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Immune checkpoint inhibitors (ICIs) activate anti-cancer immunity by blocking T cell checkpoint molecules such as programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Although ICIs induce some durable responses in various cancer patients, they also have disadvantages, including low response rates, the potential for severe side effects, and high treatment costs. Therefore, selection of patients who can benefit from ICI treatment is critical, and identification of biomarkers is essential to improve the efficiency of ICIs. In this review, we provide updated information on established predictive biomarkers (tumor programmed death-ligand 1 [PD-L1] expression, DNA mismatch repair deficiency, microsatellite instability high, and tumor mutational burden) and potential biomarkers currently under investigation such as tumor-infiltrated and peripheral lymphocytes, gut microbiome, and signaling pathways related to DNA damage and antigen presentation. In particular, this review aims to summarize the current knowledge of biomarkers, discuss issues, and further explore future biomarkers., Competing Interests: Declaration of interests M.-C.H. is a co-inventor on the methodology for de-glycosylation staining., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Programmed death-1 (PD-1)/PD ligand-1 (PD-L1)-mediated immune escape contributes to cancer development and has been targeted as an anti-cancer strategy. Here, we show that inhibition of the RNA helicase DDX3 increased CD8 + T cell infiltration in syngeneic oral squamous cell carcinoma tumors. DDX3 knockdown compromised interferon-γ-induced PD-L1 expression and, in particular, reduced the level of cell-surface PD-L1. DDX3 promoted surface PD-L1 expression by recruiting the adaptor protein 2 (AP2) complex to the 3' UTR of PD-L1 mRNA. DDX3 depletion or 3' UTR truncation increased the binding of the coatomer protein complexes to PD-L1, leading to its intracellular accumulation. Therefore, this 3' UTR-dependent mechanism may counteract cellular negative effects on surface trafficking of PD-L1. Finally, pharmaceutic disruption of DDX3's interaction with AP2 reduced surface PD-L1 expression, supporting that the DDX3-AP2 pathway routes PD-L1 to the cell surface. Targeting DDX3 to modulate surface trafficking of immune checkpoint proteins may provide a potential strategy for cancer immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Immune checkpoint blockade (ICB) is a promising therapy for solid tumors, but its effectiveness depends on biomarkers that are not precise. Here, we utilized genome-wide association study to investigate the association between genetic variants and tumor mutation burden to interpret ICB response. We identified 16 variants (p < 5 × 10 -8 ) probed to 17 genes on 9 chromosomes. Subsequent analysis of one of the most significant loci in 19q13.11 suggested that the rs111308825 locus at the enhancer is causal, as its A allele impairs KLF2 binding, leading to lower carbohydrate sulfotransferase 8 (CHST8) expression. Breast cancer cells expressing CHST8 suppress T cell activation, and Chst8 loss attenuates tumor growth in a syngeneic mouse model. Further investigation revealed that programmed death-ligand 1 (PD-L1) and its homologs could be sulfated by CHST8, resulting in M2-like macrophage enrichment in the tumor microenvironment. Finally, we confirmed that low-CHST8 tumors have better ICB response, supporting the genetic effect and clinical value of rs111308825 for ICB efficacy prediction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The CD58-CD2 axis regulates T cell-mediated cancer immunity, but little is known about the regulation of CD58. In two recent papers pubished in Cancer Cell, Miao et al. and Ho et al. define a mechanism of CD58 regulation by CMTM6 and show an unexpected yin-yang link between PD-L1 and CD58., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Obesity, in which the functional importance of small nucleolar RNAs (snoRNAs) remains elusive, correlates with risk for many cancer types. Here, we identify that the serum copies of adipocyte-expressed SNORD46 correlate with body mass index (BMI), and serum SNORD46 antagonizes interleukin-15 (IL-15) signaling. Mechanically, SNORD46 binds IL-15 via G11, and G11A (a mutation that significantly enhances binding affinity) knockin drives obesity in mice. Functionally, SNORD46 blocks IL-15-induced, FER kinase-dependent phosphorylation of platelet glycoprotein 4 (CD36) and monoglyceride lipase (MGLL) in adipocytes, leading to inhibited lipolysis and browning. In natural killer (NK) cells, SNORD46 suppresses the IL-15-dependent autophagy, leading to reduced viability of obese NK. SNORD46 power inhibitors exhibit anti-obesity effects, concurring with improved viability of obese NK and anti-tumor immunity of CAR-NK cell therapy. Hence, our findings