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Abstract Background Bladder cancer (BLCA) is a common malignancy characterized by dysregulated transcription and a lack of effective therapeutic targets. In this study, we aimed to identify and evaluate novel targets with clinical potential essential for tumor growth in BLCA. Methods CRISPR-Cas9 screening was used to identify transcription factors essential for bladder cancer cell viability. The biological functions of KLF16 in bladder cancer were investigated both in vitro and in vivo. The regulatory mechanism between KLF16 and MYC was elucidated through a series of analyses, including RNA sequencing, quantitative polymerase chain reaction (qPCR), RNA immunoprecipitation, Western blotting, Mass spectrometry, Dual-luciferase reporter assays, Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing, OptoDroplets assays, and RNA stability assay. The clinical relevance of KLF16 and MYC in bladder cancer was evaluated through analyses of public databases and immunohistochemistry. Results Krüppel-like factor 16 (KLF16) was essential for BLCA cell viability. Elevated expression of KLF16 was observed in bladder cancer tissues, and higher expression levels of KLF16 were correlated with poor progression-free survival (PFS) and cancer-specific survival (CSS) probabilities in BLCA patients. Mechanistically, KLF16 mRNA competed with the mRNA of dual-specificity phosphatase 16 (DUSP16) for binding to the RNA-binding protein, WW domain binding protein 11 (WBP11), resulting in destabilization of the DUSP16 mRNA. This, in turn, led to activation of ERK1/2, which stabilized the MYC protein. Furthermore, KLF16 interacted with MYC to form nuclear condensates, thereby enhancing MYC’s transcriptional activity. Additionally, MYC transcriptionally upregulated KLF16, creating a positive feedback loop between KLF16 and MYC that amplified their oncogenic functions. Targeting this loop with bromodomain inhibitors, such as OTX015 and ABBV-744, suppressed the transcription of both KLF16 and MYC, resulting in reduced BLCA cell viability and tumor growth, as well as increased sensitivity to chemotherapy. Conclusions Our study revealed the crucial role of the KLF16/MYC regulatory axis in modulating tumor growth and chemotherapy sensitivity in BLCA, suggesting that combining bromodomain inhibitors, such as OTX015 or ABBV-744, with DDP or gemcitabine could be a promising therapeutic intervention for BLCA patients.

Abstract Microvesicles (MVs) containing proteins, nucleic acid or organelles are shed from the plasma membrane. Although the mechanisms of MV budding are well elucidated, the connection between endosomal trafficking and MV formation remains poorly understood. In this report, RAB22A is revealed to be crucial for EGFR‐containing MVs formation by the RAB GTPase family screening. RAB22A recruits TBC1D2B, a GTPase‐activating protein (GAP) of RAB7A, to inactivate RAB7A, thus preventing EGFR from being transported to late endosomes and lysosomes. RAB22A also engages SH3BP5L, a guanine‐nucleotide exchange factor (GEF) of RAB11A, to activate RAB11A on early endosomes. Consequently, EGFR is recycled to the cell surface and packaged into MVs. Furthermore, EGFR can phosphorylate RAB22A at Tyr136, which in turn promotes EGFR‐containing MVs formation. Our findings illustrate that RAB22A acts as a sorter on early endosomes to sort EGFR to recycling endosomes for MV shedding by both activating RAB11A and inactivating RAB7A.

Abstract Bladder cancer (BC) is one of the most common tumors characterized by a high rate of relapse and a lack of targeted therapy. Here, YEATS domain‐containing protein 4 (YEATS4) is an essential gene for BC cell viability using CRISPR‐Cas9 library screening is reported, and that HUWE1 is an E3 ligase responsible for YEATS4 ubiquitination and proteasomal degradation by the Protein Stability Regulators Screening Assay. KAT8‐mediated acetylation of YEATS4 impaired its interaction with HUWE1 and consequently prevented its ubiquitination and degradation. The protein levels of YEATS4 and KAT8 are positively correlated and high levels of these two proteins are associated with poor overall survival in BC patients. Importantly, suppression of YEATS4 acetylation with the KAT8 inhibitor MG149 decreased YEATS4 acetylation, reduced cell viability, and sensitized BC cells to cisplatin treatment. The findings reveal a critical role of the KAT8/YEATS4 axis in both tumor growth and cisplatin sensitivity in BC cells, potentially generating a novel therapeutic strategy for BC patients.

