METTL16 antagonizes MRE11-mediated DNA end resection and confers synthetic lethality to PARP inhibition in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Characterization of genetic alterations will improve our understanding and therapies for this disease. Here, we report that PDAC with elevated expression of METTL16, one of the 'writers' of RNA N6-methyladenosine modification, may benefit from poly-(ADP-ribose)-polymerase inhibitor (PARPi) treatment. Mechanistically, METTL16 interacts with MRE11 through RNA and this interaction inhibits MRE11's exonuclease activity in a methyltransferase-independent manner, thereby repressing DNA end resection. Upon DNA damage, ATM phosphorylates METTL16 resulting in a conformational change and autoinhibition of its RNA binding. This dissociates the METTL16-RNA-MRE11 complex and releases inhibition of MRE11. Concordantly, PDAC cells with high METTL16 expression show increased sensitivity to PARPi, especially when combined with gemcitabine. Thus, our findings reveal a role for METTL16 in homologous recombination repair and suggest that a combination of PARPi with gemcitabine could be an effective treatment strategy for PDAC with elevated METTL16 expression.

MET Amplification Attenuates Lung Tumor Response to Immunotherapy by Inhibiting STING.

Immune checkpoint blockade (ICB) has revolutionized cancer therapy. However, the response of patients to ICB is difficult to predict. Here, we examined 81 patients with lung cancer under ICB treatment and found that patients with MET amplification were resistant to ICB and had a poor progression-free survival. Tumors with MET amplifications had significantly decreased STING levels and antitumor T-cell infiltration. Furthermore, we performed deep single-cell RNA sequencing on more than 20,000 single immune cells and identified an immunosuppressive signature with increased subsets of XIST- and CD96-positive exhausted natural killer (NK) cells and decreased CD8+ T-cell and NK-cell populations in patients with MET amplification. Mechanistically, we found that oncogenic MET signaling induces phosphorylation of UPF1 and downregulates tumor cell STING expression via modulation of the 3'-UTR length of STING by UPF1. Decreased efficiency of ICB by MET amplification can be overcome by inhibiting MET. SIGNIFICANCE: We suggest that the combination of MET inhibitor together with ICB will overcome ICB resistance induced by MET amplification. Our report reveals much-needed information that will benefit the treatment of patients with primary MET amplification or EGFR-tyrosine kinase inhibitor resistant-related MET amplification.This article is highlighted in the In This Issue feature, p. 2659.

Elevation of EIF4G1 promotes non-small cell lung cancer progression by activating mTOR signalling.

Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1), as the key component of the transcription initiation factor complex EIF4F, is significantly upregulated in multiple solid tumours, including lung cancer. However, the function and mechanism of EIF4G1 in the regulation of non-small-cell lung cancer (NSCLC) remain unclear. Here, using the clinical samples and the comprehensive survival analysis platforms Kaplan-Meier plotter, we observed aberrant upregulation of EIF4G1 in NSCLC tissues; furthermore, high expression of EIF4G1 showed association with low differentiation of lung cancer cells and poor overall survival in NSCLC patients. Non-small-cell lung cancer cell line A549 and H1703 stably infected with EIF4G1 shRNA were used to determine the function of EIF4G1 in regulating cell proliferation and tumorigenesis in vitro and in vivo. The results demonstrated that EIF4G1 promoted the G1/S transition of the cell cycle and tumour cell proliferation in non-small cell lung cancer. Mechanistically, EIF4G1 was found to regulate the expression and phosphorylation of mTOR (Ser2448), which mediates the tumorigenesis-promoting function of EIF4G1. The inhibition of mTOR attenuated the EIF4G1-induced development and progression of tumours. These findings demonstrated that EIF4G1 is a new potential molecular target for the clinical treatment of non-small cell lung cancer.

Ribonucleotide Reductase Inhibitor 3-AP Induces Oncogenic Virus Infected Cell Death and Represses Tumor Growth.

Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of several human malignancies, particularly Kaposi's Sarcoma (KS), which preferentially arise in immunocompromised patients such as HIV+ subpopulation while still lacking of effective therapeutic options. We recently found that the ribonucleotide reductase (RR) subunit M2 is potentially regulated by the key oncogenic HGF/c-MET pathway in KSHV-related lymphoma cells. One of RR inhibitor, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) effectively induced apoptosis of KSHV+ lymphomas and suppressed tumor progression in vivo. In the current study, we found that 3-AP treatment selectively inhibited the proliferation of KSHV-infected endothelial cells, the major cellular components of KS, through inducing DNA damage, reducing the levels of intracellular iron and reactive oxygen species (ROS) and increasing viral lytic gene expression. By using a KS-like nude mouse model, we found that 3-AP treatment significantly suppressed KSHV induced tumorigenesis in vivo. Taken together, our data demonstrate targeting RR by 3-AP may represent a promising strategy for improving the treatment of KS in future.