demonstrate the functional importance of snoRNAs in obesity and the utility of snoRNA power inhibitors for antagonizing obesity-associated immune resistance., Competing Interests: Declaration of interests M.A.C. reports grants and personal fees from ImmunoGenesis, Inc. and personal fees from Alligator Bioscience, Inc.; ImmunOS, Inc.; Oncoresponse, Inc.; Nurix, Inc.; Aptevo, Inc.; Kineta, Inc.; Xencor, Inc.; Agenus, Inc.; Adagene, Inc.; and Astrazeneca, Inc. outside the submitted work. K.R. and the University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Takeda Pharmaceutical and Affimed GmbH. K.R. participates on the Scientific Advisory Board for GemoAb, AvengeBio, Virogin Biotech, GSK, Caribou Biosciences, Navan Technologies, and Bayer. B.G. is an inventor on patent applications involving targeting ferroptosis in cancer therapy and reports personal fees from Guidepoint Global, Cambridge Solutions, and NGM Bio., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Remote tumors disrupt the bone marrow (BM) ecosystem (BME), eliciting the overproduction of BM-derived immunosuppressive cells. However, the underlying mechanisms remain poorly understood. Herein, we characterized breast and lung cancer-induced BME shifts pre- and post-tumor removal. Remote tumors progressively lead to osteoprogenitor (OP) expansion, hematopoietic stem cell dislocation, and CD41 - granulocyte-monocyte progenitor (GMP) aggregation. The tumor-entrained BME is characterized by co-localization between CD41 - GMPs and OPs. OP ablation abolishes this effect and diminishes abnormal myeloid overproduction. Mechanistically, HTRA1 carried by tumor-derived small extracellular vesicles upregulates MMP-13 in OPs, which in turn induces the alterations in the hematopoietic program. Importantly, these effects persist post-surgery and continue to impair anti-tumor immunity. Conditional knockout or inhibition of MMP-13 accelerates immune reinstatement and restores the efficacies of immunotherapies. Therefore, tumor-induced systemic effects are initiated by OP-GMP crosstalk that outlasts tumor burden, and additional treatment is required to reverse these effects for optimal therapeutic efficacy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dampening tumor growth by converting tumor-associated macrophages (TAMs) from M2/repair-types to M1/kill-types is of high interest. Here, we show that cryptotanshinone (CPT) can function as an antitumor immune modulator that switches TAMs from an M2 to an M1 phenotype, leading to tumor regression. An orthotopic triple-negative breast cancer (TNBC) implantation model was used to determine the role and mechanism of CPT in suppressing M1-to-M2 repolarization of TAMs. Co-culturing TNBC cells with CPT-treated macrophages reduced TNBC proliferation and motility, while in TNBC orthotopic mouse models, CPT treatment inhibited breast tumor formation. Moreover, we identified that CPT inhibits mitochondrial oxidative phosphorylation and mitochondrial fusion via autophagy and transcriptional activation of the apoptosis signal-regulating kinase 1 (ASK1) pathway. Suppression of ASK1 downregulates autophagy and abolishes CPT-induced effects upon TAMs. In addition, CPT inhibits M2 macrophage differentiation and causes TRAF6 auto-ubiquitination-dependent activation of the ASK1, leading to M1 polarization. On the contrary, in M1 macrophage, CPT increases interaction of ASK1 and TRAF6 which induces ASK1 ubiquitination and degradation. Intriguingly, CPT plays opposite roles in the M1 and M2 phenotype. Our findings help to illuminate a previously unrecognized antitumor mechanism of CPT and suggest that this natural compound offers a macrophage-based approach for cancer immunotherapy., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Immunotherapy via PD-1/PD-L1 blockade is a promising strategy to eradicate cancer cells. However, the PD-L1 pathological level is inconsistent with the therapeutic response and is not a reliable biomarker to stratify patients for anti-PD-1/PD-L1 therapy. Here, we describe patient sample deglycosylation in an immunohistochemistry (IHC) assay to resolve this challenge. This protocol facilitates antigen retrieval by removing N -glycans from surface antigens on formalin-fixed paraffin-embedded (FFPE) tissue slides and can be applied in medical pathology for multiple cancer types. For complete details on the use and execution of this profile, please refer to Lee et al. (2019)., Competing Interests: H.-H.L., Y.-N.W., and M.-C.H. are listed as inventors on a patent application (International Patent Application No. PCT/US2019/036073 based on U.S. Provisional Patent Application No. 62/681,929, entitled “Detection of Immune Checkpoint Molecules by Deglycosylation”) submitted by The University of Texas MD Anderson Cancer Center. All other authors declare no non-financial or financial competing interests., (© 2022 The Author(s).)