Abstract Background Clear cell renal cell carcinoma (ccRCC) is the most lethal renal cancer. An overwhelming increase of patients experience tumor progression and unfavorable prognosis. However, the molecular events underlying ccRCC tumorigenesis and metastasis remain unclear. Therefore, uncovering the underlying mechanisms will pave the way for developing novel therapeutic targets for ccRCC. In this study, we sought to investigate the role of mitofusin‐2 (MFN2) in supressing ccRCC tumorigenesis and metastasis. Methods The expression pattern and clinical significance of MFN2 in ccRCC were analyzed by using the Cancer Genome Atlas datasets and samples from our independent ccRCC cohort. Both in vitro and in vivo experiments, including cell proliferation, xenograft mouse models and transgenic mouse model, were used to determine the role of MFN2 in regulating the malignant behaviors of ccRCC. RNA‐sequencing, mass spectrum analysis, co‐immunoprecipitation, bio‐layer interferometry and immunofluorescence were employed to elucidate the molecular mechanisms for the tumor‐supressing role of MFN2. Results we reported a tumor‐suppressing pathway in ccRCC, characterized by mitochondria‐dependent inactivation of epidermal growth factor receptor (EGFR) signaling. This process was mediated by the outer mitochondrial membrane (OMM) protein MFN2. MFN2 was down‐regulated in ccRCC and associated with favorable prognosis of ccRCC patients. in vivo and in vitro assays demonstrated that MFN2 inhibited ccRCC tumor growth and metastasis by suppressing the EGFR signaling pathway. In a kidney‐specific knockout mouse model, loss of MFN2 led to EGFR pathway activation and malignant lesions in kidney. Mechanistically, MFN2 preferably binded small GTPase Rab21 in its GTP‐loading form, which was colocalized with endocytosed EGFR in ccRCC cells. Through this EGFR‐Rab21‐MFN2 interaction, endocytosed EGFR was docked to mitochondria and subsequently dephosphorylated by the OMM‐residing tyrosine‐protein phosphatase receptor type J (PTPRJ). Conclusions Our findings uncover an important non‐canonical mitochondria‐dependent pathway regulating EGFR signaling by the Rab21‐MFN2‐PTPRJ axis, which contributes to the development of novel therapeutic strategies for ccRCC.

Abstract Immune checkpoint inhibitors (ICIs) show promise as second‐line treatment for advanced bladder cancer (BLCA); however, their responsiveness is limited by the immune evasion mechanisms in tumor cells. This study conduct a Cox regression analysis to screen mRNA‐binding proteins and reveals an association between Ras GTPase‐activating protein‐binding protein 1 (G3BP1) and diminished effectiveness of ICI therapy in patients with advanced BLCA. Subsequent investigation demonstrates that G3BP1 enhances immune evasion in BLCA cells by downregulating major histocompatibility complex class I (MHC‐I) through phosphoinositide 3‐kinase (PI3K)/Akt signaling activation. Mechanistically, G3BP1 interacts with splicing factor synergistic lethal with U5 snRNA 7 (SLU7) to form a complex with poly(A)‐binding protein cytoplasmic 1 and eukaryotic translation initiation factor 4 gamma 1. This complex stabilizes the closed‐loop structure of the mRNAs of class IA PI3Ks and consequently facilitates their translation and stabilization, thereby activating PI3K/Akt signaling to downregulate MHC‐I. Consistently, targeting G3BP1 with epigallocatechin gallate (EGCG) impedes immune evasion and sensitizes BLCA cells to anti‐programmed cell death (PD)‐1 antibodies in mice. Thus, G3BP1 and SLU7 collaboratively contribute to immune evasion in BLCA, indicating that EGCG is a precision therapeutic agent to enhance the effectiveness of anti‐PD‐1 therapy.

Abstract Background Natural killer/T-cell lymphoma (NKTL) is a rare type of aggressive and heterogeneous non-Hodgkin's lymphoma (NHL) with a poor prognosis and limited therapeutic options. Therefore, there is an urgent need to exploit potential novel therapeutic targets for the treatment of NKTL. Histone deacetylase (HDAC) inhibitor chidamide was recently approved for treating relapsed/refractory peripheral T-cell lymphoma (PTCL) patients. However, its therapeutic efficacy in NKTL remains unclear. Methods We performed a phase II clinical trial to evaluate the efficacy of chidamide in 28 relapsed/refractory NKTL patients. Integrative transcriptomic, chromatin profiling analysis and functional studies were performed to identify potential predictive biomarkers and unravel the mechanisms of resistance to chidamide. Immunohistochemistry (IHC) was used to validate the predictive biomarkers in tumors from the clinical trial. Results We demonstrated that chidamide is effective in treating relapsed/refractory NKTL patients, achieving an overall response and complete response rate of 39 and 18%, respectively. In vitro studies showed that hyperactivity of JAK-STAT signaling in NKTL cell lines was associated with the resistance to chidamide. Mechanistically, our results revealed that aberrant JAK-STAT signaling remodels the chromatin and confers resistance to chidamide. Subsequently, inhibition of JAK-STAT activity could overcome resistance to chidamide by reprogramming the chromatin from a resistant to sensitive state, leading to synergistic anti-tumor effect in vitro and in vivo. More importantly, our clinical data demonstrated that combinatorial therapy with chidamide and JAK inhibitor ruxolitinib is effective against chidamide-resistant NKTL. In addition, we identified TNFRSF8 (CD30), a downstream target of the JAK-STAT pathway, as a potential biomarker that could predict NKTL sensitivity to chidamide. Conclusions Our study suggests that chidamide, in combination with JAK-STAT inhibitors, can be a novel targeted therapy in the standard of care for NKTL. Trial registration: ClinicalTrials.gov, NCT02878278. Registered 25 August 2016, https://clinicaltrials.gov/ct2/show/NCT02878278