Regulation of Virus-Associated Lymphoma Growth and Gene Expression by Bacterial Quorum-Sensing Molecules.

Kaposi's sarcoma-associated herpesvirus (KSHV) can cause several human cancers, including primary effusion lymphoma (PEL), which frequently occur in immunocompromised patients. KSHV-infected patients often suffer from polymicrobial infections caused by opportunistic bacterial pathogens. Therefore, it is crucial to understand how these coinfecting microorganisms or their secreted metabolites may affect KSHV infection and the pathogenesis of virus-associated malignancies. Quorum sensing (QS), a cell density-based intercellular communication system, employs extracellular diffusible signaling molecules to regulate bacterial virulence mechanisms in a wide range of bacterial pathogens, such as Pseudomonas aeruginosa, which is one of the most common opportunistic microorganisms found in immunocompromised individuals. In this study, we evaluated and compared the influence on PEL growth and the host/viral interactome of the major QS signaling molecules [N-(3-oxododecanoyl)-l-homoserine lactone (OdDHL), N-butyrylhomoserine lactone (BHL), and 2-heptyl-3-hydroxy-4-quinolone (PQS)] in conditioned medium from wild-type (wt) and QS mutant laboratory strains as well as clinical isolates of P. aeruginosa Our data indicate that P. aeruginosa coinfection may facilitate virus dissemination and establishment of new infection and further promote tumor development through effectively inducing viral lytic gene expression by its QS systems.IMPORTANCE Currently, most studies about KSHV infection and/or virus-associated malignancies depend on pure culture systems or immunodeficient animal models. However, the real situation should be much more complicated in KSHV-infected immunocompromised patients due to frequent polymicrobial infections. It is important to understand the interaction of KSHV and coinfecting microorganisms, especially opportunistic bacterial pathogens. Here we report for the first time that P. aeruginosa and its quorum-sensing signaling molecules display a complicated impact on KSHV-associated lymphoma growth as well as the intracellular host/viral gene expression profile. Our data imply that targeting of coinfecting pathogens is probably necessary during treatment of virus-associated malignancies in these immunocompromised patients.

Targeting EIF4F complex in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) accounts for about 85-90% of lung cancer cases, which represents the leading cause of cancer-related death in the world. The majority of lung cancer patients doesn't respond well to conventional chemo-/radio-therapeutic regimens and have a poor prognosis. The recent introduction of targeted therapy and immunotherapy gives new hopes to NSCLC patients, but their outcome/prognosis is far from satisfactory. The translation initiation EIF4F complex has been shown to play important roles in cancer progression, but its functional role and therapeutic effect in lung cancers especially NSCLC remain largely unknown. In this current review, we summarize recent findings regarding the role of EIF4F complex in NSCLC progression and targeted therapy potentials. We also discuss the unanswered questions and future directions in this field.

KSHV co-infection down-regulates HPV16 E6 and E7 from cervical cancer cells.

High-risk human papillomavirus (HPV) infection is the etiological agent of some malignancies such as cervical, oral and oropharyngeal cancers. Kaposi sarcoma-associated herpesvirus (KSHV) represents a principal causative agent of several human cancers arising in those immunocompromised patients. Interestingly, KSHV DNA has been detected in the oral cavity and the female genital tract, although its detection rate in cervical samples is very low and few reports are about KSHV/HPV co-infection. Therefore, it remains unclear about the role of KSHV co-infection in the development of HPV-related neoplasias. In the current study, we report that HPV16-integrated cervical cancer cell-line SiHa is susceptible to KSHV latent infection and replication. We also have found that KSHV infection or viral latent proteins are capable of reducing HPV16 E6/E7 expression through the manipulation of cellular microRNA function. Array analysis indicates that KSHV infection induces some inflammatory cytokines/chemokines production as well as up-regulates a series of interferon-induced genes expression, which may facilitate host immune defense system attacking these co-infected cells and clearance of viruses. Together, our data have provided possible explanations for very low detection rate of KSHV shedding as well as of KSHV/HPV co-infection in cervical samples and/or cervical cancer cells.