Anti-angiogenic therapies, such as anti-VEGF antibodies (AVAs), have shown promise in clinical settings. However, adaptive resistance to such therapies occurs frequently. We use orthotopic ovarian cancer models with AVA-adaptive resistance to investigate the underlying mechanisms. Genomic profiling of AVA-resistant tumors guides us to endothelial p130cas. We find that bevacizumab induces cleavage of VEGFR2 in endothelial cells by caspase-10 and that VEGFR2 fragments internalize into the nucleus and autophagosomes. Nuclear VEGFR2 and p130cas fragments, together with TNKS1BP1 (tankyrase-1-binding protein), initiate endothelial cell death. Blockade of autophagy in AVA-resistant endothelial cells retains VEGFR2 at the membrane with bevacizumab treatment. Targeting host p130cas with RGD (Arg-Gly-Asp)-tagged nanoparticles or genomic ablation of vascular p130cas in p130cas flox/flox Tie2 Cre mice significantly extends the survival of mice with AVA-resistant ovarian tumors. Higher vascular p130cas is associated with shorter survival of individuals with ovarian cancer. Our findings identify opportunities for new strategies to overcome adaptive resistance to AVA therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Canonically, gasdermin D (GSDMD) cleavage by caspase-1 through inflammasome signaling triggers immune cell pyroptosis (ICP) as a host defense against pathogen infection. However, cancer cell pyroptosis (CCP) was recently discovered to be activated by distinct molecular mechanisms in which GSDMB, GSDMC, and GSDME, rather than GSDMD, are the executioners. Moreover, instead of inflammatory caspases, apoptotic caspases and granzymes are required for gasdermin protein cleavage to induce CCP. Sufficient accumulation of protease-cleaved gasdermin proteins is the prerequisite for CCP. Inflammation induced by ICP or CCP results in diametrically opposite effects on antitumor immunity because of the differential duration and released cellular contents, leading to contrary effects on therapeutic outcomes. Here, we focus on the distinct mechanisms of ICP and CCP and discuss the roles of ICP and CCP in inflammation and antitumor immunity, representing actionable targets., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Increased DNA replication and metastasis are hallmarks of cancer progression, while deregulated proliferation often triggers sustained replication stresses in cancer cells. How cancer cells overcome the growth stress and proceed to metastasis remains largely elusive. Proliferating cell nuclear antigen (PCNA) is an indispensable component of the DNA replication machinery. Here, we show that phosphorylation of PCNA on tyrosine 211 (pY211-PCNA) regulates DNA metabolism and tumor microenvironment. Abrogation of pY211-PCNA blocks fork processivity, resulting in biogenesis of single-stranded DNA (ssDNA) through a MRE11-dependent mechanism. The cytosolic ssDNA subsequently induces inflammatory cytokines through a cyclic GMP-AMP synthetase (cGAS)-dependent cascade, triggering an anti-tumor immunity by natural killer (NK) cells to suppress distant metastasis. Expression of pY211-PCNA is inversely correlated with cytosolic ssDNA and associated with poor survival in patients with cancer. Our results pave the way to biomarkers and therapies exploiting immune responsiveness to target metastatic cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Aberrant energy status contributes to multiple metabolic diseases, including obesity, diabetes, and cancer, but the underlying mechanism remains elusive. Here, we report that ketogenic-diet-induced changes in energy status enhance the efficacy of anti-CTLA-4 immunotherapy by decreasing PD-L1 protein levels and increasing expression of type-I interferon (IFN) and antigen presentation genes. Mechanistically, energy deprivation activates AMP-activated protein kinase (AMPK), which in turn, phosphorylates PD-L1 on Ser283, thereby disrupting its interaction with CMTM4 and subsequently triggering PD-L1 degradation. In addition, AMPK phosphorylates EZH2, which disrupts PRC2 function, leading to enhanced IFNs and antigen presentation gene expression. Through these mechanisms, AMPK agonists or ketogenic diets enhance the efficacy of anti-CTLA-4 immunotherapy and improve the overall survival rate in syngeneic mouse tumor models. Our findings reveal a pivotal role for AMPK in regulating the immune response to immune-checkpoint blockade and advocate for combining ketogenic diets or AMPK agonists with anti-CTLA4 immunotherapy to combat cancer., Competing Interests: Declaration of interests G.J.F. has patents/pending royalties on the PD-1 pathway from Roche, Merck, Bristol-Myers-Squibb, EMD-Serono, Boehringer-Ingelheim, AstraZeneca, Dako, and Novartis. G.J.F. has served on advisory boards for Roche, Bristol-Myers-Squibb, Xios, Origimed, Triursus, iTeos, NextPoint, IgM, Jubilant, Geode, and GV20. G.J.F. has equity in Nextpoint, Triursus, Xios, iTeos, IgM, Geode, and GV20. W.W. is a co-founder and consultant for ReKindle Therapeutics. The other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

N 6 -methyladenosine (m 6 A) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of m 6 A methylation in mRNA metabolism has been well documented recently, regulation of the m 6 A machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the m 6 A deposition. We find that ERK phosphorylates METTL3 at S43/S50/S525 and WTAP at S306/S341, followed by deubiquitination by USP5, resulting in stabilization of the m 6 A methyltransferase complex. Lack of METTL3/WTAP phosphorylation reduces decay of m 6 A-labeled pluripotent factor transcripts and traps mouse embryonic stem cells in the pluripotent state. The same phosphorylation can also be found in ERK-activated human cancer cells and contribute to tumorigenesis. Our study reveals an unrecognized function of ERK in regulating m 6 A methylation., Competing Interests: Declaration of Interests C.H. is a scientific founder and member of the scientific advisory board of Accent Therapeutics, Inc., (Published by Elsevier Inc.)

Here, we show that tumor ADORA1 deletion suppresses cell growth in human melanoma cell lines in vitro and tumor development in vivo in immune-deficient xenografts. However, this deletion induces the upregulation of PD-L1 levels, which inactivates cocultured T cells in vitro, compromises anti-tumor immunity in vivo, and reduces anti-tumor efficacy in an immune-competent mouse model. Functionally, PD-1 mAb treatment enhances the efficacy of ADORA1-deficient or ADORA1 antagonist-treated melanoma and NSCLC immune-competent mouse models. Mechanistically, we identify ATF3 as the factor transcriptionally upregulating PD-L1 expression. Tumor ATF3 deletion improves the effect of ADORA1 antagonist treatment of melanoma and NSCLC xenografts. We observe higher ADORA1, lower ATF3, and lower PD-L1 expression levels in tumor tissues from nonresponders among PD-1 mAb-treated NSCLC patients., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The engagement of programmed cell death protein 1 (PD-1; encoded by the PDCD1 gene) receptor expressed on activated T cells and its ligand, programmed death-ligand 1 (PD-L1; encoded by the CD274 gene), is a major co-inhibitory checkpoint signaling that controls T cell activities. Various types of cancers express high levels of PD-L1 and exploit PD-L1/PD-1 signaling to evade T cell immunity. Blocking the PD-L1/PD-1 pathway has consistently shown remarkable anti-tumor effects in patients with advanced cancers and is recognized as the gold standard for developing new immune checkpoint blockade (ICB) and combination therapies. However, the response rates of anti-PD-L1 have been limited in several solid tumors. Therefore, furthering our understanding of the regulatory mechanisms of PD-L1 can bring substantial benefits to patients with cancer by improving the efficacy of current PD-L1/PD-1 blockade or other ICBs. In this review, we provide current knowledge of PD-L1 regulatory mechanisms at the transcriptional, posttranscriptional, post-translational, and extracellular levels, and discuss the implications of these findings in cancer diagnosis and immunotherapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Reactivation of T cell immunity by PD-1/PD-L1 immune checkpoint blockade has been shown to be a promising cancer therapeutic strategy. However, PD-L1 immunohistochemical readout is inconsistent with patient response, which presents a clinical challenge to stratify patients. Because PD-L1 is heavily glycosylated, we developed a method to resolve this by removing the glycan moieties from cell surface antigens via enzymatic digestion, a process termed sample deglycosylation. Notably, deglycosylation significantly improves anti-PD-L1 antibody binding affinity and signal intensity, resulting in more accurate PD-L1 quantification and prediction of clinical outcome. This proposed method of PD-L1 antigen retrieval may provide a practical and timely approach to reduce false-negative patient stratification for guiding anti-PD-1/PD-L1 therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multiple mechanisms of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been identified in EGFR-mutant non-small cell lung cancer (NSCLC); however, recurrent resistance to EGFR TKIs due to the heterogeneous mechanisms underlying resistance within a single patient remains a major challenge in the clinic. Here, we report a role of nuclear protein kinase Cδ (PKCδ) as a common axis across multiple known TKI-resistance mechanisms. Specifically, we demonstrate that TKI-inactivated EGFR dimerizes with other membrane receptors implicated in TKI resistance to promote PKCδ nuclear translocation. Moreover, the level of nuclear PKCδ is associated with TKI response in patients. The combined inhibition of PKCδ and EGFR induces marked regression of resistant NSCLC tumors with EGFR mutations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The adaptor protein CARD9 links detection of fungi by surface receptors to the activation of the NF-κB pathway. Mice deficient in CARD9 exhibit dysbiosis and are more susceptible to colitis. Here we examined the impact of Card9 deficiency in the development of colitis-associated colon cancer (CAC). Treatment of Card9 -/- mice with AOM-DSS resulted in increased tumor loads as compared to WT mice and in the accumulation of myeloid-derived suppressor cells (MDSCs) in tumor tissue. The impaired fungicidal functions of Card9 -/- macrophages led to increased fungal loads and variation in the overall composition of the intestinal mycobiota, with a notable increase in C. tropicalis. Bone marrow cells incubated with C. tropicalis exhibited MDSC features and suppressive functions. Fluconazole treatment suppressed CAC in Card9 -/- mice and was associated with decreased MDSC accumulation. The frequency of MDSCs in tumor tissues of colon cancer patients correlated positively with fungal burden, pointing to the relevance of this regulatory axis in human disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Although EZH2 enzymatic inhibitors have shown antitumor effects in EZH2-mutated lymphoma and ARID1A-mutated ovarian cancer, many cancers do not respond because EZH2 can promote cancer independently of its histone methyltransferase activity. Here we identify ZRANB1 as the EZH2 deubiquitinase. ZRANB1 binds, deubiquitinates, and stabilizes EZH2. Depletion of ZRANB1 in breast cancer cells results in EZH2 destabilization and growth inhibition. Systemic delivery of ZRANB1 small interfering RNA (siRNA) leads to marked antitumor and antimetastatic effects in preclinical models of triple-negative breast cancer (TNBC). Intriguingly, a small-molecule inhibitor of ZRANB1 destabilizes EZH2 and inhibits the viability of TNBC cells. In patients with breast cancer, ZRANB1 levels correlate with EZH2 levels and poor survival. These findings suggest the therapeutic potential for targeting the EZH2 deubiquitinase ZRANB1., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pancreatic ribonuclease (RNase) is a secreted enzyme critical for host defense. We discover an intrinsic RNase function, serving as a ligand for epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase (RTK), in pancreatic ductal adenocarcinoma (PDAC). The closely related bovine RNase A and human RNase 5 (angiogenin [ANG]) can trigger oncogenic transformation independently of their catalytic activities via direct association with EGFR. Notably, high plasma ANG level in PDAC patients is positively associated with response to EGFR inhibitor erlotinib treatment. These results identify a role of ANG as a serum biomarker that may be used to stratify patients for EGFR-targeted therapies, and offer insights into the ligand-receptor relationship between RNase and RTK families., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring β-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy., (Published by Elsevier Inc.)

Sustained energy starvation leads to activation of AMP-activated protein kinase (AMPK), which coordinates energy status with numerous cellular processes including metabolism, protein synthesis, and autophagy. Here, we report that AMPK phosphorylates the histone methyltransferase EZH2 at T311 to disrupt the interaction between EZH2 and SUZ12, another core component of the polycomb repressive complex 2 (PRC2), leading to attenuated PRC2-dependent methylation of histone H3 at Lys27. As such, PRC2 target genes, many of which are known tumor suppressors, were upregulated upon T311-EZH2 phosphorylation, which suppressed tumor cell growth both in cell culture and mouse xenografts. Pathologically, immunohistochemical analyses uncovered a positive correlation between AMPK activity and pT311-EZH2, and higher pT311-EZH2 correlates with better survival in both ovarian and breast cancer patients. Our finding suggests that AMPK agonists might be promising sensitizers for EZH2-targeting cancer therapies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Nuclear receptors recruit multiple coactivators sequentially to activate transcription. This "ordered" recruitment allows different coactivator activities to engage the nuclear receptor complex at different steps of transcription. Estrogen receptor (ER) recruits steroid receptor coactivator-3 (SRC-3) primary coactivator and secondary coactivators, p300/CBP and CARM1. CARM1 recruitment lags behind the binding of SRC-3 and p300 to ER. Combining cryo-electron microscopy (cryo-EM) structure analysis and biochemical approaches, we demonstrate that there is a close crosstalk between early- and late-recruited coactivators. The sequential recruitment of CARM1 not only adds a protein arginine methyltransferase activity to the ER-coactivator complex, it also alters the structural organization of the pre-existing ERE/ERα/SRC-3/p300 complex. It induces a p300 conformational change and significantly increases p300 HAT activity on histone H3K18 residues, which, in turn, promotes CARM1 methylation activity on H3R17 residues to enhance transcriptional activity. This study reveals a structural role for a coactivator sequential recruitment and biochemical process in ER-mediated transcription., (Copyright © 2017 Elsevier Inc. All rights reserved.)

UBE2O is localized in the 17q25 locus, which is known to be amplified in human cancers, but its role in tumorigenesis remains undefined. Here we show that Ube2o deletion in MMTV-PyVT or TRAMP mice profoundly impairs tumor initiation, growth, and metastasis, while switching off the metabolic reprogramming of tumor cells. Mechanistically, UBE2O specifically targets AMPKα2 for ubiquitination and degradation, and thereby promotes activation of the mTOR-HIF1α pathway. Notably, inactivation of AMPKα2, but not AMPKα1, abrogates the tumor attenuation caused by UBE2O loss, while treatment with rapamycin or inhibition of HIF1α ablates UBE2O-dependent tumor biology. Finally, pharmacological blockade of UBE2O inhibits tumorigenesis through the restoration of AMPKα2, suggesting the UBE2O-AMPKα2 axis as a potential cancer therapeutic target., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pro-inflammatory cytokines produced in the tumor microenvironment lead to eradication of anti-tumor immunity and enhanced tumor cell survival. In the current study, we identified tumor necrosis factor alpha (TNF-α) as a major factor triggering cancer cell immunosuppression against T cell surveillance via stabilization of programmed cell death-ligand 1 (PD-L1). We demonstrated that COP9 signalosome 5 (CSN5), induced by NF-κB p65, is required for TNF-α-mediated PD-L1 stabilization in cancer cells. CSN5 inhibits the ubiquitination and degradation of PD-L1. Inhibition of CSN5 by curcumin diminished cancer cell PD-L1 expression and sensitized cancer cells to anti-CTLA4 therapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using single-particle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

MicroRNAs (miRNA) are mostly downregulated in cancer. However, the mechanism underlying this phenomenon and the precise consequence in tumorigenesis remain obscure. Here we show that ERK suppresses pre-miRNA export from the nucleus through phosphorylation of exportin-5 (XPO5) at T345/S416/S497. After phosphorylation by ERK, conformation of XPO5 is altered by prolyl isomerase Pin1, resulting in reduction of pre-miRNA loading. In liver cancer, the ERK-mediated XPO5 suppression reduces miR-122, increases microtubule dynamics, and results in tumor development and drug resistance. Analysis of clinical specimens further showed that XPO5 phosphorylation is associated with poor prognosis for liver cancer patients. Our study reveals a function of ERK in miRNA biogenesis and suggests that modulation of miRNA export has potential clinical implications., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Receptor tyrosine kinase (RTK) recycling is of critical importance for RTK signaling and cancer, yet the process is poorly understood. In this issue, Ye et al. identify GOLM1 as a cargo adaptor that drives hepatocellular carcinoma metastasis by promoting EGFR recycling and provide insights into how this process is regulated., (Copyright © 2016. Published by Elsevier Inc.)

While recombinant human erythropoietin (rhEpo) has been widely used to treat anemia in cancer patients, concerns about its adverse effects on patient survival have emerged. A lack of correlation between expression of the canonical EpoR and rhEpo's effects on cancer cells prompted us to consider the existence of an alternative Epo receptor. Here, we identified EphB4 as an Epo receptor that triggers downstream signaling via STAT3 and promotes rhEpo-induced tumor growth and progression. In human ovarian and breast cancer samples, expression of EphB4 rather than the canonical EpoR correlated with decreased disease-specific survival in rhEpo-treated patients. These results identify EphB4 as a critical mediator of erythropoietin-induced tumor progression and further provide clinically significant dimension to the biology of erythropoietin., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The mesoderm- and epithelial-mesenchymal transition-associated transcription factor FOXC1 is specifically overexpressed in basal-like breast cancer (BLBC), but its biochemical function is not understood. Here, we demonstrate that FOXC1 controls cancer stem cell (CSC) properties enriched in BLBC cells via activation of Smoothened (SMO)-independent Hedgehog (Hh) signaling. This non-canonical activation of Hh is specifically mediated by Gli2. Furthermore, we show that the N-terminal domain of FOXC1 (aa 1-68) binds directly to an internal region (aa 898-1168) of Gli2, enhancing the DNA-binding and transcription-activating capacity of Gli2. FOXC1 expression correlates with that of Gli2 and its targets in human breast cancers. Moreover, FOXC1 overexpression reduces sensitivity to anti-Hedgehog (Hh) inhibitors in BLBC cells and xenograft tumors. Together, these findings reveal FOXC1-mediated non-canonical Hh signaling that determines the BLBC stem-like phenotype and anti-Hh sensitivity, supporting inhibition of FOXC1 pathways as potential approaches for improving BLBC treatment., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

In vitro modeling of human disease has recently become feasible with induced pluripotent stem cell (iPSC) technology. Here, we established patient-derived iPSCs from a Li-Fraumeni syndrome (LFS) family and investigated the role of mutant p53 in the development of osteosarcoma (OS). LFS iPSC-derived osteoblasts (OBs) recapitulated OS features including defective osteoblastic differentiation as well as tumorigenic ability. Systematic analyses revealed that the expression of genes enriched in LFS-derived OBs strongly correlated with decreased time to tumor recurrence and poor patient survival. Furthermore, LFS OBs exhibited impaired upregulation of the imprinted gene H19 during osteogenesis. Restoration of H19 expression in LFS OBs facilitated osteoblastic differentiation and repressed tumorigenic potential. By integrating human imprinted gene network (IGN) into functional genomic analyses, we found that H19 mediates suppression of LFS-associated OS through the IGN component DECORIN (DCN). In summary, these findings demonstrate the feasibility of studying inherited human cancer syndromes with iPSCs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Transforming growth factor β (TGF-β) functions as a tumor suppressor in premalignant cells but as a metastasis promoter in cancer cells. The dichotomous functions of TGF-β are proposed to be dictated by different partners of its downstream effector Smads. However, the mechanism for the contextual changes of Smad partners remained undefined. Here, we demonstrate that 14-3-3ζ destabilizes p53, a Smad partner in premalignant mammary epithelial cells, by downregulating 14-3-3σ, thus turning off TGF-β's tumor suppression function. Conversely, 14-3-3ζ stabilizes Gli2 in breast cancer cells, and Gli2 partners with Smads to activate PTHrP and promote TGF-β-induced bone metastasis. The 14-3-3ζ-driven contextual changes of Smad partners from p53 to Gli2 may serve as biomarkers and therapeutic targets of TGF-β-mediated cancer progression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

lncRNAs are known to regulate a number of different developmental and tumorigenic processes. Here, we report a role for lncRNA BCAR4 in breast cancer metastasis that is mediated by chemokine-induced binding of BCAR4 to two transcription factors with extended regulatory consequences. BCAR4 binding of SNIP1 and PNUTS in response to CCL21 releases the SNIP1's inhibition of p300-dependent histone acetylation, which in turn enables the BCAR4-recruited PNUTS to bind H3K18ac and relieve inhibition of RNA Pol II via activation of the PP1 phosphatase. This mechanism activates a noncanonical Hedgehog/GLI2 transcriptional program that promotes cell migration. BCAR4 expression correlates with advanced breast cancers, and therapeutic delivery of locked nucleic acids (LNAs) targeting BCAR4 strongly suppresses breast cancer metastasis in mouse models. The findings reveal a disease-relevant lncRNA mechanism consisting of both direct coordinated protein recruitment and indirect regulation of transcription factors.