Summary: Dysregulation of transcription is a hallmark of cancer, including bladder cancer (BLCA). CRISPR-Cas9 screening using a lentivirus library with single guide RNAs (sgRNAs) targeting human transcription factors and chromatin modifiers is used to reveal genes critical for the proliferation and survival of BLCA cells. As a result, the nuclear transcription factor Y subunit gamma (NFYC)-37, but not NFYC-50, is observed to promote cell proliferation and tumor growth in BLCA. Mechanistically, NFYC-37 interacts with CBP and SREBP2 to activate mevalonate pathway transcription, promoting cholesterol biosynthesis. However, NFYC-50 recruits more of the arginine methyltransferase CARM1 than NFYC-37 to methylate CBP, which prevents the CBP-SREBP2 interaction and subsequently inhibits the mevalonate pathway. Importantly, statins targeting the mevalonate pathway can suppress NFYC-37-induced cell proliferation and tumor growth, indicating the need for conducting a clinical trial with statins for treating patients with BLCA and high NFYC-37 levels, as most patients with BLCA have high NFYC-37 levels.

Abstract The combination of immune checkpoint blockade (ICB) with chemotherapy significantly improves clinical benefit of cancer treatment. Since chemotherapy is often associated with adverse events, concomitant treatment with drugs managing side effects of chemotherapy is frequently used in the combination therapy. However, whether these ancillary drugs could impede immunotherapy remains unknown. Here, we showed that ∆9-tetrahydrocannabinol (THC), the key ingredient of drugs approved for the treatment of chemotherapy-caused nausea, reduced the therapeutic effect of PD-1 blockade. The endogenous cannabinoid anandamide (AEA) also impeded antitumor immunity, indicating an immunosuppressive role of the endogenous cannabinoid system (ECS). Consistently, high levels of AEA in the sera were associated with poor overall survival in cancer patients. We further found that cannabinoids impaired the function of tumor-specific T cells through CNR2. Using a knock-in mouse model expressing a FLAG-tagged Cnr2 gene, we discovered that CNR2 binds to JAK1 and inhibits the downstream STAT signaling in T cells. Taken together, our results unveiled a novel mechanism of the ECS-mediated suppression on T-cell immunity against cancer, and suggest that cannabis and cannabinoid drugs should be avoided during immunotherapy.

Prevalent copy number alteration is the most prominent genetic characteristic associated with ovarian cancer (OV) development, but its role in immune evasion has not been fully elucidated. In this study, we identified RAD21, a key component of the cohesin complex, as a frequently amplified oncogene that could modulate immune response in OV. Through interrogating the RAD21-regulated transcriptional program, we found that RAD21 directly interacts with YAP/TEAD4 transcriptional corepressors and recruits the NuRD complex to suppress interferon (IFN) signaling. In multiple clinical cohorts, RAD21 overexpression is inversely correlated with IFN signature gene expression in OV. We further demonstrated in murine syngeneic tumor models that RAD21 ablation potentiated anti–PD-1 efficacy with increased intratumoral CD8+ T cell effector activity. Our study identifies a RAD21–YAP/TEAD4–NuRD corepressor complex in immune modulation, and thus provides a potential target and biomarker for precision immunotherapy in OV.

Abstract It remains unknown for decades how some of the therapeutic fusion proteins positive in a small percentage of cancer cells account for patient outcome. Here, we report that osteosarcoma Rab22a-NeoF1 fusion protein, together with its binding partner PYK2, is sorted into exosomes by HSP90 via its KFERQ-like motif (RVLFLN142). The exosomal Rab22a-NeoF1 fusion protein facilitates the pulmonary pre-metastatic niche formation by recruiting bone marrow-derived macrophages. The exosomal PYK2 activates RhoA in its negative recipient osteosarcoma cells and induces signal transducer and activator of transcription 3 activation in its recipient macrophages to increase M2 phenotype. Consequently, lung metastases of its recipient osteosarcoma cells are promoted by this exosomal Rab22a-NeoF1 fusion protein, and this event can be targeted by disrupting its interaction with PYK2 using a designed internalizing RGD peptide.

