SARS-CoV-2 down-regulates ACE2 through lysosomal degradation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes its Spike (S) glycoprotein to bind to the angiotensin-converting enzyme 2 (ACE2) receptor for cellular entry. ACE2 is a critical negative regulator of the renin-angiotensin system and plays a protective role in preventing tissue injury. Expression of ACE2 has been shown to decrease upon infection by SARS-CoV. However, whether SARS-CoV-2 down-regulates ACE2 and the underlying mechanism and biological impact of this down-regulation have not been well defined. Here we show that the SARS-CoV-2 infection down-regulates ACE2 in vivo in an animal model, and in cultured cells in vitro, by inducing clathrin- and AP2-dependent endocytosis, leading to its degradation in the lysosome. SARS-CoV-2 S-treated cells and ACE2 knockdown cells exhibit similar alterations in downstream gene expression, with a pattern indicative of activated cytokine signaling that is associated with respiratory distress and inflammatory diseases often observed in COVID-19 patients. Finally, we have identified a soluble ACE2 fragment with a stronger binding to SARS-CoV-2 S that can efficiently block ACE2 down-regulation and viral infection. Thus, our study suggests that ACE2 down-regulation represents an important mechanism underlying SARS-CoV-2-associated pathology, and blocking this process could be a promising therapeutic strategy.

Poly(ADP-ribosylation) of P-TEFb by PARP1 disrupts phase separation to inhibit global transcription after DNA damage.

DNA damage shuts down genome-wide transcription to prevent transcriptional mutagenesis and to initiate repair signalling, but the mechanism to stall elongating RNA polymerase II (Pol II) is not fully understood. Central to the DNA damage response, poly(ADP-ribose) polymerase 1 (PARP1) initiates DNA repair by translocating to the lesions where it catalyses protein poly(ADP-ribosylation). Here we report that PARP1 inhibits Pol II elongation by inactivating the transcription elongation factor P-TEFb, a CDK9-cyclin T1 (CycT1) heterodimer. After sensing damage, the activated PARP1 binds to transcriptionally engaged P-TEFb and modifies CycT1 at multiple positions, including histidine residues that are rarely used as an acceptor site. This prevents CycT1 from undergoing liquid-liquid phase separation that is required for CDK9 to hyperphosphorylate Pol II and to stimulate elongation. Functionally, poly(ADP-ribosylation) of CycT1 promotes DNA repair and cell survival. Thus, the P-TEFb-PARP1 signalling plays a protective role in transcription quality control and genomic stability maintenance after DNA damage.

HEXIM1 controls P-TEFb processing and regulates drug sensitivity in triple-negative breast cancer.

The positive transcription elongation factor b (P-TEFb), composed of CDK9 and cyclin T, stimulates transcriptional elongation by RNA polymerase (Pol) II and regulates cell growth and differentiation. Recently, we demonstrated that P-TEFb also controls the expression of EMT regulators to promote breast cancer progression. In the nucleus, more than half of P-TEFb are sequestered in the inactive-state 7SK snRNP complex. Here, we show that the assembly of the 7SK snRNP is preceded by an intermediate complex between HEXIM1 and P-TEFb that allows transfer of the kinase active P-TEFb from Hsp90 to 7SK snRNP for its suppression. Down-regulation of HEXIM1 locks P-TEFb in the Hsp90 complex, keeping it in the active state to enhance breast cancer progression, but also rendering the cells highly sensitive to Hsp90 inhibition. Because HEXIM1 is often down-regulated in human triple-negative breast cancer (TNBC), these cells are particularly sensitive to Hsp90 inhibition. Our study provides a mechanistic explanation for the increased sensitivity of TNBC to Hsp90 inhibition.

Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression.