Posttranslational modifications of histones play fundamental roles in many biological functions. Specifically, histone H4-K20 methylation is critical for DNA synthesis and repair. However, little is known about how these functions are regulated by the upstream stimuli. Here, we identify a tyrosine phosphorylation site at Y72 of histone H4, which facilitates recruitment of histone methyltransferases (HMTases), SET8 and SUV4-20H, to enhance its K20 methylation, thereby promoting DNA synthesis and repair. Phosphorylation-defective histone H4 mutant is deficient in K20 methylation, leading to reduced DNA synthesis, delayed cell cycle progression, and decreased DNA repair ability. Disrupting the interaction between epidermal growth factor receptor (EGFR) and histone H4 by Y72 peptide significantly reduced tumor growth. Furthermore, EGFR expression clinically correlates with histone H4-Y72 phosphorylation, H4-K20 monomethylation, and the Ki-67 proliferation marker. These findings uncover a mechanism by which EGFR transduces signal to chromatin to regulate DNA synthesis and repair., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Many mutant p53 proteins (mutp53s) exert oncogenic gain-of-function (GOF) properties, but the mechanisms mediating these functions remain poorly defined. We show here that GOF mutp53s inhibit AMP-activated protein kinase (AMPK) signaling in head and neck cancer cells. Conversely, downregulation of GOF mutp53s enhances AMPK activation under energy stress, decreasing the activity of the anabolic factors acetyl-CoA carboxylase and ribosomal protein S6 and inhibiting aerobic glycolytic potential and invasive cell growth. Under conditions of energy stress, GOF mutp53s, but not wild-type p53, preferentially bind to the AMPKα subunit and inhibit AMPK activation. Given the importance of AMPK as an energy sensor and tumor suppressor that inhibits anabolic metabolism, our findings reveal that direct inhibition of AMPK activation is an important mechanism through which mutp53s can gain oncogenic function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Epigenetic regulation plays a critical role in glioblastoma (GBM) tumorigenesis. However, how microRNAs (miRNAs) and cytokines cooperate to regulate GBM tumor progression is still unclear. Here, we show that interleukin-6 (IL-6) inhibits miR142-3p expression and promotes GBM propagation by inducing DNA methyltransferase 1-mediated hypermethylation of the miR142-3p promoter. Interestingly, miR142-3p also suppresses IL-6 secretion by targeting the 3' UTR of IL-6. In addition, miR142-3p also targets the 3' UTR and suppresses the expression of high-mobility group AT-hook 2 (HMGA2), leading to inhibition of Sox2-related stemness. We further show that HMGA2 enhances Sox2 expression by directly binding to the Sox2 promoter. Clinically, GBM patients whose tumors present upregulated IL-6, HMGA2, and Sox2 protein expressions and hypermethylated miR142-3p promoter also demonstrate poor survival outcome. Orthotopic delivery of miR142-3p blocks IL-6/HMGA2/Sox2 expression and suppresses stem-like properties in GBM-xenotransplanted mice. Collectively, we discovered an IL-6/miR142-3p feedback-loop-dependent regulation of GBM malignancy that could be a potential therapeutic target., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Oncogenic activation of Ras proteins due to missense mutations is frequently detected in human cancers but rarely in breast cancer. In this issue of Cancer Cell, McLaughlin and colleagues report that ablation of the GasGAP gene, RASAL2, is an alternative mechanism by which Ras becomes activated in breast cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Skp2 E3 ligase is overexpressed in numerous human cancers and plays a critical role in cell-cycle progression, senescence, metabolism, cancer progression, and metastasis. In the present study, we identified a specific Skp2 inhibitor using high-throughput in silico screening of large and diverse chemical libraries. This Skp2 inhibitor selectively suppresses Skp2 E3 ligase activity, but not activity of other SCF complexes. It also phenocopies the effects observed upon genetic Skp2 deficiency, such as suppressing survival and Akt-mediated glycolysis and triggering p53-independent cellular senescence. Strikingly, we discovered a critical function of Skp2 in positively regulating cancer stem cell populations and self-renewal ability through genetic and pharmacological approaches. Notably, Skp2 inhibitor exhibits potent antitumor activities in multiple animal models and cooperates with chemotherapeutic agents to reduce cancer cell survival. Our study thus provides pharmacological evidence that Skp2 is a promising target for restricting cancer stem cell and cancer progression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Epidermal growth factor receptor (EGFR) initiates a signaling cascade that leads to DNA synthesis and cell proliferation, but its role in regulating DNA replication licensing is unclear. Here, we show that activated EGFR phosphorylates the p56 isoform of Lyn, p56(Lyn), at Y32, which then phosphorylates MCM7, a licensing factor critical for DNA replication, at Y600 to increase its association with other minichromosome maintenance complex proteins, thereby promoting DNA synthesis complex assembly and cell proliferation. Both p56(Lyn) Y32 and MCM7 Y600 phosphorylation are enhanced in proliferating cells and correlated with poor survival of breast cancer patients. These results establish a signaling cascade in which EGFR enhances MCM7 phosphorylation