Abstract Background Overexpression of Aurora‐A (AURKA) is a feature of breast cancer and associates with adverse prognosis. The selective Aurora‐A inhibitor alisertib (MLN8237) has recently demonstrated promising antitumor responses as a single agent in various cancer types but its phase III clinical trial was reported as a failure since MLN8237 did not show an apparent effect in prolonging the survival of patients. Thus, identification of potential targets that could enhance the activity of MLN8237 would provide a rationale for drug combination to achieve better therapeutic outcome. Methods Here, we conducted a systematic synthetic lethality CRISPR/Cas9 screening of 507 kinases using MLN8237 in breast cancer cells and identified a number of targetable kinases that displayed synthetic lethality interactions with MLN8237. Then, we performed competitive growth assays, colony formation assays, cell viability assays, apoptosis assays, and xenograft murine model to evaluate the synergistic therapeutic effects of Haspin (GSG2) depletion or inhibition with MLN8237. For mechanistic studies, immunofluorescence was used to detect the state of microtubules and the localization of Aurora‐B and mitotic centromere‐associated kinesin (MCAK). Results Among the hits, we observed that Haspin depletion or inhibition marginally inhibited breast cancer cell growth but could substantially enhance the killing effects of MLN8237. Mechanistic studies showed that co‐treatment with Aurora‐A and Haspin inhibitors abolished the recruitment of Aurora‐B and mitotic centromere‐associated kinesin (MCAK) to centromeres which were associated with excessive microtubule depolymerization, kinetochore‐microtubule (KT‐MT) attachment failure, and severe mitotic catastrophe. We further showed that the combination of MLN8237 and the Haspin inhibitor CHR‐6494 synergistically reduced breast cancer cell viability and significantly inhibited both in vitro and in vivo tumor growth. Conclusions These findings establish Haspin as a synthetic lethal target and demonstrate CHR‐6494 as a potential combinational drug for promoting the therapeutic effects of MLN8237 on breast cancer.

Macrophages constitute a major immune component in tumor tissues, but how these cells adapt to and survive in the nutrient-depleted and lactic acid–induced acidic tumor microenvironments is not yet fully understood. Here, we found that levels of carbonic anhydrase XII (CA12) expression were significantly and selectively upregulated on macrophages in human hepatocellular carcinoma (HCC). Transient glycolytic activation of peritumoral monocytes induced sustained expression of CA12 on tumor-infiltrating macrophages via autocrine cytokines and HIF1α pathways. On the one hand, CA12 mediated the survival of macrophages in relatively acidic tumor microenvironments, while on the other hand, it induced macrophage production of large amounts of C-C motif chemokine ligand 8 (CCL8), which enhanced cancer cell epithelial-mesenchymal transition (EMT) and facilitated tumor metastasis. Consistently, the accumulation of CA12+ macrophages in tumor tissues was associated with increased tumor metastatic potential and reduced survival of patients with HCC. Selective targeting of tumor-infiltrating macrophages with a CA12 inhibitor reduced tumor growth in mice and was sufficient to synergistically enhance the therapeutic efficacy of immune-checkpoint blockade. We suggest that CA12 activity is a previously unappreciated mechanism regulating the accumulation and functions of macrophages in tumor microenvironments and therefore represents a selective vulnerability that could be exploited in future designs for antitumor immunotherapeutic strategies.

Objective: Osteosarcoma is the most common primary malignant bone tumor. However, the survival of patients with osteosarcoma has remained unchanged during the past 30 years, owing to a lack of efficient therapeutic targets. Methods: We constructed a kinome-targeting CRISPR-Cas9 library containing 507 kinases and 100 nontargeting controls and screened the potential kinase targets in osteosarcoma. The CRISPR screening sequencing data were analyzed with the Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) Python package. The functional data were applied in the 143B cell line through lenti-CRISPR-mediated gene knockout. The clinical significance of kinases in the survival of patients with osteosarcoma was analyzed in the R2: Genomics Analysis and Visualization Platform. Results: We identified 53 potential kinase targets in osteosarcoma. Among these targets, we analyzed 3 kinases, TRRAP, PKMYT1, and TP53RK, to validate their oncogenic functions in osteosarcoma. PKMYT1 and TP53RK showed higher expression in osteosarcoma than in normal bone tissue, whereas TRRAP showed no significant difference. High expression of all 3 kinases was associated with relatively poor prognosis in patients with osteosarcoma. Conclusions: Our results not only offer potential therapeutic kinase targets in osteosarcoma but also provide a paradigm for functional genetic screening by using a CRISPR-Cas9 library, including target design, library construction, screening workflow, data analysis, and functional validation. This method may also be useful in potentially accelerating drug discovery for other cancer types.

PD-L1 is highly expressed in triple-negative breast cancers (TNBC). Here, the authors show that nucleophosmin 1 (NPM1) transcriptionally activates PD-L1 expression and inhibits T cell activity in TNBC.

Osteosarcoma is an aggressive tumour and little is known the mechanisms underpinning its highly metastatic nature. Here, the authors highlight a role for the CK1α/CBX4 axis in driving metastasis, suggesting that this pathway might be targeted for therapeutic benefit.

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s13046-021-02204-1

Abstract Background MNAT1 (menage a trois 1, MAT1), a cyclin-dependent kinase-activating kinase (CAK) complex, high expresses in various cancers and is involved in cancer pathogenesis. However, mechanisms underlying its regulation in carcinogenesis are unclear. Methods The tissue microarray of colorectal cancer (CRC) was used to evaluate MNAT1 expressions in CRC tissues using immunohistochemistry, CRC cell lines were also detected MNAT1 expression using Western-blotting. MNAT1 and shMNAT1 vectors were constructed, and transfected into CRC cells. Cell growths of the transfected cells were observed using MTT and colony formation. The affects of MNAT1 on p53 expression were analyzed using Western-blotting and Real-time PCR. Immunoprecipitation assay was used to analyze the interaction p53 and MNAT1, and Western-blotting was used to test the effects of MNAT1 on p53 downstream molecules. The apoptosis of CRC cells with MNAT1 or shMNAT1 were analyzed using flow cytometry. BABL/c athymic nude mice were used to observe the effect of MNAT1 on CRC cell growth in vivo. Results MNAT1 was found to be overexpressed in CRC tissues and cells, and MNAT1 expressions in CRC tissue samples were associated with CRC carcinogenesis and poor patient outcomes. MNAT1-knockin increased CRC cell growth and colony formation, and MNAT1-knockdown dramatically decreased cell motility and invasion. MNAT1 physically interacted with p53, MNAT1 also increased the interaction of MDM2 with p53. Strikingly, MNAT1 mediated p53 ubiquitin-degradation. MNAT1 shortened p53 half-life, and ectopic MNAT1 expression decreased p53 protein stability. Moreover, MNAT1 induced RAD51 and reduced p21, cleaved-caspase3, cleaved-PARP and BAX expression. MNAT1 inhibited CRC cell apoptosis. shMANT1 decreased tumor growths in nude mice following p53 increase. Conclusion MNAT1 binds to p53, mediates p53 ubiquitin-degradation through MDM2, increases cell growth and decreases cell apoptosis, and finally promotes CRC malignance. MNAT1 binding to p53 and mediating p53 ubiquitin-degradation axis represents a novel molecular joint in the p53 pathway.

Abstract Background Metastasis is the major cause of treatment failure in patients with nasopharyngeal carcinoma (NPC). We previously reported that TEL2, a negative regulator of SERPINE1, could inhibit NPC metastasis to lymph nodes. Method A series of in vivo and in vitro assays were performed to elucidate the regulation between Snail and TEL2. TEL2 expression was analyzed in three representative NPC cell lines expressing low levels of Snail (S26, 6-10B, HK1) and two cell lines expressing high levels of Snail (S18, 5-8F). Luciferase and chromatin immunoprecipitation assays were used to analyze the interaction between Snail and TEL2. The roles of the Snail/TEL2 pathway in cell migration and invasion of NPC cells were examined using transwell assays. Metastasis to the lungs was examined using nude mouse receiving NPC cells injection through the tail vein. Results Ectopic Snail expression down-regulated TEL2 at the mRNA and protein levels, whereas knockdown of Snail using short hairpin RNA up-regulated TEL2. Luciferase and chromatin immunoprecipitation assays indicated that Snail binds directly to the TEL2 promoter. Ectopic Snail expression enhanced migration and invasion of NPC cells, and such effects were mitigated by TEL2 overexpression. TEL2 overexpression also attenuated hypoxia-induced cell migration and invasion, and increased the number of metastatic pulmonary nodules. Snail overexpression reduced the number of metastatic pulmonary nodules. Conclusions TEL2 is a novel target of Snail and suppresses Snail-induced migration, invasion and metastasis in NPC.

Nuclear receptor coactivator 3 (NCOA3) is a transcriptional coactivator that has elevated expression in multiple tumor types, including colorectal cancer (CRC). However, the molecular mechanisms that regulate the tumorigenic functions of NCOA3 in CRC remain largely unknown. In this study, we aimed to discover and identify the novel regulatory proteins of NCOA3 and explore their mechanisms of action. Immunoprecipitation (IP) coupled with mass spectrometry (IP‐MS) analysis was used to detect, identify, and verify the proteins that interacted with NCOA3 in CRC cells. The biological functions of the candidate proteins and the underlying molecular mechanism were investigated in CRC cells and mouse model in vitro and in vivo. The clinical significance of NCOA3 and its interaction partner protein in CRC patients was also studied. We identified mitotic arrest deficient 2‐like protein 2 (MAD2L2, also known as MAD2B or REV7), with two signal peptide sequences of LIPLK and EVYPVGIFQK, to be an interaction partner of NCOA3. Overexpression of MAD2L2 suppressed the proliferation, migration, and clonogenicity of CRC cells by inducing the degradation of NCOA3. The mechanism study showed that increased MAD2L2 expression in CRC cells activated p38, which was required for the phosphorylation of NCOA3 that led to its ubiquitination and degradation by the proteasome. Moreover, we found that MAD2L2 predicted favorable prognosis in CRC patients. We have discovered a novel role of MAD2L2 in the regulation of NCOA3 degradation and proposed that MAD2L2 serves as a tumor suppressor in CRC.

