Pharmacological tumor PDL1 depletion with chlorambucil treats ovarian cancer and melanoma: improves antitumor immunity and renders anti-PDL1-resistant tumors anti-PDL1-sensitive through NK cell effects.

BACKGROUND: Tumor intracellular programmed cell death ligand-1 (PDL1) mediates pathologic signals that regulate clinical treatment responses distinctly from surface-expressed PDL1 targeted by αPDL1 immune checkpoint blockade antibodies. METHODS: We performed a drug screen for tumor cell PDL1 depleting drugs that identified Food and Drug Administration (FDA)-approved chlorambucil and also 9-[2-(phosphonomethoxy)ethyl] guanine. We used in vitro and in vivo assays to evaluate treatment and signaling effects of pharmacological tumor PDL1 depletion focused on chlorambucil as FDA approved, alone or plus αPDL1. RESULTS: PDL1-expressing mouse and human ovarian cancer lines and mouse melanoma were more sensitive to chlorambucil-mediated proliferation inhibition in vitro versus corresponding genetically PDL1-depleted lines. Orthotopic peritoneal PDL1-expressing ID8agg ovarian cancer and subcutaneous B16 melanoma tumors were more chlorambucil-sensitive in vivo versus corresponding genetically PDL1-depleted tumors. Chlorambucil enhanced αPDL1 efficacy in tumors otherwise αPDL1-refractory, and improved antitumor immunity and treatment efficacy in a natural killer cell-dependent manner alone and plus αPDL1. Chlorambucil-mediated PDL1 depletion was relatively tumor-cell selective in vivo, and treatment efficacy was preserved in PDL1KO hosts, demonstrating tumor PDL1-specific treatment effects. Chlorambucil induced PDL1-dependent immunogenic tumor cell death which could help explain immune contributions. Chlorambucil-mediated PDL1 reduction mechanisms were tumor cell-type-specific and involved transcriptional or post-translational mechanisms, including promoting PDL1 ubiquitination through the GSK3ß/ß-TRCP pathway. Chlorambucil-mediated tumor cell PDL1 depletion also phenocopied genetic PDL1 depletion in reducing tumor cell mTORC1 activation and tumor initiating cell content, and in augmenting autophagy, suggesting additional treatment potential. CONCLUSIONS: Pharmacological tumor PDL1 depletion with chlorambucil targets tumor-intrinsic PDL1 signaling that mediates treatment resistance, especially in αPDL1-resistant tumors, generates PDL1-dependent tumor immunogenicity and inhibits tumor growth in immune-dependent and independent manners. It could improve treatment efficacy of selected agents in otherwise treatment-refractory, including αPDL1-refractory cancers, and is rapidly clinically translatable.

PRC2-Mediated Epigenetic Suppression of Type I IFN-STAT2 Signaling Impairs Antitumor Immunity in Luminal Breast Cancer.

The immunosuppressive tumor microenvironment in some cancer types, such as luminal breast cancer, supports tumor growth and limits therapeutic efficacy. Identifying approaches to induce an immunostimulatory environment could help improve cancer treatment. Here, we demonstrate that inhibition of cancer-intrinsic EZH2 promotes antitumor immunity in estrogen receptor α-positive (ERα+) breast cancer. EZH2 is a component of the polycomb-repressive complex 2 (PRC2) complex, which catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3). A 53-gene PRC2 activity signature was closely associated with the immune responses of ERα+ breast cancer cells. The stimulatory effects of EZH2 inhibition on immune surveillance required specific activation of type I IFN signaling. Integrative analysis of PRC2-repressed genes and genome-wide H3K27me3 landscape revealed that type I IFN ligands are epigenetically silenced by H3K27me3. Notably, the transcription factor STAT2, but not STAT1, mediated the immunostimulatory functions of type I IFN signaling. Following EZH2 inhibition, STAT2 was recruited to the promoters of IFN-stimulated genes even in the absence of the cytokines, suggesting the formation of an autocrine IFN-STAT2 axis. In patients with luminal breast cancer, high levels of EZH2 and low levels of STAT2 were associated with the worst antitumor immune responses. Collectively, this work paves the way for the development of an effective therapeutic strategy that may reverse immunosuppression in cancer. SIGNIFICANCE: Inhibition of EZH2 activates a type I IFN-STAT2 signaling axis and provides a therapeutic strategy to stimulate antitumor immunity and therapy responsiveness in immunologically cold luminal breast cancer.

