USP7 substrates identified by proteomics analysis reveal the specificity of USP7.

Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most extensively studied DUBs, since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC, and PTEN. Thus, USP7 is a promising drug target. However, systematic identification of USP7 substrates has not yet been performed. In this study, we carried out proteome profiling with label-free quantification in control and single/double-KO cells of USP7and its closest homolog, USP47 Our proteome profiling for the first time revealed the proteome changes caused by USP7 and/or USP47 depletion. Combining protein profiling, transcriptome analysis, and tandem affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates that includes known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. We further showed that MGA deletion reduced cell proliferation, similar to what was observed in cells with USP7 deletion. In conclusion, our proteome-wide analysis uncovered potential USP7 substrates, providing a resource for further functional studies.

DePARylation is critical for S phase progression and cell survival.

Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.

Long noncoding RNA Malat1 protects against osteoporosis and bone metastasis.

MALAT1, one of the few highly conserved nuclear long noncoding RNAs (lncRNAs), is abundantly expressed in normal tissues. Previously, targeted inactivation and genetic rescue experiments identified MALAT1 as a suppressor of breast cancer lung metastasis. On the other hand, Malat1-knockout mice are viable and develop normally. On a quest to discover the fundamental roles of MALAT1 in physiological and pathological processes, we find that this lncRNA is downregulated during osteoclastogenesis in humans and mice. Remarkably, Malat1 deficiency in mice promotes osteoporosis and bone metastasis of melanoma and mammary tumor cells, which can be rescued by genetic add-back of Malat1. Mechanistically, Malat1 binds to Tead3 protein, a macrophage-osteoclast-specific Tead family member, blocking Tead3 from binding and activating Nfatc1, a master regulator of osteoclastogenesis, which results in the inhibition of Nfatc1-mediated gene transcription and osteoclast differentiation. Notably, single-cell transcriptome analysis of clinical bone samples reveals that reduced MALAT1 expression in pre-osteoclasts and osteoclasts is associated with osteoporosis and metastatic bone lesions. Altogether, these findings identify Malat1 as a lncRNA that protects against osteoporosis and bone metastasis.

DePARylation is critical for S phase progression and cell survival.

Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S-phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.

Extracellular Vesicles (EVs) in Tumor Diagnosis and Therapy.

In recent years, extracellular vesicles (EVs) have gained significant attention due to their tremendous potential for clinical applications. EVs play a crucial role in various aspects, including tumorigenesis, drug resistance, immune escape, and reconstruction of the tumor microenvironment. Despite the growing interest in EVs, many questions still need to be addressed before they can be practically applied in clinical settings. This paper aims to review EVs' isolation methods, structure research, the roles of EVs in tumorigenesis and their mechanisms in multiple types of tumors, their potential application in drug delivery, and the expectations for their future in clinical research.

Long noncoding RNA Malat1 inhibits Tead3-Nfatc1-mediated osteoclastogenesis to suppress osteoporosis and bone metastasis.

MALAT1, one of the few highly conserved nuclear long noncoding RNAs (IncRNAs), is abundantly expressed in normal tissues. Previously, targeted inactivation and genetic rescue experiments identified MALAT1 as a suppressor of breast cancer lung metastasis. On the other hand, Malat1-knockout mice are viable and develop normally. On a quest to discover new roles of MALAT1 in physiological and pathological processes, we found that this lncRNA is downregulated during osteoclastogenesis in humans and mice. Notably, Malat1 deficiency in mice promotes osteoporosis and bone metastasis, which can be rescued by genetic add-back of Malat1. Mechanistically, Malat1 binds to Tead3 protein, a macrophage-osteoclast-specific Tead family member, blocking Tead3 from binding and activating Nfatc1, a master regulator of osteoclastogenesis, which results in the inhibition of Nfatc1-mediated gene transcription and osteoclast differentiation. Altogether, these findings identify Malat1 as a lncRNA that suppresses osteoporosis and bone metastasis.