and DNA replication through Lyn phosphorylation in human cancer cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The epithelial-mesenchymal transition (EMT) enhances cancer invasiveness and confers tumor cells with cancer stem cell (CSC)-like characteristics. We show that the Snail-G9a-Dnmt1 complex, which is critical for E-cadherin promoter silencing, is also required for the promoter methylation of fructose-1,6-biphosphatase (FBP1) in basal-like breast cancer (BLBC). Loss of FBP1 induces glycolysis and results in increased glucose uptake, macromolecule biosynthesis, formation of tetrameric PKM2, and maintenance of ATP production under hypoxia. Loss of FBP1 also inhibits oxygen consumption and reactive oxygen species production by suppressing mitochondrial complex I activity; this metabolic reprogramming results in an increased CSC-like property and tumorigenicity by enhancing the interaction of β-catenin with T-cell factor. Our study indicates that the loss of FBP1 is a critical oncogenic event in EMT and BLBC., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Recurrence and metastasis result in a poor prognosis for breast cancer patients. Recent studies have demonstrated that microRNAs (miRNAs) play vital roles in the development and metastasis of breast cancer. In this study, we investigated the therapeutic potential of miR-34a in breast cancer. We found that miR-34a is downregulated in breast cancer cell lines and tissues, compared with normal cell lines and the adjacent nontumor tissues, respectively. To explore the therapeutic potential of miR-34a, we designed a targeted miR-34a expression plasmid (T-VISA-miR-34a) using the T-VISA system, and evaluated its antitumor effects, efficacy, mechanism of action, and systemic toxicity. T-VISA-miR-34a induced robust, persistent expression of miR-34a, and dramatically suppressed breast cancer cell growth, migration, and invasion in vitro by downregulating the protein expression levels of the miR-34a target genes E2F3, CD44, and SIRT1. In an orthotopic mouse model of breast cancer, intravenous injection of T-VISA-miR-34a:liposomal complex nanoparticles significantly inhibited tumor growth, prolonged survival, and did not induce systemic toxicity. In conclusion, T-VISA-miR-34a lead to robust, specific overexpression of miR-34a in breast cancer cells and induced potent antitumor effects in vitro and in vivo. T-VISA-miR-34a may provide a potentially useful, specific, and safe-targeted therapeutic approach for breast cancer.

Many signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Akt kinase plays a central role in cell growth, metabolism, and tumorigenesis. The TRAF6 E3 ligase orchestrates IGF-1-mediated Akt ubiquitination and activation. Here, we show that Akt ubiquitination is also induced by activation of ErbB receptors; unexpectedly, and in contrast to IGF-1 induced activation, the Skp2 SCF complex, not TRAF6, is a critical E3 ligase for ErbB-receptor-mediated Akt ubiquitination and membrane recruitment in response to EGF. Skp2 deficiency impairs Akt activation, Glut1 expression, glucose uptake and glycolysis, and breast cancer progression in various tumor models. Moreover, Skp2 overexpression correlates with Akt activation and breast cancer metastasis and serves as a marker for poor prognosis in Her2-positive patients. Finally, Skp2 silencing sensitizes Her2-overexpressing tumors to Herceptin treatment. Our study suggests that distinct E3 ligases are utilized by diverse growth factors for Akt activation and that targeting glycolysis sensitizes Her2-positive tumors to Herceptin treatment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Esophageal adenocarcinoma (EAC) is the most prevalent esophageal cancer type in the United States. The TNF-α/mTOR pathway is known to mediate the development of EAC. Additionally, aberrant activation of Gli1, downstream effector of the Hedgehog (HH) pathway, has been observed in EAC. In this study, we found that an activated mTOR/S6K1 pathway promotes Gli1 transcriptional activity and oncogenic function through S6K1-mediated Gli1 phosphorylation at Ser84, which releases Gli1 from its endogenous inhibitor, SuFu. Moreover, elimination of S6K1 activation by an mTOR pathway inhibitor enhances the killing effects of the HH pathway inhibitor. Together, our results established a crosstalk between the mTOR/S6K1 and HH pathways, which provides a mechanism for SMO-independent Gli1 activation and also a rationale for combination therapy for EAC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Proinflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Constitutive Kras and NF-κB activation is identified as signature alterations in pancreatic ductal adenocarcinoma (PDAC). However, how NF-κB is activated in PDAC is not yet understood. Here, we report that pancreas-targeted IKK2/β inactivation inhibited NF-κB activation and PDAC development in Kras(G12D) and Kras(G12D);Ink4a/Arf(F/F) mice, demonstrating a mechanistic link between IKK2/β and Kras(G12D) in PDAC inception. Our findings reveal that Kras(G12D)-activated AP-1 induces IL-1α, which, in turn, activates NF-κB and its target genes IL-1α and p62, to initiate IL-1α/p62 feedforward loops for inducing and sustaining NF-κB activity. Furthermore, IL-1α overexpression correlates with Kras mutation, NF-κB activity, and poor survival in PDAC patients. Therefore, our findings demonstrate the mechanism by which IKK2/β/NF-κB is activated by Kras(G12D) through dual feedforward loops of IL-1α/p62., (Copyright © 2012 Elsevier Inc. All rights reserved.)