Up-regulation of tumor suppressor genes by exogenous dhC16-Cer contributes to its anti-cancer activity in primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a rare and highly aggressive B-cell malignancy with Kaposi's sarcoma-associated herpesvirus (KSHV) infection, while lack of effective therapies. Our recent data indicated that targeting the sphingolipid metabolism by either sphingosine kinase inhibitor or exogenous ceramide species induces PEL cell apoptosis and suppresses tumor progression in vivo. However, the underlying mechanisms for these exogenous ceramides "killing" PEL cells remain largely unknown. Based on the microarray analysis, we found that exogenous dhC16-Cer treatment affected the expression of many cellular genes with important functions within PEL cells such as regulation of cell cycle, cell survival/proliferation, and apoptosis/anti-apoptosis. Interestingly, we found that a subset of tumor suppressor genes (TSGs) was up-regulated from dhC16-Cer treated PEL cells. One of these elevated TSGs, Thrombospondin-1 (THBS1) was required for dhC16-Cer induced PEL cell cycle arrest. Moreover, dhC16-Cer up-regulation of THBS1 was through the suppression of multiple KSHV microRNAs expression. Our data demonstrate that exogenous ceramides display anti-cancer activities for PEL through regulation of both host and oncogenic virus factors.

Genomic analysis of xCT-regulatory network in KSHV + primary effusion lymphomas.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of primary effusion lymphoma (PEL), a rapidly progressing malignancy mostly arising in HIV-infected patients Chen et al. (2007) [1]. Even under conventional chemotherapy, PEL continues to portend nearly 100% mortality within several months, which urgently requires novel therapeutic strategies. We have previously demonstrated that targeting xCT, an amino acid transporter for cystine/glutamate exchange, induces significant PEL cell apoptosis through regulation of multiple host and viral factors [2]. More importantly, one of xCT selective inhibitors, Sulfasalazine (SASP), effectively prevents PEL tumor progression in an immune-deficient xenograft model [2]. In the current study, we use Illumina microarray to explore the profile of genes altered by SASP treatment within 3 KSHV + PEL cell-lines, and discover that many genes involved in oxidative stress/antioxidant defense system, apoptosis/anti-apoptosis/cell death, and cellular response to unfolded proteins/topologically incorrect proteins are potentially regulated by xCT Dai et al. (2015) [3]. The microarray original data have been submitted to Gene Expression Omnibus (GEO) database (Accession number: GSE65418).

Functional role of eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) in NSCLC.

Eukaryotic translation initiation factor 4 gamma 1(EIF4G1) is related to tumorigenesis and tumor progression. However, its role and the underlying mechanisms in the regulation of tumor development in non-small cell lung cancers (NSCLC) remain largely unknown. Here we report that the levels of EIF4G1 expression are much higher in NSCLC cell lines and tumor tissues than those in the normal lung cells and adjacent normal tissues from the same patients. Using shRNA to knock down EIF4G1 expression stably, we found EIF4G1 required for NSCLC cell proliferation, anchorage-independent growth, migration and invasion. Furthermore, silencing of EIF4G1 induces NSCLC cell apoptosis and causes G0/G1 cell cycle arrest. To identify the partner protein network of EIF4G1 in NSCLC cells, we found that Ubiquitin-specific protease 10 (USP10) can directly interacts with EIF4G1, while acting as a negative regulator for EIF4G1-mediated functions. Together, our results indicate that EIF4G1 functions as an oncoprotein during NSCLC development, which may represent a novel and promising therapeutic target in lung cancer.

Targeting HGF/c-MET induces cell cycle arrest, DNA damage, and apoptosis for primary effusion lymphoma.

Kaposi sarcoma-associated herpesvirus (KSHV) is a principal causative agent of primary effusion lymphoma (PEL) with a poor prognosis in immunocompromised patients. However, it still lacks effective treatment which urgently requires the identification of novel therapeutic targets for PEL. Here, we report that the hepatocyte growth factor (HGF)/c-MET pathway is highly activated by KSHV in vitro and in vivo. The selective c-MET inhibitor, PF-2341066, can induce PEL apoptosis through cell cycle arrest and DNA damage, and suppress tumor progression in a xenograft murine model. By using microarray analysis, we identify many novel genes that are potentially controlled by HGF/c-MET within PEL cells. One of the downstream candidates, ribonucleoside-diphosphate reductase subunit M2 (RRM2), also displays the promising therapeutic value for PEL treatment. Our findings provide the framework for development of HGF/c-MET-focused therapy and implementation of clinical trials for PEL patients.