LIFR recruits HGF-producing neutrophils to promote liver injury repair and regeneration.

The molecular links between tissue repair and tumorigenesis remain elusive. Here, we report that loss of the liver tumor suppressor Lifr in mouse hepatocytes impairs the recruitment and activity of reparative neutrophils, resulting in the inhibition of liver regeneration after partial hepatectomy or toxic injuries. On the other hand, overexpression of LIFR promotes liver repair and regeneration after injury. Interestingly, LIFR deficiency or overexpression does not affect hepatocyte proliferation ex vivo or in vitro . In response to physical or chemical damage to the liver, LIFR from hepatocytes promotes the secretion of the neutrophil chemoattractant CXCL1 (which binds CXCR2 to recruit neutrophils) and cholesterol in a STAT3-dependent manner. Cholesterol, in turn, acts on the recruited neutrophils to secrete hepatocyte growth factor (HGF) to accelerate hepatocyte proliferation and regeneration. Altogether, our findings reveal a LIFR-STAT3- CXCL1-CXCR2 axis and a LIFR-STAT3-cholesterol-HGF axis that mediate hepatic damage- induced crosstalk between hepatocytes and neutrophils to repair and regenerate the liver.

SLC7A11 expression level dictates differential responses to oxidative stress in cancer cells.

The cystine transporter solute carrier family 7 member 11 (SLC7A11; also called xCT) protects cancer cells from oxidative stress and is overexpressed in many cancers. Here we report a surprising finding that, whereas moderate overexpression of SLC7A11 is beneficial for cancer cells treated with H2O2, a common oxidative stress inducer, its high overexpression dramatically increases H2O2-induced cell death. Mechanistically, high cystine uptake in cancer cells with high overexpression of SLC7A11 in combination with H2O2 treatment results in toxic buildup of intracellular cystine and other disulfide molecules, NADPH depletion, redox system collapse, and rapid cell death (likely disulfidptosis). We further show that high overexpression of SLC7A11 promotes tumor growth but suppresses tumor metastasis, likely because metastasizing cancer cells with high expression of SLC7A11 are particularly susceptible to oxidative stress. Our findings reveal that SLC7A11 expression level dictates cancer cells' sensitivity to oxidative stress and suggests a context-dependent role for SLC7A11 in tumor biology.

Methionine restriction promotes cGAS activation and chromatin untethering through demethylation to enhance antitumor immunity.

Cyclic GMP-AMP synthase (cGAS) is the major sensor for cytosolic DNA and activates type I interferon signaling and plays an essential role in antitumor immunity. However, it remains unclear whether the cGAS-mediated antitumor activity is affected by nutrient status. Here, our study reports that methionine deprivation enhances cGAS activity by blocking its methylation, which is catalyzed by methyltransferase SUV39H1. We further show that methylation enhances the chromatin sequestration of cGAS in a UHRF1-dependent manner. Blocking cGAS methylation enhances cGAS-mediated antitumor immunity and suppresses colorectal tumorigenesis. Clinically, cGAS methylation in human cancers correlates with poor prognosis. Thus, our results indicate that nutrient stress promotes cGAS activation via reversible methylation, and suggest a potential therapeutic strategy for targeting cGAS methylation in cancer treatment.

CRISPR-Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames.

Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR-Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the 'dark' proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.

Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance.