TAZ promotes growth, development and tumorigenesis by regulating the expression of target genes. However, the manner in which TAZ orchestrates the transcriptional responses is poorly defined. Here we demonstrate that TAZ forms nuclear condensates through liquid-liquid phase separation to compartmentalize its DNA-binding cofactor TEAD4, coactivators BRD4 and MED1, and the transcription elongation factor CDK9 for transcription. TAZ forms phase-separated droplets in vitro and liquid-like nuclear condensates in vivo, and this ability is negatively regulated by Hippo signalling through LATS-mediated phosphorylation and is mediated by the coiled-coil (CC) domain. Deletion of the TAZ CC domain or substitution with the YAP CC domain prevents the phase separation of TAZ and its ability to induce the expression of TAZ-specific target genes. Thus, we identify a mechanism of transcriptional activation by TAZ and demonstrate that pathway-specific transcription factors also engage the phase-separation mechanism for efficient and specific transcriptional activation.

The regulatory protein SnoN antagonizes activin/Smad2 protein signaling and thereby promotes adipocyte differentiation and obesity in mice.

Ski-related oncogene SnoN (SnoN or SKIL) regulates multiple signaling pathways in a tissue- and developmental stage-dependent manner and has broad functions in embryonic angiogenesis, mammary gland alveologenesis, cancer, and aging. Here, we report that SnoN also plays a critical role in white adipose tissue (WAT) development by regulating mesenchymal stem cell (MSC) self-renewal and differentiation. We found that SnoN promotes MSC differentiation in the adipocyte lineage by antagonizing activin A/Smad2, but not TGFß/Smad3 signaling. Mice lacking SnoN or expressing a mutant SnoN defective in binding to the Smads were protected from high-fat diet-induced obesity and insulin resistance, and MSCs lacking a functional SnoN exhibited defective differentiation. We further demonstrated that activin, via Smad2, appears to be the major regulator of WAT development in vivo We also noted that activin A is abundantly expressed in WAT and adipocytes through an autocrine mechanism and promotes MSC self-renewal and inhibits adipogenic differentiation by inducing expression of the gene encoding the homeobox transcription factor Nanog. Of note, SnoN repressed activin/Smad2 signaling and activin A expression, enabling expression of adipocyte-specific transcription factors and promoting adipogenic differentiation. In conclusion, our study has revealed that SnoN plays an important in vivo role in adipocyte differentiation and WAT development in vivo by decreasing activity in the activin/Smad2 signaling pathway.

Signaling Cross Talk between TGF-ß/Smad and Other Signaling Pathways.

Cytokines of the transforming growth factor ß (TGF-ß) family, including TGF-ßs, bone morphogenic proteins (BMPs), activins, and Nodal, play crucial roles in embryonic development and adult tissue homeostasis by regulating cell proliferation, survival, and differentiation, as well as stem-cell self-renewal and lineage-specific differentiation. Smad proteins are critical downstream mediators of these signaling activities. In addition to regulating the transcription of direct target genes of TGF-ß, BMP, activin, or Nodal, Smad proteins also participate in extensive cross talk with other signaling pathways, often in a cell-type- or developmental stage-specific manner. These combinatorial signals often produce context-, time-, and location-dependent biological outcomes that are critical for development. This review discusses recent progress in our understanding of the cross talk between Smad proteins and signaling pathways of Wnt, Notch, Hippo, Hedgehog (Hh), mitogen-activated protein (MAP), kinase, phosphoinositide 3-kinase (PI3K)-Akt, nuclear factor κB (NF-κB), and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways.

SnoN Antagonizes the Hippo Kinase Complex to Promote TAZ Signaling during Breast Carcinogenesis.

SnoN regulates multiple signaling pathways, including TGF-ß/Smad and p53, and displays both pro-oncogenic and anti-oncogenic activities in human cancer. We have observed previously that both its intracellular localization and expression levels are sensitive to cell density, suggesting that it may crosstalk with Hippo signaling. Here we report that, indeed, SnoN interacts with multiple components of the Hippo pathway to inhibit the binding of Lats2 to TAZ and the subsequent phosphorylation of TAZ, leading to TAZ stabilization. Consistently, SnoN enhances the transcriptional and oncogenic activities of TAZ, and reducing SnoN decreases TAZ expression as well as malignant progression of breast cancer cells. Interestingly, SnoN itself is downregulated by Lats2 that is activated by the Scribble basolateral polarity protein. Thus, SnoN is a critical component of the Hippo regulatory network that receives signals from the tissue architecture and polarity to coordinate the activity of intracellular signaling pathways.