Genomic analysis of xCT-mediated regulatory network: Identification of novel targets against AIDS-associated lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of primary effusion lymphoma (PEL), a rapidly progressing malignancy mostly arising in HIV-infected patients. Even under conventional chemotherapy, PEL continues to portend nearly 100% mortality within several months, which urgently requires novel therapeutic strategies. We have previously demonstrated that targeting xCT, an amino acid transporter for cystine/glutamate exchange, induces significant PEL cell apoptosis through regulation of multiple host and viral factors. More importantly, one of xCT selective inhibitors, Sulfasalazine (SASP), effectively prevents PEL tumor progression in an immune-deficient xenograft model. In the current study, we use Illumina microarray to explore the profile of genes altered by SASP treatment within 3 KSHV(+) PEL cell-lines, and discover that many genes involved in oxidative stress/antioxidant defense system, apoptosis/anti-apoptosis/cell death, and cellular response to unfolded proteins/topologically incorrect proteins are potentially regulated by xCT. We further validate 2 downstream candidates, OSGIN1 (oxidative stress-induced growth inhibitor 1) and XRCC5 (X-ray repair cross-complementing protein 5), and evaluate their functional relationship with PEL cell survival/proliferation and chemoresistance, respectively. Together, our data indicate that targeting these novel xCT-regulated downstream genes may represent a promising new therapeutic strategy against PEL and/or other AIDS-related lymphoma.

Lipoteichoic acid (LTA) and lipopolysaccharides (LPS) from periodontal pathogenic bacteria facilitate oncogenic herpesvirus infection within primary oral cells.

Kaposi's sarcoma (KS) remains the most common tumor arising in patients with HIV/AIDS, and involvement of the oral cavity represents one of the most common clinical manifestations of this tumor. HIV infection incurs an increased risk for periodontal diseases and oral carriage of a variety of bacteria. Whether interactions involving pathogenic bacteria and oncogenic viruses in the local environment facilitate replication or maintenance of these viruses in the oral cavity remains unknown. In the current study, our data indicate that pretreatment of primary human oral fibroblasts with two prototypical pathogen-associated molecular patterns (PAMPs) produced by oral pathogenic bacteria-lipoteichoic acid (LTA) and lipopolysaccharide (LPS), increase KSHV entry and subsequent viral latent gene expression during de novo infection. Further experiments demonstrate that the underlying mechanisms induced by LTA and/or LPS include upregulation of cellular receptor, increasing production of reactive oxygen species (ROS), and activating intracellular signaling pathways such as MAPK and NF-κB, and all of which are closely associated with KSHV entry or gene expression within oral cells. Based on these findings, we hope to provide the framework of developing novel targeted approaches for treatment and prevention of oral KSHV infection and KS development in high-risk HIV-positive patients.

Targeting xCT, a cystine-glutamate transporter induces apoptosis and tumor regression for KSHV/HIV-associated lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of primary effusion lymphoma (PEL), which represents a rapidly progressing malignancy arising in HIV-infected patients. Conventional chemotherapy for PEL treatment induces unwanted toxicity and is ineffective--PEL continues to portend nearly 100% mortality within a period of months, which requires novel therapeutic strategies. The amino acid transporter, xCT, is essential for the uptake of cystine required for intracellular glutathione (GSH) synthesis and for maintaining the intracellular redox balance. Inhibition of xCT induces growth arrest in a variety of cancer cells, although its role in virus-associated malignancies including PEL remains unclear. In the current study, we identify that xCT is expressed on the surface of patient-derived KSHV+ PEL cells, and targeting xCT induces caspase-dependent cell apoptosis. Further experiments demonstrate the underlying mechanisms including host and viral factors: reducing intracellular GSH while increasing reactive oxygen species (ROS), repressing cell-proliferation-related signaling, and inducing viral lytic genes. Using an immune-deficient xenograft model, we demonstrate that an xCT selective inhibitor, Sulfasalazine (SASP), prevents PEL tumor progression in vivo. Together, our data provide innovative and mechanistic insights into the role of xCT in PEL pathogenesis, and the framework for xCT-focused therapies for AIDS-related lymphoma in future.