In response to DNA double-strand breaks (DSBs), repair proteins are recruited to the damaged sites. Ubiquitin signaling plays a critical role in coordinating protein recruitment during the DNA damage response. Here, we find that the microRNA biogenesis factor DGCR8 promotes tumor resistance to X-ray radiation independently of its Drosha-binding ability. Upon radiation, the kinase ATM and the deubiquitinase USP51 mediate the activation and stabilization of DGCR8 through phosphorylation and deubiquitination. Specifically, radiation-induced ATM-dependent phosphorylation of DGCR8 at serine 677 facilitates USP51 to bind, deubiquitinate, and stabilize DGCR8, which leads to the recruitment of DGCR8 and DGCR8's binding partner RNF168 to MDC1 and RNF8 at DSBs. This, in turn, promotes ubiquitination of histone H2A, repair of DSBs, and radioresistance. Altogether, these findings reveal the non-canonical function of DGCR8 in DSB repair and suggest that radiation treatment may result in therapy-induced tumor radioresistance through ATM- and USP51-mediated activation and upregulation of DGCR8.

A targetable LIFR-NF-κB-LCN2 axis controls liver tumorigenesis and vulnerability to ferroptosis.

The growing knowledge of ferroptosis has suggested the role and therapeutic potential of ferroptosis in cancer, but has not been translated into effective therapy. Liver cancer, primarily hepatocellular carcinoma (HCC), is highly lethal with limited treatment options. LIFR is frequently downregulated in HCC. Here, by studying hepatocyte-specific and inducible Lifr-knockout mice, we show that loss of Lifr promotes liver tumorigenesis and confers resistance to drug-induced ferroptosis. Mechanistically, loss of LIFR activates NF-κB signaling through SHP1, leading to upregulation of the iron-sequestering cytokine LCN2, which depletes iron and renders insensitivity to ferroptosis inducers. Notably, an LCN2-neutralizing antibody enhances the ferroptosis-inducing and anticancer effects of sorafenib on HCC patient-derived xenograft tumors with low LIFR expression and high LCN2 expression. Thus, anti-LCN2 therapy is a promising way to improve liver cancer treatment by targeting ferroptosis.

Expression of SHH signaling pathway components in the developing human lung.

The Sonic hedgehog (Shh) cascade is crucial for the patterning of the early lung morphogenesis in mice, but its role in the developing human lung remains to be determined. In the present study, the expression patterns of SHH signaling pathway components, including SHH, PTCH1, SMO, GLI1, GLI2 and GLI3 were examined by in situ hybridization and immunohistochemistry, and compared with the equivalent patterns in mice. Our results showed that, as in mice, SHH was expressed in the epithelium of the developing human lung. However, SHH receptors (PTCH1 and SMO) and SHH signaling effectors (GLI1-3) were strongly detected in the human lung epithelium, but weakly in the mesenchyme, slightly different from their expressions in mice. Furthermore, the expression levels of SHH signaling pathway genes in human lung, but not that of GLI1, were subsequently downregulated at the canalicular stage evaluated by real-time PCR, coincident with a decline in the developing murine lung. In conclusion, in spite of slight differences, the considerable similarities of gene expression in human and mice suggest that conserved molecular networks regulate mammalian lung development.

Challenges and Strategies in Ascribing Functions to Long Noncoding RNAs.

Long noncoding RNAs (lncRNAs) are involved in many physiological and pathological processes, such as development, aging, immunity, and cancer. Mechanistically, lncRNAs exert their functions through interaction with proteins, genomic DNA, and other RNA, leading to transcriptional and post-transcriptional regulation of gene expression, either in cis or in trans; it is often difficult to distinguish between these two regulatory mechanisms. A variety of approaches, including RNA interference, antisense oligonucleotides, CRISPR-based methods, and genetically engineered mouse models, have yielded abundant information about lncRNA functions and underlying mechanisms, albeit with many discrepancies. In this review, we elaborate on the challenges in ascribing functions to lncRNAs based on the features of lncRNAs, including the genomic location, copy number, domain structure, subcellular localization, stability, evolution, and expression pattern. We also describe a framework for the investigation of lncRNA functions and mechanisms of action. Rigorous characterization of cancer-implicated lncRNAs is critical for the identification of bona fide anticancer targets.

Ubiquitination of Rheb governs growth factor-induced mTORC1 activation.