Three-dimensional Mammary Epithelial Cell Morphogenesis Model for Analysis of TGFß Signaling.

Culturing mammary epithelial cells in laminin-rich extracellular matrices (three dimensional or 3D culture) offers significant advantages over that in the conventional two-dimensional (2D) tissue culture system in that it takes into considetation the impact of extracellular matrix (ECM) microenvironment on the proliferation, survival, and differentiation of mammary epithelial cells. When grown in the 3D culture, untransformed mammary epithelial cells undergo morphogenesis to form a multicellular and polarized acini-like structure that functionally mimics the differentiated alveoli in the pregnancy mammary gland. This process is subjected to regulation by many growth factors and cytokines. The transforming growth factor-ß (TGFß) is a multipotent cytokine that regulates multiple aspects of development and tumorigenesis. In addition to its effects on epithelial cell proliferation, survival, and differentiation, it is also a potent regulator of the cell-matrix interaction. Thus, the 3D culture model may recapitulate the complex in vivo epithelial cell microenvironment and allow us to fully evaluate the role of TGFß signaling in multiple aspects of normal and cancerous cell behavior. In this chapter we provide detailed protocols for growing mammary epithelial cells in the 3D Matrigel for analysis of signaling pathways.

Compensatory induction of MYC expression by sustained CDK9 inhibition via a BRD4-dependent mechanism.

CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.

Ski regulates Hippo and TAZ signaling to suppress breast cancer progression.

Ski, the transforming protein of the avian Sloan-Kettering retrovirus, inhibits transforming growth factor-ß (TGF-ß)/Smad signaling and displays both pro-oncogenic and anti-oncogenic activities in human cancer. Inhibition of TGF-ß signaling is likely responsible for the pro-oncogenic activity of Ski. We investigated the mechanism(s) underlying the tumor suppressor activity of Ski and found that Ski suppressed the activity of the Hippo signaling effectors TAZ and YAP to inhibit breast cancer progression. TAZ and YAP are transcriptional coactivators that can contribute to cancer by promoting proliferation, tumorigenesis, and cancer stem cell expansion. Hippo signaling activates the the Lats family of kinases, which phosphorylate TAZ and YAP, resulting in cytoplasmic retention and degradation and inhibition of their transcriptional activity. We showed that Ski interacted with multiple components of the Hippo pathway to facilitate activation of Lats2, resulting in increased phosphorylation and subsequent degradation of TAZ. Ski also promoted the degradation of a constitutively active TAZ mutant that is not phosphorylated by Lats, suggesting the existence of a Lats2-independent degradation pathway. Finally, we showed that Ski repressed the transcriptional activity of TAZ by binding to the TAZ partner TEAD and recruiting the transcriptional co-repressor NCoR1 to the TEAD-TAZ complex. Ski effectively reversed transformation and epithelial-to-mesenchyme transition in cultured breast cancer cells and metastasis in TAZ-expressing xenografted tumors. Thus, Ski inhibited the function of TAZ through multiple mechanisms in human cancer cells.

LARP7 suppresses P-TEFb activity to inhibit breast cancer progression and metastasis.

Transcriptional elongation by RNA polymerase (Pol) II is essential for gene expression during cell growth and differentiation. The positive transcription elongation factor b (P-TEFb) stimulates transcriptional elongation by phosphorylating Pol II and antagonizing negative elongation factors. A reservoir of P-TEFb is sequestered in the inactive 7SK snRNP where 7SK snRNA and the La-related protein LARP7 are required for the integrity of this complex. Here, we show that P-TEFb activity is important for the epithelial-mesenchymal transition (EMT) and breast cancer progression. Decreased levels of LARP7 and 7SK snRNA redistribute P-TEFb to the transcriptionally active super elongation complex, resulting in P-TEFb activation and increased transcription of EMT transcription factors, including Slug, FOXC2, ZEB2, and Twist1, to promote breast cancer EMT, invasion, and metastasis. Our data provide the first demonstration that the transcription elongation machinery plays a key role in promoting breast cancer progression by directly controlling the expression of upstream EMT regulators.