Abstract Background Acute myeloid leukemia (AML) is one of the most common types of adult acute leukemia. Standard chemotherapies can induce complete remission in selected patients; however, a majority of patients eventually relapse and succumb to the disease. Thus, the development of novel therapeutics for AML is urgently needed. Human C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein, and its expression is restricted to myeloid cells and the majority of AML blasts. Moreover, CLL-1 is expressed in leukemia stem cells (LSCs), but absent in hematopoietic stem cells (HSCs), which may provide a potential therapeutic target for AML treatment. Methods We tested the expression of CLL-1 antigen on peripheral blood cells and bone marrow cells in healthy donor and AML patients. Then, we developed a chimeric antigen receptor (CAR) containing a CLL1-specific single-chain variable fragment, in combination with CD28, 4-1BB costimulatory domains, and CD3-ζ signaling domain. We further investigate the function of CLL-1 CAR-T cells. Results The CLL-1 CAR-T cells specifically lysed CLL-1+ cell lines as well as primary AML patient samples in vitro. Strong anti-leukemic activity was observed in vivo by using a xenograft model of disseminated AML. Importantly, CLL-1+ myeloid progenitor cells and mature myeloid cells were specifically eliminated by CLL-1 CAR-T cells, while normal HSCs were not targeted due to the lack of CLL-1 expression. Conclusions CLL-1 CAR-T represents a promising immunotherapy for the treatment of AML.

Abstract Background N-myc (and STAT) interactor (NMI) plays vital roles in tumor growth, progression, and metastasis. In this study, we identified NMI as a potential tumor suppressor in lung cancer and explored its molecular mechanism involved in lung cancer progression. Methods Human lung cancer cell lines and a mouse xenograft model was used to study the effect of NMI on tumor growth. The expression of NMI, COX-2 and relevant signaling proteins were examined by Western blot. Tissue microarray immunohistochemical analysis was performed to assess the correlation between NMI and COX-2 expression in lung cancer patients. Results NMI was highly expressed in normal lung cells and tissues, but lowly expressed in lung cancer cells and tissues. Overexpression of NMI induced apoptosis, suppressed lung cancer cell growth and migration, which were mediated by up-regulation of the cleaved caspase-3/9 and down-regulation of phosphorylated PI3K/AKT, MMP2/MMP9, β-cadherin, and COX-2/PGE2. In contrast, knockdown of NMI promoted lung cancer cell colony formation and migration, which were correlated with the increased expression of phosphorylated PI3K/AKT, MMP2/MMP9, β-cadherin and COX-2/PGE2. Further study showed that NMI suppressed COX-2 expression through inhibition of the p50/p65 NF-κB acetylation mediated by p300. The xenograft lung cancer mouse models also confirmed the NMI-mediated suppression of tumor growth by inhibiting COX-2 signaling. Moreover, tissue microarray immunohistochemical analysis of lung adenocarcinomas also demonstrated a negative correlation between NMI and COX-2 expression. Kaplan-Meier analysis indicated that the patients with high level of NMI had a significantly better prognosis. Conclusions Our study showed that NMI suppressed tumor growth by inhibiting PI3K/AKT, MMP2/MMP9, COX-2/PGE2 signaling pathways and p300-mediated NF-κB acetylation, and predicted a favorable prognosis in human lung adenocarcinomas, suggesting that NMI was a potential tumor suppressor in lung cancer.

HOPX is a transcription factor epigenetically silenced in several cancers. Here the authors, by analysing methylation profiles, identify HOPX as a suppressor of metastasis in nasopharyngeal carcinoma: mechanistically HOPX inhibitsSNAILtranscription through deacetylation-mediated silencing.

Melanoma is one of the most malignant and aggressive cancers with high cancer-related deaths. However, it is unclear whether Ku80 regulates tumor growth in human melanoma. In this study, we screened a siRNA library targeting 6024 human genes and identified Ku80 as a potential therapeutic target in melanoma cells. Knockdown of Ku80 significantly suppressed melanoma cell proliferation and induced apoptosis, as well as enhanced the antitumor effect of melatonin in melanoma in vitro and in vivo. Overexpression of Ku80, however, promoted melanoma growth and increased the insensitivity of melanoma cells to melatonin. Mechanistically, we found that Ku80 bound to the PDK1 promoter and activated the transcription of PDK1. Moreover, we showed that the binding of Ku80 at the PDK-1 promoter was HIF1-α dependent, and melatonin degraded HIF1-α in melanoma cells. Furthermore, clinical data revealed that the expression of Ku80 and PDK-1 proteins were positively correlated and elevated in the tumor tissues of melanoma patients, and high expression of Ku80 predicted a poor prognosis in melanoma. Collectively, our study demonstrated that Ku80 promoted melanoma growth and regulated antitumor activity of melatonin by targeting HIF1-α dependent PDK-1 signaling pathway, suggesting that Ku80 may be a potential molecular target for melanoma treatment. Keywords: Ku80, PDK-1, Melatonin, HIF1-α, Melanoma