Mechanistic target of rapamycin mTOR complex 1 (mTORC1) plays a key role in the integration of various environmental signals to regulate cell growth and metabolism. mTORC1 is recruited to the lysosome where it is activated by its interaction with GTP-bound Rheb GTPase. However, the regulatory mechanism of Rheb activity remains largely unknown. Here, we show that ubiquitination governs the nucleotide-bound status of Rheb. Lysosome-anchored E3 ligase RNF152 catalyzes Rheb ubiquitination and promotes its binding to the TSC complex. EGF enhances the deubiquitination of Rheb through AKT-dependent USP4 phosphorylation, leading to the release of Rheb from the TSC complex. Functionally, ubiquitination of Rheb is linked to mTORC1-mediated signaling and  consequently regulates tumor growth. Thus, we propose a mechanistic model whereby Rheb-mediated mTORC1 activation is dictated by a dynamic opposing act between Rheb ubiquitination and deubiquitination that are catalyzed by RNF152 and USP4 respectively.

AMPK Promotes SPOP-Mediated NANOG Degradation to Regulate Prostate Cancer Cell Stemness.

NANOG is an essential transcriptional factor for the maintenance of embryonic stem cells (ESCs) and cancer stem cells (CSCs) in prostate cancer (PCa). However, the regulation mechanism of NANOG protein stability in cancer progression is still elusive. Here, we report that NANOG is degraded by SPOP, a frequently mutated tumor suppressor of PCa. Cancer-associated mutations of SPOP or the mutation of NANOG at S68Y abrogates the SPOP-mediated NANOG degradation, leading to elevated PCa cancer stemness and poor prognosis. In addition, SPOP-mediated NANOG degradation is controlled by the AMPK-BRAF signal axis through the phosphorylation of NANOG at Ser68, which blocked the interaction between SPOP and NANOG. Thus, our study provides a regulation mechanism of PCa stemness controlled by phosphorylation-mediated NANOG stability, which helps to identify novel drug targets and improve therapeutic strategy for PCa.

SET1A-Mediated Mono-Methylation at K342 Regulates YAP Activation by Blocking Its Nuclear Export and Promotes Tumorigenesis.

YAP, a key effector of Hippo pathway, is activated by its translocation from cytoplasm to nucleus to regulate gene expression and promote tumorigenesis. Although the mechanism by which YAP is suppressed in cytoplasm has been well-studied, how the activated YAP is sequestered in the nucleus remains unknown. Here, we demonstrate that YAP is a nucleocytoplasmic shuttling protein and its nuclear export is controlled by SET1A-mediated mono-methylation of YAP at K342, which disrupts the binding of YAP to CRM1. YAP mimetic methylation knockin mice are more susceptible to colorectal tumorigenesis. Clinically, YAP K342 methylation is reversely correlated with cancer survival. Collectively, our study identifies SET1A-mediated mono-methylation at K342 as an essential regulatory mechanism for regulating YAP activity and tumorigenesis.

p38 inhibition provides anti-DNA virus immunity by regulation of USP21 phosphorylation and STING activation.

Stimulator of IFN genes (STING) is a central adaptor protein that mediates the innate immune responses to DNA virus infection. Although ubiquitination is essential for STING function, how the ubiquitination/deubiquitination system is regulated by virus infection to control STING activity remains unknown. In this study, we found that USP21 is an important deubiquitinating enzyme for STING and that it negatively regulates the DNA virus-induced production of type I interferons by hydrolyzing K27/63-linked polyubiquitin chain on STING. HSV-1 infection recruited USP21 to STING at late stage by p38-mediated phosphorylation of USP21 at Ser538. Inhibition of p38 MAPK enhanced the production of IFNs in response to virus infection and protected mice from lethal HSV-1 infection. Thus, our study reveals a critical role of p38-mediated USP21 phosphorylation in regulating STING-mediated antiviral functions and identifies p38-USP21 axis as an important pathway that DNA virus adopts to avoid innate immunity responses.