Metabolic profiling reveals PAFAH1B3 as a critical driver of breast cancer pathogenicity.

Many studies have identified metabolic pathways that underlie cellular transformation, but the metabolic drivers of cancer progression remain less well understood. The Hippo transducer pathway has been shown to confer malignant traits on breast cancer cells. In this study, we used metabolic mapping platforms to identify biochemical drivers of cellular transformation and malignant progression driven through RAS and the Hippo pathway in breast cancer and identified platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3) as a key metabolic driver of breast cancer pathogenicity that is upregulated in primary human breast tumors and correlated with poor prognosis. Metabolomic profiling suggests that PAFAH1B3 inactivation attenuates cancer pathogenicity through enhancing tumor-suppressing signaling lipids. Our studies provide a map of altered metabolism that underlies breast cancer progression and put forth PAFAH1B3 as a critical metabolic node in breast cancer.

SnoN facilitates ALK1-Smad1/5 signaling during embryonic angiogenesis.

In endothelial cells, two type I receptors of the transforming growth factor ß (TGF-ß) family, ALK1 and ALK5, coordinate to regulate embryonic angiogenesis in response to BMP9/10 and TGF-ß. Whereas TGF-ß binds to and activates ALK5, leading to Smad2/3 phosphorylation and inhibition of endothelial cell proliferation and migration, BMP9/10 and TGF-ß also bind to ALK1, resulting in the activation of Smad1/5. SnoN is a negative regulator of ALK5 signaling through the binding and repression of Smad2/3. Here we uncover a positive role of SnoN in enhancing Smad1/5 activation in endothelial cells to promote angiogenesis. Upon ligand binding, SnoN directly bound to ALK1 on the plasma membrane and facilitated the interaction between ALK1 and Smad1/5, enhancing Smad1/5 phosphorylation. Disruption of this SnoN-Smad interaction impaired Smad1/5 activation and up-regulated Smad2/3 activity. This resulted in defective angiogenesis and arteriovenous malformations, leading to embryonic lethality at E12.5. Thus, SnoN is essential for TGF-ß/BMP9-dependent biological processes by its ability to both positively and negatively modulate the activities of Smad-dependent pathways.

Ether lipid generating enzyme AGPS alters the balance of structural and signaling lipids to fuel cancer pathogenicity.

Aberrant lipid metabolism is an established hallmark of cancer cells. In particular, ether lipid levels have been shown to be elevated in tumors, but their specific function in cancer remains elusive. We show here that the metabolic enzyme alkylglyceronephosphate synthase (AGPS), a critical step in the synthesis of ether lipids, is up-regulated across multiple types of aggressive human cancer cells and primary tumors. We demonstrate that ablation of AGPS in cancer cells results in reduced cell survival, cancer aggressiveness, and tumor growth through altering the balance of ether lipid, fatty acid, eicosanoid, and fatty acid-derived glycerophospholipid metabolism, resulting in an overall reduction in the levels of several oncogenic signaling lipids. Taken together, our results reveal that AGPS, in addition to maintaining ether lipids, also controls cellular utilization of fatty acids, favoring the generation of signaling lipids necessary for promoting the aggressive features of cancer.

Cancer cells incorporate and remodel exogenous palmitate into structural and oncogenic signaling lipids.

De novo lipogenesis is considered the primary source of fatty acids for lipid synthesis in cancer cells, even in the presence of exogenous fatty acids. Here, we have used an isotopic fatty acid labeling strategy coupled with metabolomic profiling platforms to comprehensively map palmitic acid incorporation into complex lipids in cancer cells. We show that cancer cells and tumors robustly incorporate and remodel exogenous palmitate into structural and oncogenic glycerophospholipids, sphingolipids, and ether lipids. We also find that fatty acid incorporation into oxidative pathways is reduced in aggressive human cancer cells, and instead shunted into pathways for generating structural and signaling lipids. Our results demonstrate that cancer cells do not solely rely on de novo lipogenesis, but also utilize exogenous fatty acids for generating lipids required for proliferation and protumorigenic lipid signaling. This article is part of a special issue entitled Lipid Metabolism in Cancer.