Abstract Background Centrosomal protein 78 (CEP78) has been characterized as a component of the centrosome required for the regulation of centrosome-related events during the cell cycle, but its role in human cancers remains unclear. This study aimed to investigate the role and the clinical value of CEP78 in colorectal cancer (CRC). Methods Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry were performed to examine CEP78 expression in CRC tissues and adjacent noncancerous tissues. The association between CEP78 expression and clinical outcomes of CRC patients was determined. The effect of CEP78 on cell growth was examined in vitro by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, colony formation, and flow cytometry assays and in vivo using a nude mouse model. Results The expression level of CEP78 was significantly lower in tumor tissues than in the adjacent normal tissues (P

The androgen receptor (AR) is required for castration-resistant prostate cancer (CRPC) progression, but the function and disease relevance of AR-bound enhancers remain unclear. Here, we identify a group of AR-regulated enhancer RNAs (e.g., PSA eRNA) that are upregulated in CRPC cells, patient-derived xenografts (PDXs), and patient tissues. PSA eRNA binds to CYCLIN T1, activates P-TEFb, and promotes cis and trans target gene transcription by increasing serine-2 phosphorylation of RNA polymerase II (Pol II-Ser2p). We define an HIV-1 TAR RNA-like (TAR-L) motif in PSA eRNA that is required for CYCLIN T1 binding. Using TALEN-mediated gene editing we further demonstrate that this motif is essential for increased Pol II-Ser2p occupancy levels and CRPC cell growth. We have uncovered a P-TEFb activation mechanism and reveal altered eRNA expression that is related to abnormal AR function and may potentially be a therapeutic target in CRPC.

Abstract The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled “A genome-scale CRISPR–Cas9 screening method for protein stability reveals novel regulators of Cdc25A,” recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR–Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.

LSD1 is a critical chromatin modulator that controls cellular pluripotency and differentiation through the demethylation of H3K4me1/2. Overexpression of LSD1 has been observed in many types of tumors and is correlated with its oncogenic effects in tumorigenesis. However, the mechanism leading to LSD1 upregulation in tumors remains unclear. Using an unbiased siRNA screening against all the human deubiquitinases, we identified USP28 as a bona fide deubiquitinase of LSD1. USP28 interacted with and stabilized LSD1 via deubiquitination. USP28 overexpression correlated with LSD1 upregulation in multiple cancer cell lines and breast tumor samples. Knockdown of USP28 resulted in LSD1 destabilization, leading to the suppression of cancer stem cell (CSC)-like characteristics in vitro and inhibition of tumorigenicity in vivo, which can be rescued by ectopic LSD1 expression. Our study reveals a critical mechanism underlying the epigenetic regulation by USP28 and provides another treatment approach against breast cancer.

Berberine (BBR), an isoquinoline derivative alkaloid isolated from Chinese herbs, has a long history of uses for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of BBR in human lung cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which BBR inhibits cell growth in human non-small-cell lung cancer (NSCLC) cells. Treatment with BBR promoted cell morphology change, inhibited cell migration, proliferation and colony formation, and induced cell apoptosis. Further molecular mechanism study showed that BBR simultaneously targeted multiple cell signaling pathways to inhibit NSCLC cell growth. Treatment with BBR inhibited AP-2α and AP-2β expression and abrogated their binding on hTERT promoters, thereby inhibiting hTERT expression. Knockdown of AP-2α and AP-2β by siRNA considerably augmented the BBR-mediated inhibition of cell growth. BBR also suppressed the nuclear translocation of p50/p65 NF-κB proteins and their binding to COX-2 promoter, causing inhibition of COX-2. BBR also downregulated HIF-1α and VEGF expression and inhibited Akt and ERK phosphorylation. Knockdown of HIF-1α by siRNA considerably augmented the BBR-mediated inhibition of cell growth. Moreover, BBR treatment triggered cytochrome-c release from mitochondrial inter-membrane space into cytosol, promoted cleavage of caspase and PARP, and affected expression of BAX and Bcl-2, thereby activating apoptotic pathway. Taken together, these results demonstrated that BBR inhibited NSCLC cell growth by simultaneously targeting AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF, PI3K/AKT, Raf/MEK/ERK and cytochrome-c/caspase signaling pathways. Our findings provide new insights into understanding the anticancer mechanisms of BBR in human lung cancer therapy.

