADMP activity. The system self-regulates because ADMP/ACVR2a/Smad1 signaling in turn represses the expression of the Nodal antagonists, reestablishing the equilibrium. In sum, this work reveals an unprecedented set of interactions operating within the organizer that is critical for embryonic patterning.

Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern.

The high mutation rate of SARS-CoV-2 leads to the emergence of multiple variants, some of which are resistant to vaccines and drugs targeting viral elements. Targeting host dependency factors, e.g. cellular proteins required for viral replication, would help prevent resistance. However, it remains unclear whether different SARS-CoV-2 variants induce conserved cellular responses and exploit the same core host factors. To this end, we compared three variants of concern and found that the host transcriptional response was conserved, differing only in kinetics and magnitude. Through CRISPR screening, we identified host genes required for infection by each variant. Most of the genes were shared by multiple variants. We validated our hits with small molecules and repurposed Food and Drug Administration-approved drugs. All the drugs were highly active against all the variants tested, including new variants that emerged during the study (Delta and Omicron). Mechanistically, we identified reactive oxygen species production as a key step in early virus replication. Antioxidants such as N-acetyl cysteine (NAC) were effective against all the variants in both human lung cells and a humanised mouse model. Our study supports the use of available antioxidant drugs, such as NAC, as a general and effective anti-COVID-19 approach.

MicroRNA control of Nodal signalling.

MicroRNAs are crucial modulators of gene expression, yet their involvement as effectors of growth factor signalling is largely unknown. Ligands of the transforming growth factor-beta superfamily are essential for development and adult tissue homeostasis. In early Xenopus embryos, signalling by the transforming growth factor-beta ligand Nodal is crucial for the dorsal induction of the Spemann's organizer. Here we report that Xenopus laevis microRNAs miR-15 and miR-16 restrict the size of the organizer by targeting the Nodal type II receptor Acvr2a. Endogenous miR-15 and miR-16 are ventrally enriched as they are negatively regulated by the dorsal Wnt/beta-catenin pathway. These findings exemplify the relevance of microRNAs as regulators of early embryonic patterning acting at the crossroads of fundamental signalling cascades.

Hydrogel-in-hydrogel live bioprinting for guidance and control of organoids and organotypic cultures.

Three-dimensional hydrogel-based organ-like cultures can be applied to study development, regeneration, and disease in vitro. However, the control of engineered hydrogel composition, mechanical properties and geometrical constraints tends to be restricted to the initial time of fabrication. Modulation of hydrogel characteristics over time and according to culture evolution is often not possible. Here, we overcome these limitations by developing a hydrogel-in-hydrogel live bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organ-like cultures. This can be achieved by crosslinking photosensitive hydrogels via two-photon absorption at any time during culture. We show that instructive hydrogels guide neural axon directionality in growing organotypic spinal cords, and that hydrogel geometry and mechanical properties control differential cell migration in developing cancer organoids. Finally, we show that hydrogel constraints promote cell polarity in liver organoids, guide small intestinal organoid morphogenesis and control lung tip bifurcation according to the hydrogel composition and shape.

Mitochondrial fission links ECM mechanotransduction to metabolic redox homeostasis and metastatic chemotherapy resistance.

Metastatic breast cancer cells disseminate to organs with a soft microenvironment. Whether and how the mechanical properties of the local tissue influence their response to treatment remains unclear. Here we found that a soft extracellular matrix empowers redox homeostasis. Cells cultured on a soft extracellular matrix display increased peri-mitochondrial F-actin, promoted by Spire1C and Arp2/3 nucleation factors, and increased DRP1- and MIEF1/2-dependent mitochondrial fission. Changes in mitochondrial dynamics lead to increased production of mitochondrial reactive oxygen species and activate the NRF2 antioxidant transcriptional response, including increased cystine uptake and glutathione metabolism. This retrograde response endows cells with resistance to oxidative stress and reactive oxygen species-dependent chemotherapy drugs. This is relevant in a mouse model of metastatic breast cancer cells dormant in the lung soft tissue, where inhibition of DRP1 and NRF2 restored cisplatin sensitivity and prevented disseminated cancer-cell awakening. We propose that targeting this mitochondrial dynamics- and redox-based mechanotransduction pathway could open avenues to prevent metastatic relapse.

A Lung Organotypic Coculture Reveals a Role for TFEB-Lysosomal Axis in the Survival of Disseminated Dormant Cancer Cells.

(1) Background: metastatic relapse following a prolonged period of disease-free survival is a common cause of mortality for many cancer patients. Disseminated dormant cancer cells (DDCCs) lie below the radar before waking up years, or even decades, after the removal of the primary tumor. This implies that they are able to survive in a latent state in a foreign environment for an extended period of time supported by intrinsic and extrinsic factors still to be elucidated. (2) Methods: we employed a coculture of DDCCs with lung epithelial cells together with RNA sequencing analysis to understand the overlap in gene transcription between in vivo and cocultured DDCCs. (3) Results: we found a significant overlap between the processes activated in DDCCs from lungs and in the coculture, as well as in alveolar type I cells in vivo and in coculture. We identified the transcription factor EB (TFEB)-lysosomal axis as a relevant process activated in DDCCs upon dissemination to the lung and confirmed the results in our lung coculture. Interestingly, breast cancer patients with a higher expression of TFEB targets show increased likelihood of developing relapses. (4) Conclusions: we propose that lysosomal accumulation following TFEB activation is an important feature of breast cancer DDCCs that might be exploited for future therapeutic interventions.

EphB6 Regulates TFEB-Lysosomal Pathway and Survival of Disseminated Indolent Breast Cancer Cells.

Late relapse of disseminated cancer cells is a common feature of breast and prostate tumors. Several intrinsic and extrinsic factors have been shown to affect quiescence and reawakening of disseminated dormant cancer cells (DDCCs); however, the signals and processes sustaining the survival of DDCCs in a foreign environment are still poorly understood. We have recently shown that crosstalk with lung epithelial cells promotes survival of DDCCs of estrogen receptor-positive (ER+) breast tumors. By using a lung organotypic system and in vivo dissemination assays, here we show that the TFEB-lysosomal axis is activated in DDCCs and that it is modulated by the pro-survival ephrin receptor EphB6. TFEB lysosomal direct targets are enriched in DDCCs in vivo and correlate with relapse in ER+ breast cancer patients. Direct coculture of DDCCs with alveolar type I-like lung epithelial cells and dissemination in the lung drive lysosomal accumulation and EphB6 induction. EphB6 contributes to survival, TFEB transcriptional activity, and lysosome formation in DDCCs in vitro and in vivo. Furthermore, signaling from EphB6 promotes the proliferation of surrounding lung parenchymal cells in vivo. Our data provide evidence that EphB6 is a key factor in the crosstalk between disseminated dormant cancer cells and the lung parenchyma and that the TFEB-lysosomal pathway plays an important role in the persistence of DDCCs.

Technical Advancements for Studying Immune Regulation of Disseminated Dormant Cancer Cells.

Metastases are a major cause of cancer-related death and despite the fact that they have been focus of intense research over the last two decades, effective therapies for patients with distant secondary lesions are still very limited. In addition, in some tumor types metastases can grow years after the patients have been declared clinically cured, indicating that disseminated cancer cells (DCCs) persist undetected for years, even decades in a quiescent state. Clinical and experimental data highlight the importance of the immune system in shaping the fitness and behaviour of DCCs. Here, we review mechanisms of survival, quiescence and outgrowth of DCCs with a special focus on immune-regulation and we highlight the latest cutting-edge techniques for modelling the biology of DCCs in vitro and for studying the metastatic niche in vivo. We believe that a wide dissemination of those techniques will boost scientific findings towards new therapies to defeat metastatic relapses in cancer patients.

In vitro Models of Breast Cancer Metastatic Dormancy.