Tumor Intrinsic PD-L1 Promotes DNA Repair in Distinct Cancers and Suppresses PARP Inhibitor-Induced Synthetic Lethality.

BRCA1-mediated homologous recombination is an important DNA repair mechanism that is the target of FDA-approved PARP inhibitors, yet details of BRCA1-mediated functions remain to be fully elucidated. Similarly, immune checkpoint molecules are targets of FDA-approved cancer immunotherapies, but the biological and mechanistic consequences of their application are incompletely understood. We show here that the immune checkpoint molecule PD-L1 regulates homologous recombination in cancer cells by promoting BRCA1 nuclear foci formation and DNA end resection. Genetic depletion of tumor PD-L1 reduced homologous recombination, increased nonhomologous end joining, and elicited synthetic lethality to PARP inhibitors olaparib and talazoparib in vitro in some, but not all, BRCA1 wild-type tumor cells. In vivo, genetic depletion of tumor PD-L1 rendered olaparib-resistant tumors sensitive to olaparib. In contrast, anti-PD-L1 immune checkpoint blockade neither enhanced olaparib synthetic lethality nor improved its efficacy in vitro or in wild-type mice. Tumor PD-L1 did not alter expression of BRCA1 or its cofactor BARD1 but instead coimmunoprecipitated with BARD1 and increased BRCA1 nuclear accumulation. Tumor PD-L1 depletion enhanced tumor CCL5 expression and TANK-binding kinase 1 activation in vitro, similar to known immune-potentiating effects of PARP inhibitors. Collectively, these data define immune-dependent and immune-independent effects of PARP inhibitor treatment and genetic tumor PD-L1 depletion. Moreover, they implicate a tumor cell-intrinsic, immune checkpoint-independent function of PD-L1 in cancer cell BRCA1-mediated DNA damage repair with translational potential, including as a treatment response biomarker. SIGNIFICANCE: PD-L1 upregulates BRCA1-mediated homologous recombination, and PD-L1-deficient tumors exhibit BRCAness by manifesting synthetic lethality in response to PARP inhibitors, revealing an exploitable therapeutic vulnerability and a candidate treatment response biomarker. See related commentary by Hanks, p. 2069.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress.

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

Correction: Systemic Delivery of MicroRNA-101 Potently Inhibits Hepatocellular Carcinoma In Vivo by Repressing Multiple Targets.

[This corrects the article DOI: 10.1371/journal.pgen.1004873.].

Regulation of telomere homeostasis and genomic stability in cancer by N 6-adenosine methylation (m6A).

The role of RNA methylation on N 6-adenosine (m6A) in cancer has been acknowledged, but the underlying mechanisms remain obscure. Here, we identified homeobox containing 1 (HMBOX1) as an authentic target mRNA of m6A machinery, which is highly methylated in malignant cells compared to the normal counterparts and subject to expedited degradation upon the modification. m6A-mediated down-regulation of HMBOX1 causes telomere dysfunction and inactivation of p53 signaling, which leads to chromosome abnormalities and aggressive phenotypes. CRISPR-based, m6A-editing tools further prove that the methyl groups on HMBOX1 per se contribute to the generation of altered cancer genome. In multiple types of human cancers, expression of the RNA methyltransferase METTL3 is negatively correlated with the telomere length but favorably with fractions of altered cancer genome, whereas HMBOX1 mRNA levels show the opposite patterns. Our work suggests that the cancer-driving genomic alterations may potentially be fixed by rectifying particular epitranscriptomic program.