Nicotine, the major component in cigarette smoke, can promote tumor growth and angiogenesis, but the precise mechanisms involved remain largely unknown. Here, we investigated the mechanism of action of nicotine in human nasopharyngeal carcinoma (NPC) cells. Nicotine significantly promoted cell proliferation in a dose and time-dependent manner in human NPC cells. The mechanism studies showed that the observed stimulation of proliferation was accompanied by the nicotine-mediated simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling. Treatment of NPC cells with nicotine markedly upregulated the expression of α7AChR and HIF-1α proteins. Transfection with a α7AChR or HIF-1α-specific siRNA or a α7AChR-selective inhibitor significantly attenuated the nicotine-mediated promotion of NPC cell proliferation. Nicotine also promoted the phosphorylation of ERK1/2 but not JNK and p38 proteins, thereby induced the activation of ERK/MAPK signaling pathway. Pretreatment with an ERK-selective inhibitor effectively reduced the nicotine-induced proliferation of NPC cells. Moreover, nicotine upregulated the expression of VEGF but suppressed the expression of PEDF at mRNA and protein levels, leading to a significant increase of the ratio of VEGF/PEDF in NPC cells. Pretreatment with a α7AChR or ERK-selective inhibitor or transfection with a HIF-1α-specific siRNA in NPC cells significantly inhibited the nicotine-induced HIF-1α expression and VEGF/PEDF ratio. These results therefore indicate that nicotine promotes proliferation of human NPC cells in vitro through simultaneous modulation of α7AChR, HIF-1α, ERK and VEGF/PEDF signaling and suggest that the related molecules such as HIF-1α might be the potential therapeutic targets for tobacco-associated diseases such as nasopharyngeal carcinomas.

BACKGROUND:Nonviral vectors are attractively used for gene therapy owing to their distinctive advantages. Our previous study has demonstrated that transfer of human IFNγ gene into nasopharyngeal carcinoma (NPC) by using a novel nonviral vector, minicircle (mc), under the control of cytomegalovirus (CMV) promoter was effective to inhibit tumor growth. However, therapies based on CMV promoter cannot express the targeted genes in cancer tissues. Previous studies indicated that the development of human NPC was closely associated with Epstein-Barr virus (EBV) and demonstrated the transcriptional enhancer function of oriP when bound by EBV protein. Therefore, the present study is to explore the targeted gene expression and the anti-tumor effect of a novel tumor-specific gene therapeutic system (mc-oriP-IFNγ) in which the transgene expression was under the transcriptional regulation of oriP promoter. METHODOLOGY/PRINCIPAL FINDINGS:Dual-luciferase reporter assay and ELISA were used to assess the expression of luciferase and IFNγ. WST assay was used to assess the cell proliferation. RT-PCR was used to detect the mRNA level of EBNA1. RNAi was used to knockdown the expression of EBNA1. NPC xenograft models in nude mice were used to investigate the targeted antitumor efficacy of mc-oriP-IFNγ. Immunohistochemistry was used to detect the expression and the activity of the IFNγ in tumor sections. Our results demonstrated that mc-oriP vectors mediated comparable gene expression and anti-proliferative effect in the EBV-positive NPC cell line C666-1 compared to mc-CMV vectors. Furthermore, mc-oriP vectors exhibited much lower killing effects on EBV-negative cell lines compared to mc-CMV vectors. The targeted expression of mc-oriP vectors was inhibited by EBNA1-siRNA in C666-1. This selective expression was corroborated in EBV-positive and -negative tumor models. CONCLUSIONS/SIGNIFICANCE:This study demonstrates the feasibility of mc-oriP-IFNγ as a safe and highly effective targeted gene therapeutic system for the treatment of EBV positive NPC.

Quercetin, a polyphenolic bioflavonoid, possesses multiple pharmacological actions including anti-inflammatory and antitumor properties. However, the precise action mechanisms of quercetin remain unclear. Here, we reported the regulatory actions of quercetin on cyclooxygenase-2 (COX-2), an important mediator in inflammation and tumor promotion, and revealed the underlying mechanisms. Quercetin significantly suppressed COX-2 mRNA and protein expression and prostaglandin (PG) E(2) production, as well as COX-2 promoter activation in breast cancer cells. Quercetin also significantly inhibited COX-2-mediated angiogenesis in human endothelial cells in a dose-dependent manner. The in vitro streptavidin-agarose pulldown assay and in vivo chromatin immunoprecipitation assay showed that quercetin considerably inhibited the binding of the transactivators CREB2, C-Jun, C/EBPβ and NF-κB and blocked the recruitment of the coactivator p300 to COX-2 promoter. Moreover, quercetin effectively inhibited p300 histone acetyltransferase (HAT) activity, thereby attenuating the p300-mediated acetylation of NF-κB. Treatment of cells with p300 HAT inhibitor roscovitine was as effective as quercetin at inhibiting p300 HAT activity. Addition of quercetin to roscovitine-treated cells did not change the roscovitine-induced inhibition of p300 HAT activity. Conversely, gene delivery of constitutively active p300 significantly reversed the quercetin-mediated inhibition of endogenous HAT activity. These results indicate that quercetin suppresses COX-2 expression by inhibiting the p300 signaling and blocking the binding of multiple transactivators to COX-2 promoter. Our findings therefore reveal a novel mechanism of action of quercetin and suggest a potential use for quercetin in the treatment of COX-2-mediated diseases such as breast cancers.