Expression profiles of SnoN in normal and cancerous human tissues support its tumor suppressor role in human cancer.

SnoN is a negative regulator of TGF-ß signaling and also an activator of the tumor suppressor p53 in response to cellular stress. Its role in human cancer is complex and controversial with both pro-oncogenic and anti-oncogenic activities reported. To clarify its role in human cancer and provide clinical relevance to its signaling activities, we examined SnoN expression in normal and cancerous human esophageal, ovarian, pancreatic and breast tissues. In normal tissues, SnoN is expressed in both the epithelium and the surrounding stroma at a moderate level and is predominantly cytoplasmic. SnoN levels in all tumor epithelia examined are lower than or similar to that in the matched normal samples, consistent with its anti-tumorigenic activity in epithelial cells. In contrast, SnoN expression in the stroma is highly upregulated in the infiltrating inflammatory cells in high-grade esophageal and ovarian tumor samples, suggesting that SnoN may potentially promote malignant progression through modulating the tumor microenvironment in these tumor types. The overall levels of SnoN expression in these cancer tissues do not correlate with the p53 status. However, in human cancer cell lines with amplification of the snoN gene, a strong correlation between increased SnoN copy number and inactivation of p53 was detected, suggesting that the tumor suppressor SnoN-p53 pathway must be inactivated, either through downregulation of SnoN or inactivation of p53, in order to allow cancer cell to proliferate and survive. These data strongly suggest that SnoN can function as a tumor suppressor at early stages of tumorigenesis in human cancer tissues.

SnoN activates p53 directly to regulate aging and tumorigenesis.

We have identified SnoN as a direct activator of p53 to accelerate aging and inhibit tumorigenesis. SnoN has been shown previously to promote proliferation and transformation by antagonizing TGFß signaling. We show that elimination of this TGFß antagonistic activity of SnoN in vivo results in accelerated aging and resistance to tumorigenesis. The SnoN knockin mice display a shortened lifespan, decreased reproductivity, osteoporosis, reduced regenerative capacity, and other aging phenotypes, similar to that found in mice expressing an active p53. These activities of SnoN rely on the ability of SnoN to activate p53. SnoN can bind directly to p53 and compete with Mdm2 for binding to p53, preventing p53 ubiquitination and degradation and additionally facilitating p53 acetylation and phosphorylation. SnoN also binds to p53 on the promoter of p53 responsive genes to promote transcription activation. This activation of p53 by SnoN is necessary for its antitumorigenic and progeria activities in vivo because elimination of one copy of p53 reverses the aging phenotypes and accelerates tumorigenesis. Thus, we have revealed a novel function of SnoN in regulating aging and tumorigenesis by directly activating p53.

SnoN regulates mammary gland alveologenesis and onset of lactation by promoting prolactin/Stat5 signaling.

Mammary epithelial cells undergo structural and functional differentiation at late pregnancy and parturition to produce and secrete milk. Both TGF-ß and prolactin pathways are crucial regulators of this process. However, how the activities of these two antagonistic pathways are orchestrated to initiate lactation has not been well defined. Here, we show that SnoN, a negative regulator of TGF-ß signaling, coordinates TGF-ß and prolactin signaling to control alveologenesis and lactogenesis. SnoN expression is induced at late pregnancy by the coordinated actions of TGF-ß and prolactin. The elevated SnoN promotes Stat5 signaling by enhancing its stability, thereby sharply increasing the activity of prolactin signaling at the onset of lactation. SnoN-/- mice display severe defects in alveologenesis and lactogenesis, and mammary epithelial cells from these mice fail to undergo proper morphogenesis. These defects can be rescued by an active Stat5. Thus, our study has identified a new player in the regulation of milk production and revealed a novel function of SnoN in mammary alveologenesis and lactogenesis in vivo through promotion of Stat5 signaling.