Delayed relapses at distant sites are a common clinical observation for certain types of cancers after removal of primary tumor, such as breast and prostate cancer. This evidence has been explained by postulating a long period during which disseminated cancer cells (DCCs) survive in a foreign environment without developing into overt metastasis. Because of the asymptomatic nature of this phenomenon, isolation, and analysis of disseminated dormant cancer cells from clinically disease-free patients is ethically and technically highly problematic and currently these data are largely limited to the bone marrow. That said, detecting, profiling and treating indolent metastatic lesions before the onset of relapse is the imperative. To overcome this major limitation many laboratories developed in vitro models of the metastatic niche for different organs and different types of cancers. In this review we focus specifically on in vitro models designed to study metastatic dormancy of breast cancer cells (BCCs). We provide an overview of the BCCs employed in the different organotypic systems and address the components of the metastatic microenvironment that have been shown to impact on the dormant phenotype: tissue architecture, stromal cells, biochemical environment, oxygen levels, cell density. A brief description of the organ-specific in vitro models for bone, liver, and lung is provided. Finally, we discuss the strategies employed so far for the validation of the different systems.

Mechanical Forces as Determinants of Disseminated Metastatic Cell Fate.

Disseminated metastatic cancer cells represent one of the most relevant causes of disease relapse and associated death for cancer patients, and a therapeutic target of the highest priority. Still, our understanding of how disseminated cancer cells survive in the foreign metastatic environment, and eventually cause metastatic outgrowth, remains rather limited. In this review we focus on the cell microenvironment as a key regulator of cell behavior at the metastatic site, and especially on the mechanical properties of the extracellular matrix and associated integrin signaling. We discuss available evidence pointing to a pervasive role of extracellular matrix (ECM) mechanical properties in regulating cancer cell proliferation and survival after dissemination, and propose that this might represent an important bottleneck for cells invading and establishing into a novel tissue. We point to the known molecular players, how these might contribute to modulate the mechanical properties of the metastatic environment, and the response of cells to these cues. Finally, we propose that emerging knowledge on the physical interaction of disseminated metastatic cells and on the downstream mechanotransduction pathways, including YAP/TAZ (Yes-associated protein-1 and WW-domain transcription activator 1) and MRTFs (Myocardin-related transcription factors), may help to identify novel approaches for therapy.

Crosstalk with lung epithelial cells regulates Sfrp2-mediated latency in breast cancer dissemination.

The process of metastasis is complex1. In breast cancer, there are frequently long time intervals between cells leaving the primary tumour and growth of overt metastases2,3. Reasons for disease indolence and subsequent transition back to aggressive growth include interactions with myeloid and fibroblastic cells in the tumour microenvironment and ongoing immune surveillance4-6. However, the signals that cause actively growing cells to enter an indolent state, thereby enabling them to survive for extended periods of time, are not well understood. Here we reveal how the behaviour of indolent breast cancer cells in the lung is determined by their interactions with alveolar epithelial cells, in particular alveolar type 1 cells. This promotes the formation of fibronectin fibrils by indolent cells that drive integrin-dependent pro-survival signals. Combined in vivo RNA sequencing and drop-out screening identified secreted frizzled-related protein 2 (SFRP2) as a key mediator of this interaction. Sfrp2 is induced in breast cancer cells by signals from lung epithelial cells and promotes fibronectin fibril formation and survival, whereas blockade of Sfrp2 expression reduces the burden of indolent disease.

The transcriptional regulator ZNF398 mediates pluripotency and epithelial character downstream of TGF-beta in human PSCs.

Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability.

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability.

Monitoring Smad Activity In Vivo Using the Xenopus Model System.

The embryo of the African clawed frog Xenopus laevis plays a central role in the field of cell and developmental biology. One of the strengths of Xenopus as model system lies in the high degree of conservation between amphibians and mammals in the molecular mechanisms controlling tissue patterning and differentiation. As such, many signaling cascades were first investigated in frog embryos and then confirmed in mouse and/or human cells. The TGF-ß signaling cascade greatly benefited from this model system. Here we review the overall logic and experimental planning for studying Smad activity in vivo in the context of Xenopus embryonic development, and provide a guide for the interpretation of the results.