Enhancer RNAs Mediate Estrogen-Induced Decommissioning of Selective Enhancers by Recruiting ERα and Its Cofactor.

The function of enhancer RNAs (eRNAs) in transcriptional regulation remains obscure. By analyzing the genome-wide nascent transcript profiles in breast cancer cells, we identify a special group of eRNAs that are essential for estrogen-induced transcriptional repression. Using eRNAs of TM4SF1 and EFEMP1 as the paradigms, we find that these RNA molecules not only stabilize promoter-enhancer interactions but also recruit liganded estrogen receptor α (ERα) to particular enhancer regions, facilitate the formation of a functional transcriptional complex, and cause gene silencing. Interestingly, ERα is shown to directly bind with eRNAs by its DNA-binding domain. These eRNAs help with the formation of a specific ERα-centered transcriptional complex and promote the association of the histone demethylase KDM2A, which dismisses RNA polymerase II from designated enhancers and suppresses the transcription of target genes. Our work demonstrates a complete mechanism underlying the action of eRNAs in modulating and refining the locus-specific transcriptional program.

Epigenetic regulation of prostate cancer: the theories and the clinical implications.

Epigenetics is the main mechanism that controls transcription of specific genes with no changes in the underlying DNA sequences. Epigenetic alterations lead to abnormal gene expression patterns that contribute to carcinogenesis and persist throughout disease progression. Because of the reversible nature, epigenetic modifications emerge as promising anticancer drug targets. Several compounds have been developed to reverse the aberrant activities of enzymes involved in epigenetic regulation, and some of them show encouraging results in both preclinical and clinical studies. In this article, we comprehensively review the up-to-date roles of epigenetics in the development and progression of prostate cancer. We especially focus on three epigenetic mechanisms: DNA methylation, histone modifications, and noncoding RNAs. We elaborate on current models/theories that explain the necessity of these epigenetic programs in driving the malignant phenotypes of prostate cancer cells. In particular, we elucidate how certain epigenetic regulators crosstalk with critical biological pathways, such as androgen receptor (AR) signaling, and how the cooperation dynamically controls cancer-oriented transcriptional profiles. Restoration of a "normal" epigenetic landscape holds promise as a cure for prostate cancer, so we concluded by highlighting particular epigenetic modifications as diagnostic and prognostic biomarkers or new therapeutic targets for treatment of the disease.

FMNL1 mediates nasopharyngeal carcinoma cell aggressiveness by epigenetically upregulating MTA1.

It has been suggested that formin-like protein 1 (FMNL1) plays an important role in the pathogenic process of several hematopoietic malignancies. In this study, we performed a series of in vivo and in vitro assays to elucidate the biological functions of FMNL1 and underlying mechanisms in human nasopharyngeal carcinoma (NPC) pathogenesis. Herein, we report that high expression of FMNL1 in NPC is positively associated with an aggressive disease and/or poor patient survival. Ectopic overexpression of FMNL1 in NPC cells substantially promoted cell invadopodia formation, epithelial-mesenchymal transition (EMT) and invasiveness, whereas depletion of FMNL1 potently suppressed NPC cells invadopodia formation, EMT, and invasive/metastatic capacities. We further show that FMNL1 could enhance NPC cell aggressiveness by increasing a key downstream target, the metastasis-associated protein 1 (MTA1) gene. Importantly, ectopic overexpression of FMNL1 in NPC cells markedly improved the binding of HDAC1 with Profilin2 in the cytoplasm and suppressed the enrichment of HDAC1 on the promoter of MTA1 and thereby, leading to an increased MTA1 transcription and expression. Furthermore, in addition to the amplification of FMNL1 gene, decreased level of miR-16 in NPCs is another critical mechanism to upregulate FMNL1 expression. These results, collectively, provide first-line of evidences that high expression of FMNL1, resulted from decreased miR-16 and/or MTA1 amplification, has a potent oncogenic role to drive the development and aggressive process of NPC by upregulating MTA1, and FMNL1 might be employed as a new prognostic biomarker and therapeutic target for human NPC.

Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2-ERα-GREB1 Transcriptional Axis.

Resistance to cancer treatment can be driven by epigenetic reprogramming of specific transcriptomes in favor of the refractory phenotypes. Here we discover that tamoxifen resistance in breast cancer is driven by a regulatory axis consisting of a master transcription factor, its cofactor, and an epigenetic regulator. The oncogenic histone methyltransferase EZH2 conferred tamoxifen resistance by silencing the expression of the estrogen receptor α (ERα) cofactor GREB1. In clinical specimens, induction of DNA methylation of a particular CpG-enriched region at the GREB1 promoter negatively correlated with GREB1 levels and cell sensitivity to endocrine agents. GREB1 also ensured proper cellular reactions to different ligands by recruiting distinct sets of ERα cofactors to cis-regulatory elements, which explains the contradictory biological effects of GREB1 on breast cancer cell growth in response to estrogen or antiestrogen. In refractory cells, EZH2-dependent repression of GREB1 triggered chromatin reallocation of ERα coregulators, converting the antiestrogen into an agonist. In clinical specimens from patients receiving adjuvant tamoxifen treatment, expression levels of EZH2 and GREB1 were correlated negatively, and taken together better predicted patient responses to endocrine therapy. Overall, our work suggests a new strategy to overcome endocrine resistance in metastatic breast cancer by targeting a particular epigenetic program.Significance: This study suggests a new strategy to overcome endocrine resistance in metastatic breast cancer by targeting a particular epigenetic program defined within. Cancer Res; 78(3); 671-84. ©2017 AACR.

Eukaryotic Initiation Factor 5A2 Contributes to the Maintenance of CD133(+) Hepatocellular Carcinoma Cells via the c-Myc/microRNA-29b Axis.

Cancer stem cells (CSCs)/cancer-initiating cells (CICs) are suggested responsible for driving cancer resistance to conventional therapies and for cancer recurrence and/or metastasis. CD133 is served as a key biomarker to identify and characterize this subpopulation of cells in hepatocellular carcinoma (HCC). Our previous study indicated that overexpression of eukaryotic initiation factor 5A2 (EIF5A2) promotes HCC cell metastasis and angiogenesis. In this study, we demonstrated that EIF5A2 might play a crucial role in CSCs regulation and investigated its potential molecular mechanisms. Using quantitative real-time polymerase chain reaction assay, we observed that the expression of EIF5A2 positively correlated with CD133 levels in a cohort of cancerous and noncancerous liver tissues and cells. Next, HCC cells with high expression of EIF5A2 have a strong capacity to form undifferentiated tumor spheres in vitro and show elevated levels of stem cell-related genes, leading to an increased ability to develop tumors when subcutaneously injected into nude mice. Furthermore, differential microRNA expression was profiling between two EIF5A2-depleted HCC cell lines and their control one identified a decreased expression of miR-29b in EIF5A2-depleted cell lines. Further functional studies illustrated that downregulated miR-29b level is responsible for EIF5A2-maintained HCC cell stemness either in vitro or in vivo. Moreover, enforced expression of EIF5A2 in HCC cells largely enhanced the binding of c-Myc on the promoter of miR-29b and downregulation of miR-29b by EIF5A2 was dependent on c-Myc. Our findings, collectively, reveal that EIF5A2 contributes to the maintenance of CD133+ HCC cells via the c-Myc/miR-29b axis. Stem Cells 2018;36:180-191.

CLDN14 is epigenetically silenced by EZH2-mediated H3K27ME3 and is a novel prognostic biomarker in hepatocellular carcinoma.

Trimethylation of lysine 27 on histone H3 (H3K27ME3) is a transcription-suppressive histone mark mediated by enhancer of zeste homolog 2 (EZH2). We have previously suggested that EZH2-mediated H3K27ME3 plays a critical oncogenic role in human hepatocellular carcinoma (HCC) aggressiveness. However, the direct downstream targets of EZH2-H3K27ME3 and the molecular mechanisms by which regulates HCC pathogenesis remain unclear. In this study, we used chromatin immunoprecipitation together with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis to assess genome-wide chromatin occupancy of H3K27ME3 in HCC cells. We identified that claudin14 (CLDN14) is a potentially direct target for EZH2-mediated H3K27ME3 in HCC. In a large cohort of clinical HCC tissues, we found that low expression of CLDN14 was significantly associated with advanced tumor stage and determined to be an independent predictor of shortened survival of HCC patients. Next, functional experiment demonstrated that depletion of CLDN14 substantially restored EZH2-silenced HCC cells motility and invasive capacities and supported cell epithelial-mesenchymal transition (EMT). Furthermore, downregulation of CLDN14 dramatically re-enhanced the wnt/ß-catenin signaling activity in EZH2-silenced HCC cells by increasing the levels of active ß-catenin and promoting the nuclear localization of ß-catenin. These results, collectively, uncover that CLDN14 is a novel direct target of EZH2-mediated H3K27ME3, and provide an explanation for the aggressive nature of HCC with downregulation of CLDN14 and the underling mechanism that links the tumor suppressor CLDN14 to the wnt/ß-catenin signaling pathway.

Anti-cancer effects of curcumin on lung cancer through the inhibition of EZH2 and NOTCH1.

Curcumin is potentially therapeutic for malignant diseases. The mechanisms of this effect might involve a combination of antioxidant, immunomodulatory, proapoptotic, and antiangiogenic activities. However, the exact mechanisms are not fully understood. In the present study, we provided evidences that curcumin suppressed the expression of enhancer of zeste homolog 2 (EZH2) in lung cancer cells both transcriptionally and post-transcriptionally. Curcumin inhibited the expression of EZH2 through microRNA (miR)-let 7c and miR-101. Curcumin decreased the expression of NOTCH1 through the inhibition of EZH2. There was a reciprocal regulation between EZH2 and NOTCH1 in lung cancer cells. These observations suggest that curcumin inhibits lung cancer growth and metastasis at least partly through the inhibition of EZH2 and NOTCH1.

The putative tumor activator ARHGEF3 promotes nasopharyngeal carcinoma cell pathogenesis by inhibiting cellular apoptosis.

Nasopharyngeal carcinoma (NPC) is one of the most prevalent forms of highly invasive malignancy in Southern China and Southeast Asia. The pathogenesis of NPC is a multistep process driven by the acquisition of numerous genetic abnormalities. We investigated the potential oncogenic role of the Rho-guanine nucleotide exchange factor 3 gene, ARHGEF3, in NPC pathogenesis. Expression levels of ARHGEF3 were frequently up-regulated in NPC cell lines and tissues. In a large cohort of clinical NPC tissues high expression of ARHGEF3 was positively associated with an increased T status, distant metastasis, and a more advanced clinical stage (P < 0.05). Survival analysis revealed that ARHGEF3 expression was a significant and independent prognosis factor for NPC patients. In NPC cell lines, knockdown of ARHGEF3 was sufficient to inhibit cell growth, motility, and invasion in vitro, whereas ectopic overexpression of ARHGEF3 substantially enhanced NPC cells tumorigenesis and metastasis in vivo. Depletion of ARHGEF3 in NPC cells dramatically promoted caspase-3 induced apoptosis and an anti-apoptosis factor, BIRC8, was identified as a critical downstream target of the ARHGEF3. Our findings suggest that increased expression of ARHGEF3 plays a critical oncogenic role in NPC pathogenesis by preventing cell apoptosis through the up-regulation of BIRC8, and ARHGEF3 might be employed as a novel prognostic marker and effective therapeutic target for human NPC.

Evidence for transcriptional interference in a dual-luciferase reporter system.

The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3'untranslated region (3'UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3'UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems.