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Lung cancers are the leading cause of cancer-related mortality worldwide, and the majority of lung cancers are non–small cell lung carcinoma (NSCLC). Overexpressed or activated EGFR has been associated with a poor prognosis in NSCLC. We previously identified a circular noncoding RNA, hsa_circ_0000190 (C190), as a negative prognostic biomarker of lung cancer. Here, we attempted to dissect the mechanistic function of C190 and test the potential of C190 as a therapeutic target in NSCLC. C190 was upregulated in both NSCLC clinical samples and cell lines. Activation of the EGFR pathway increased C190 expression through a MAPK/ERK-dependent mechanism. Transient and stable overexpression of C190 induced ERK1/2 phosphorylation, proliferation, and migration in vitro and xenograft tumor growth in vivo. RNA sequencing and Expression2Kinases (X2K) analysis indicated that kinases associated with cell-cycle and global translation are involved in C190-activated networks, including CDKs and p70S6K, which were further validated by immunoblotting. CRISPR/Cas13a-mediated knockdown of C190 decreased proliferation and migration of NSCLC cells in vitro and suppressed tumor growth in vivo. TargetScan and CircInteractome databases predicted that C190 targets CDKs by sponging miR-142-5p. Analysis of clinical lung cancer samples showed that C190, CDK1, and CDK6 expressions were significantly higher in advanced-stage lung cancer than in early-stage lung cancer. In summary, C190 is directly involved in EGFR–MAPK–ERK signaling and may serve as a potential therapeutic target for the treatment of NSCLC. Significance: The circRNA C190 is identified as a mediator of multiple pro-oncogenic signaling pathways in lung cancer and can be targeted to suppress tumor progression.

Transglutaminase 2 (TG2) is a key epidermal squamous cell carcinoma cancer cell survival protein. However, how TG2 maintains the aggressive cancer phenotype is not well understood. The present studies show that TG2, which is highly expressed in epidermal cancer stem–like cells (ECS cells), maintains hepatocyte growth factor (HGF) signaling to drive an aggressive ECS cell cancer phenotype. Inhibiting TG2 reduces MET tyrosine kinase receptor expression and activity and attenuates the cancer cell phenotype. Moreover, inhibition of TG2 or HGF/MET function reduces downstream MEK1/2 and ERK1/2 activity, and this is associated with reduced cancer cell spheroid formation, invasion, and migration, and reduced stem and EMT marker expression. Treatment of TG2 knockdown cells with HGF partially restores the aggressive cancer phenotype, confirming that MET signaling is downstream of TG2. MET knockout reduces ERK1/2 signaling, doubles the time to initial tumor appearance, and reduces overall tumor growth. These findings suggest that TG2 maintains HGF/MET and MAPK (MEK1/2 and ERK1/2) signaling to drive the aggressive ECS cell cancer phenotype and tumor formation, and that TG2-dependent MET signaling may be a useful anti-cancer target. Implications: TG2 is an important epidermal squamous cell carcinoma stem cell survival protein. We show that TG2 activates an HGF/MET, MEK1/2 ERK1/2 signaling cascade that maintains the aggressive cancer phenotype.

Activation of Wnt signaling is among the earliest events in colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding β-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations affecting APC, a major β-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct β-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that MYC suppresses DKK3 transcription through one of miR-17-92 cluster miRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in Apc-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration/invasion in vitro and metastasis in a syngeneic orthotopic mouse colon cancer model. Implications: Our results suggest that the MYC→miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feed-forward oncogenic loop.

2021 The Authors., Melanoma is the deadliest skin cancer with a very poor prognosis in advanced stages. Although targeted and immune therapies have improved survival, not all patients benefit from these treatments. The mitogen-activated protein kinase ERK5 supports the growth of melanoma cells in vitro and in vivo. However, ERK5 inhibition results in cell-cycle arrest rather than appreciable apoptosis. To clarify the role of ERK5 in melanoma growth, we performed transcriptomic analyses following ERK5 knockdown in melanoma cells expressing BRAFV600E and found that cellular senescence was among the most affected processes. In melanoma cells expressing either wild-type or mutant (V600E) BRAF, both genetic and pharmacologic inhibition of ERK5 elicited cellular senescence, as observed by a marked increase in senescence-associated β-galactosidase activity and p21 expression. In addition, depletion of ERK5 from melanoma cells resulted in increased levels of CXCL1, CXCL8, and CCL20, proteins typically involved in the senescence-associated secretory phenotype. Knockdown of p21 suppressed the induction of cellular senescence by ERK5 blockade, pointing to p21 as a key mediator of this process. In vivo, ERK5 knockdown or inhibition with XMD8–92 in melanoma xenografts promoted cellular senescence. Based on these results, small-molecule compounds targeting ERK5 constitute a rational series of prosenescence drugs that may be exploited for melanoma treatment., The work in E. Rovida’s lab was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC, IG-15282 and IG-21349), by Ente Fondazione Cassa di Risparmio di Firenze (ECRF), and Universita degli Studi di Firenze (Fondo di Ateneo ex-60%). A. Tubita was supported by a “Carlo Zanotti” Fondazione Italiana per la Ricerca sul Cancro (FIRC)-AIRC fellowship (ID-23847).

High-risk human papillomaviruses (HPV), exemplified by HPV16/18, are causally linked to human cancers of the anogenital tract, skin, and upper aerodigestive tract. Previously, we identified Ecdysoneless (ECD) protein, the human homolog of the Drosophila ecdysoneless gene, as a novel HPV16 E6–interacting protein. Here, we show that ECD, through its C-terminal region, selectively binds to high-risk but not to low-risk HPV E6 proteins. We demonstrate that ECD is overexpressed in cervical and head and neck squamous cell carcinoma (HNSCC) cell lines as well as in tumor tissues. Using The Cancer Genome Atlas dataset, we show that ECD mRNA overexpression predicts shorter survival in patients with cervical and HNSCC. We demonstrate that ECD knockdown in cervical cancer cell lines led to impaired oncogenic behavior, and ECD co-overexpression with E7 immortalized primary human keratinocytes. RNA-sequencing analyses of SiHa cells upon ECD knockdown showed to aberrations in E6/E7 RNA splicing, as well as RNA splicing of several HPV oncogenesis–linked cellular genes, including splicing of components of mRNA splicing machinery itself. Taken together, our results support a novel role of ECD in viral and cellular mRNA splicing to support HPV-driven oncogenesis. Implications: This study links ECD overexpression to poor prognosis and shorter survival in HNSCC and cervical cancers and identifies a critical role of ECD in cervical oncogenesis through regulation of viral and cellular mRNA splicing.

Previous studies have demonstrated that glucocorticoid receptor β (GRβ) functions as an oncoprotein, regulating the malignant phenotypes and stem-like cell maintaining in human glioblastoma (GBM). Of the glucocorticoid receptor (GR) isoforms, GRβ and GRα are highly homologous, though the mechanism underlying the distinct functions of these two isoforms in GBM has not been clarified. Here by establishing a carboxyl-terminal (COOH-terminal) deletion mutant, we determined that GRβ can be ubiquitinated. We also found that its COOH terminal is essential for this ubiquitination. The mutation of a lysine to arginine at residue 733 (K733R) blocked the ubiquitination of GRβ, indicating that K733 is a key site for ubiquitination. Using K733R to establish nonubiquitinated GRβ, we demonstrated that ubiquitination not only regulates the stability and nuclear translocation of GRβ, but is also a vital mechanism for its oncogenic functions in vitro and in vivo. Protein interaction assay further indicated that ubiquitin-specific protease 49 (USP49) is a GRβ-binding protein and the interaction depends on GRβ ubiquitination. USP49 knockdown resulted in a decrease of cell proliferation, invasion, and an increase of cell apoptosis. More importantly, USP49 knockdown increased ubiquitination and amplified the oncogenic effects of GRβ, confirming the decisive role of ubiquitination on GRβ carcinogenicity. Taken together, these findings established that ubiquitination is a vial process for GRβ the execution of oncogenic functions in GBM and that the K733 site is crucial for ubiquitination of GRβ. Implications: This work is the first identify of the activation GRβ by a single lysine point-mediated ubiquitination and proteasome degradation, which determines its oncogenic functions in GBM.

Uterine leiomyosarcoma (ULMS) is a malignancy, which arises from the uterine smooth muscle. Because of its rarity, aggressive nature, and extremely poor prognosis, the molecular mechanisms driving ULMS remain elusive. To identify candidate cancer genes (CCG) driving ULMS, we conducted an in vivo Sleeping Beauty (SB) transposon mutagenesis screen in uterine myometrium–specific, PTEN knockout, KRAS mutant (PTEN KO/KRAS) mice. ULMS quickly developed in SB PTEN KO/KRAS mice, but not in PTEN KO/KRAS mice, demonstrating the critical importance of SB mutagenesis for driving ULMS in this model. Subsequent sequencing of SB insertion sites in these tumors identified 19 ULMS CCGs that were significantly enriched in known cancer genes. Among them, Zfp217 and Sfmbt2 functioned at early stages of tumor initiation and appeared to be oncogenes. Expression of ZNF217, the human homolog of ZFP217, was shown to be elevated in human ULMS compared with paired normal uterine smooth muscle, where it negatively correlated with patient prognosis. Inhibition of ZNF217 suppressed, whereas overexpression induced, proliferation, survival, migration, and stemness of human ULMS. In a second ex vivo ULMS SB metastasis screen, three CCGs were identified that may drive ULMS metastasis to the lung. One of these CCGs, Nrd1 (NRDC in humans), showed stronger expression in human metastatic tumors compared with primary ULMS and negatively associated with patient survival. NRDC knockdown impaired migration and adhesion without affecting cell proliferation, whereas overexpression had the opposite effect. Together, these results reveal novel mechanism driving ULMS tumorigenesis and metastasis and identify ZNF217 and NRDC as potential targets for ULMS therapy. Significance: An in vivo Sleeping Beauty transposon mutagenesis screen identifies candidate cancer genes that drive initiation and progression of uterine leiomyosarcoma and may serve as therapeutic targets.

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. Significance: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.

Esophageal squamous cell carcinoma (ESCC) often recurs after chemoradiotherapy, and the prognosis of ESCC after chemoradiotherapy has not improved over the past few decades. The mutation process in chemoradiotherapy-resistant clones and the functional relevance of genetic alterations remain unclear. To address these problems, we performed whole-exome sequencing of 52 tumor samples from 33 patients with ESCC who received radiotherapy combined with 5-fluorouracil/platinum. In multiregion analyses of pretreatment and locally recurrent lesions from five cases, most driver gene-altered clones remained under chemoradiotherapy selection pressure, while few driver gene alterations were acquired at recurrence. The mutation signatures of recurrent ESCC, including increased deletion frequency and platinum dose-dependent base substitution signatures, were substantially different from those of primary ESCC and reflected the iatrogenic impacts of chemoradiotherapy. Single-region analysis of 28 pretreatment tumors indicated that focal copy-number gain at the MYC locus was significantly associated with poor progression-free survival and overall survival after chemoradiotherapy. MYC gain remained throughout the chemoradiotherapy course and potentially contributes to intrinsic resistance to chemoradiotherapy. Consistent with these findings, MYC copy number and mRNA and protein levels in ESCC cell lines correlated positively with resistance to radiotherapy, and MYC knockdown improved sensitivity to radiotherapy. Overall, these data characterize the clonal evolution process induced by chemoradiotherapy and clinically relevant associations for genetic alterations in ESCC. These findings increase our understanding of therapeutic resistance and support the rationale for precision chemoradiotherapy. Significance: Whole-exome sequencing reveals the genetic evolution of ESCC during chemoradiotherapy, highlighting MYC gain in pretreatment tumors as a potential marker of therapy resistance.

Genomic alterations are crucial for the development and progression of human cancers. Copy-number gains found in genes encoding metabolic enzymes may induce triple-negative breast cancer (TNBC) adaptation. However, little is known about how metabolic enzymes regulate TNBC metastasis. Using our previously constructed multiomic profiling of a TNBC cohort, we identified decaprenyl diphosphate synthase subunit 1 (PDSS1) as an essential gene for TNBC metastasis. PDSS1 expression was significantly upregulated in TNBC tissues compared with adjacent normal tissues and was positively associated with poor survival among patients with TNBC. PDSS1 knockdown inhibited TNBC cell migration, invasion, and distant metastasis. Mechanistically, PDSS1, but not a catalytically inactive mutant, positively regulated the cellular level of coenzyme Q10 (CoQ10) and intracellular calcium levels, thereby inducing CAMK2A phosphorylation, which is essential for STAT3 phosphorylation in the cytoplasm. Phosphorylated STAT3 entered the nucleus, promoting oncogenic STAT3 signaling and TNBC metastasis. STAT3 phosphorylation inhibitors (e.g., Stattic) effectively blocked PDSS1-induced cell migration and invasion in vitro and tumor metastasis in vivo. Taken together, our study highlights the importance of targeting the previously uncharacterized PDSS1/CAMK2A/STAT3 oncogenic signaling axis, expanding the repertoire of precision medicine in TNBC. Significance: A novel metabolic gene PDSS1 is highly expressed in triple-negative breast cancer tissues and contributes to metastasis, serving as a potential therapeutic target for combating metastatic disease.

Purpose: Treatment with KRASG12C inhibitors such as sotorasib can produce substantial regression of tumors in some patients with non–small cell lung cancer (NSCLC). These patients require alternative treatment after acquiring resistance to the inhibitor. The mechanisms underlying this acquired resistance are unclear. The purpose of this study was to identify the mechanisms underlying acquired sotorasib resistance, and to explore potential treatments for rescuing patients with sotorasib-resistant KRASG12C NSCLC cells. Experimental Design: Clones of sotorasib-sensitive KRASG12C NSCLC H23 cells exposed to different concentrations of sotorasib were examined using whole-genomic transcriptome analysis, multiple receptor kinase phosphorylation analysis, and gene copy-number evaluation. The underlying mechanisms of resistance were investigated using immunologic examination, and a treatment aimed at overcoming resistance was tested in vitro and in vivo. Results: Unbiased screening detected subclonal evolution of MET amplification in KRASG12C NSCLC cells that had developed resistance to sotorasib in vitro. MET knockdown using small interfering RNA (siRNA) restored susceptibility to sotorasib in these resistant cells. MET activation by its amplification reinforced RAS cycling from its inactive form to its active form. In addition to RAS-mediated MEK–ERK induction, MET induced AKT activation independently of RAS. Crizotinib, a MET inhibitor, restored sensitivity to sotorasib by eliminating RAS–MEK–ERK as well as AKT signaling. MET/KRASG12C dual inhibition led to tumor shrinkage in sotorasib-resistant xenograft mice. Conclusions: MET amplification leads to the development of resistance to KRASG12C inhibitors in NSCLC. Dual blockade of MET and KRASG12C could be a treatment option for MET-amplified, KRASG12C-mutated NSCLC.

Expression of programmed death-ligand 1 (PD-L1) on cancer cells is a critical mechanism contributing to immunosuppression and immune escape. PD-L1 expression may also affect therapeutic outcomes of epidermal growth factor receptor (EGFR)-targeted therapy (e.g., with osimertinib/AZD9291) against EGFR-mutant non–small cell lung cancers (NSCLC) and can even be altered during the treatment albeit with largely undefined mechanisms. This study primarily focuses on elucidating the mechanism by which osimertinib induces PD-L1 degradation in addition to validating osimertinib's effect on decreasing PD-L1 expression in EGFR-mutant NSCLC cells and tumors. Osimertinib and other EGFR inhibitors effectively decreased PD-L1 levels primarily in EGFR-mutant NSCLCs and xenografted tumors. Osimertinib not only decreased PD-L1 mRNA expression, but also prompted proteasomal degradation of PD-L1 protein, indicating both transcriptional and posttranslational mechanisms accounting for osimertinib-induced reduction of PD-L1. Knockdown of β-TrCP or inhibition of GSK3 failed to prevent PD-L1 reduction induced by osimertinib. Rather, knockdown of membrane-associated RING-CH 8 (MARCH8) that encodes a membrane-bound E3 ubiquitin ligase rescued osimertinib-induced PD-L1 reduction. Furthermore, manipulation of MARCH8 expression accordingly altered PD-L1 degradation rate. Critically, MARCH8 interacted with PD-L1 through its N-terminal region and also ubiquitinated PD-L1 in cells. Collectively, these results strongly suggest that MARCH8 is a previously undiscovered E3 ubiquitin ligase responsible for PD-L1 degradation including osimertinib-induced PD-L1 degradation, establishing a novel connection between MARCH8 and PD-L1 regulation. Implications: This study has demonstrated a previously undiscovered function of MARCH8 in mediating PD-L1 degradation induced by EGFR inhibitors in EGFR-mutant NSCLC cells, establishing a novel connection between MARCH8 and PD-L1 regulation.

Controlling cell proliferation is critical for organism development, tissue homeostasis, disease, and regeneration. IQGAP3 has been shown to be required for proper cell proliferation and migration, and is associated to a number of cancers. Moreover, its expression is inversely correlated with the overall survival rate in the majority of cancers. Here, we show that IQGAP3 expression is elevated in cervical cancer and that in these cancers IQGAP3 high expression is correlated with an increased lethality. Furthermore, we demonstrate that IQGAP3 is a target of YAP, a regulator of cell cycle gene expression. IQGAP3 knockdown resulted in an increased percentage of HeLa cells in S phase, delayed progression through mitosis, and caused multipolar spindle formation and consequentially aneuploidy. Protein–protein interaction studies revealed that IQGAP3 interacts with MMS19, which is known in Drosophila to permit, by competitive binding to Xpd, Cdk7 to be fully active as a Cdk-activating kinase (CAK). Notably, IQGAP3 knockdown caused decreased MMS19 protein levels and XPD knockdown partially rescued the reduced proliferation rate upon IQGAP3 knockdown. This suggests that IQGAP3 modulates the cell cycle via the MMS19/XPD/CAK axis. Thus, in addition to governing proliferation and migration, IQGAP3 is a critical regulator of mitotic progression and genome stability. Implications: Our data indicate that, while IQGAP3 inhibition might be initially effective in decreasing cancer cell proliferation, this approach harbors the risk to promote aneuploidy and, therefore, the formation of more aggressive cancers.

Long noncoding RNAs (lncRNA) have been shown to play critical regulatory roles in the onset and progression of human cancers. However, the functions of a large proportion of lncRNAs are still unexplored. Here we describe a novel lncRNA, CTD-2245E15.3, that promotes lung tumorigenesis by regulating the anabolic enzymes acetyl-CoA carboxylase 1 (ACC1, encoded by the ACACA gene) and pyruvate carboxylase (PC). Differentially expressed lncRNAs between non–small cell lung cancer (NSCLC) and paired adjacent nontumor tissues were identified by a microarray and validated using quantitative real-time polymerase chain reaction. CTD-2245E15.3 was significantly upregulated in NSCLC and was mainly located in the cytoplasm. Knockdown of CTD-2245E15.3 by specific antisense oligonucleotides suppressed cell growth in vitro and in vivo, largely due to cell-cycle arrest and induction of apoptosis. Overexpression of CTD-2245E15.3 in an orthotopic model of lung cancer led to a significant increase in total tumor burden. CTD-2245E15.3 exerted its oncogenic function by binding ACC1 and PC, which are key anabolic factors for biomolecule synthesis in rapidly proliferating tumor cells. Knockdown of CTD-2245E15.3 increased phosphorylation of ACC1 at an inhibitory site for enzymatic activity and promoted PC degradation via ubiquitination. Supplements of palmitate or oxaloacetate, products of ACC1 and PC, alleviated the suppression of cell growth caused by loss of CTD-2245E15.3. These findings reveal the important role of CTD-2245E15.3 as an oncogenic lncRNA in the anabolic process for tumor growth. Significance: These findings demonstrate a novel lncRNA CTD-2245E15.3 that binds and positively regulates anabolic enzymes ACC1 and PC to promote tumor growth.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML. Significance: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML. See related commentary by Li and Melnick, p. 198.

Long noncoding RNAs (lncRNA) are involved in tumorigenesis and drug resistance. However, the roles and underlying mechanisms of lncRNAs in colorectal cancer are still unknown. In this work, through transcriptomic profiling analysis of 21 paired tumor and normal samples, we identified a novel colorectal cancer–related lncRNA, MNX1-AS1. MNX1-AS1 expression was significantly upregulated in colorectal cancer and associated with poor prognosis. In vitro and in vivo gain- and loss-of-function experiments showed that MNX1-AS1 promotes the proliferation of colorectal cancer cells. MNX1-AS1 bound to and activated Y-box-binding protein 1 (YB1), a multifunctional RNA/DNA-binding protein, and prevented its ubiquitination and degradation. A marked overlap between genes that are differentially expressed in MNX1-AS1 knockdown cells and transcriptional targets of YB1 was observed. YB1 knockdown mimicked the loss of viability phenotype observed upon depletion of MNX1-AS1. In addition, MYC bound the promoter of the MNX1-AS1 locus and activated its transcription. In vivo experiments showed that ASO inhibited MNX1-AS1, which suppressed the proliferation of colorectal cancer cells in both cell-based and patient-derived xenograft models. Collectively, these findings suggest that the MYC–MNX1-AS1–YB1 axis might serve as a potential biomarker and therapeutic target in colorectal cancer. Significance: This study highlights the discovery of a novel colorectal cancer biomarker and therapeutic target, MNX1-AS1, a long noncoding RNA that drives proliferation via a MYC/MNX1-AS1/YB1 signaling pathway.

Active IFNγ signaling is a common feature of tumors responding to PD-1 checkpoint blockade. IFNγ exhibits both anti- and protumor activities. Here, we show that the treatment of lung adenocarcinoma cells with IFNγ led to a rapid increase of ZEB1 expression and a significant change in epithelial-to-mesenchymal transition (EMT)-associated gene expression pattern. Moreover, functional analyses show that IFNγ promoted cell migration in vitro and metastasis in vivo. We demonstrate that ZEB1 is required for IFNγ-promoted EMT, cell migration, and metastasis, as RNAi-mediated knockdown of ZEB1 abrogated EMT, cell migration, and metastasis induced by IFNγ. We show that IFNγ induced upregulation of JMJD3 significantly reduced H3K27 trimethylation in the promoter of the ZEB1 gene, which led to activation of ZEB1 gene transcription. IFNγ-induced JMJD3 expression was JAK1/2-STAT1 dependent. Inhibition of JMJD3 abrogated IFNγ-induced ZEB1 expression. IFNγ-induced ZEB1 also reduced miR-200 expression. Downregulation of ZEB1 increased miR-200 expression, which led to a reduction of PD-L1 expression induced by IFNγ. It is worth noting that knockdown of ZEB1 did not affect IFNγ-mediated antiproliferation and induction of CXCL9 and CXCL10. Thus, downregulation of ZEB1 may prevent the protumor activity of IFNγ while retaining its antitumor function. This study expands our understanding of IFNγ-mediated signaling and helps to identify therapeutic targets to improve current immunotherapies.Implications:IFNγ increases ZEB1 expression in a STAT1-JMJD3 dependent manner, and consequently promotes cancer cell aggressiveness. This study provides a potential target to minimize the procancer effect of IFNγ while preserving its antitumor function.

The long noncoding RNA (lncRNA) SAMMSON is required for human melanoma cell growth and survival. However, whether SAMMSON regulates the response of mutant BRAF melanoma cells to RAF inhibitors remains unknown. In this work, we showed that SAMMSON is rapidly induced upon inhibition of ERK signaling, and SAMMSON overexpression conferred resistance to vemurafenib-induced cytotoxicity in melanoma cells. SOX10 mediated transcriptional induction of SAMMSON by vemurafenib, and SOX10 sumoylation at K55 was essential for this function. In addition, depletion of SAMMSON activated p53 signaling, which is dependent on the SAMMSON-interacting protein CARF. Depletion of SAMMSON sensitized mutant BRAF melanoma cells to RAF inhibitors in vitro and in vivo, while CARF knockdown reversed the enhanced sensitivity. In summary, these findings suggest that SAMMSON may function as a new mediator of adaptive resistance to RAF inhibitors in melanoma by modulating CARF-p53 signaling. Significance: This study highlights the role of a SAMMSON/CARF/p53 signaling axis in modulating the adaptive resistance of mutant BRAF melanoma to RAF inhibitors.

Ferroptosis is a new form of regulated cell death resulting from the accumulation of lipid-reactive oxygen species. A growing number of studies indicate ferroptosis as an important tumor suppressor mechanism having therapeutic potential in cancers. Previously, we identified TAZ, a Hippo pathway effector, regulates ferroptosis in renal and ovarian cancer cells. Because YAP (Yes-associated protein 1) is the one and only paralog of TAZ, sharing high sequence similarity and functional redundancy with TAZ, we tested the potential roles of YAP in regulating ferroptosis. Here, we provide experimental evidence that YAP removal confers ferroptosis resistance, whereas overexpression of YAP sensitizes cancer cells to ferroptosis. Furthermore, integrative analysis of transcriptome reveals S-phase kinase-associated protein 2 (SKP2), an E3 ubiquitin ligase, as a YAP direct target gene that regulates ferroptosis. We found that the YAP knockdown represses the expression of SKP2. Importantly, the genetic and chemical inhibitions of SKP2 robustly protect cells from ferroptosis. In addition, knockdown of YAP or SKP2 abolishes the lipid peroxidation during erastin-induced ferroptosis. Collectively, our results indicate that YAP, similar to TAZ, is a determinant of ferroptosis through regulating the expression of SKP2. Therefore, our results support the connection between Hippo pathway effectors and ferroptosis with significant therapeutic implications. Implications: This study reveals that YAP promotes ferroptosis by regulating SKP2, suggesting novel therapeutic options for YAP-driven tumors.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues. Implications: BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF-mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (TF), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. Significance: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities. This article is highlighted in the In This Issue feature, p. 521

Purpose: Glioblastomas (GBMs), neoplasms derived from glia and neuroglial progenitor cells, are the most common and lethal malignant primary brain tumors diagnosed in adults, with a median survival of 14 months. GBM tumorigenicity is often driven by genetic aberrations in receptor tyrosine kinases, such as amplification and mutation of EGFR. Experimental Design: Using a Drosophila glioma model and human patient–derived GBM stem cells and xenograft models, we genetically and pharmacologically tested whether the YAP and TAZ transcription coactivators, effectors of the Hippo pathway that promote gene expression via TEA domain (TEAD) cofactors, are key drivers of GBM tumorigenicity downstream of oncogenic EGFR signaling. Results: YAP and TAZ are highly expressed in EGFR-amplified/mutant human GBMs, and their knockdown in EGFR-amplified/mutant GBM cells inhibited proliferation and elicited apoptosis. Our results indicate that YAP/TAZ-TEAD directly regulates transcription of SOX2, C-MYC, and EGFR itself to create a feedforward loop to drive survival and proliferation of human GBM cells. Moreover, the benzoporphyrin derivative verteporfin, a disruptor of YAP/TAZ-TEAD–mediated transcription, preferentially induced apoptosis of cultured patient-derived EGFR-amplified/mutant GBM cells, suppressed expression of YAP/TAZ transcriptional targets, including EGFR, and conferred significant survival benefit in an orthotopic xenograft GBM model. Our efforts led us to design and initiate a phase 0 clinical trial of Visudyne, an FDA-approved liposomal formulation of verteporfin, where we used intraoperative fluorescence to observe verteporfin uptake into tumor cells in GBM tumors in human patients. Conclusions: Together, our data suggest that verteporfin is a promising therapeutic agent for EGFR-amplified and -mutant GBM.

CTDP1 (C-terminal domain phosphatase 1) is the only phosphatase domain with a BRCT-domain and known to be associated with transcription and mitosis in eukaryotic systems. Our previous research showed that CTDP1 participates in the DNA damage response by mediating interstrand DNA repair through the Fanconi anemia pathway. This study also revealed that CTDP1 is an essential gene in breast cancer cell lines but not in normal breast derived epithelial MCF10A cells. This project aims to identify the changes in transcripts, kinases and interactors leading to the cell death induced by CTDP1 knockdown in the triple-negative breast cancer cell lines. Since RNA Pol II is one of the major targets of CTDP1 phosphatase activity, we initially hypothesized that the cellular changes were mediated by transcriptional regulation. The global transcriptomic profiling of shCTDP1 MDA-MB-231 cells detected more than 29000 gene products, where 1000 and 616 genes were found consistently up- and down-regulated, respectively. The downregulation of cell cycle-associated genes CCNA2, TOP2A and POLA1 in the MDA-MB-231 cells were confirmed with q-RT-PCR. While the upregulated genes did not show enrichment in the Reactome pathway database, the downregulated genes were significantly associated with DNA damage response, mitosis and transcription, which is consistent with the known functions of CTDP1. The transcription factor binding motifs analysis with iRegulon reveals that the E2F1 transcription activity decreased as CTDP1 was knocked down in MDA-MB-231. The subsequent cell cycle analysis supports this prediction and showed G1 arrest as CTDP1 is knocked down in MDA-MB-231 and MDA-MB-453, but not in MCF10A. A kinase array analysis was also conducted to further identify kinases activated or deactivated as CTDP1 is knocked down. A nested network analysis of the predicted transcription factors, kinases and CTDP1 interactors is used to identify potential pathways contributing to cell death of the triple-negative breast cancer cells upon loss of CTDP1 expression. Citation Format: Henry Chun Hin Law, Nicholas Woods. A transcriptome approach to reveal CTDP1 knockdown induced G1 cell cycle arrest in triple negative breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-49.

Despite recent promising advances in targeted therapies and immunotherapies, patients with melanoma incur substantial mortality. In particular, inhibitors targeting BRAF-mutant melanoma can lead to resistance, and no targeted therapies exist for NRAS-mutant melanoma, motivating the search for additional therapeutic targets and vulnerable pathways. Here we identify a regulator of Wnt/β-catenin signaling, PLEKHA4, as a factor required for melanoma proliferation and survival. PLEKHA4 knockdown in vitro decreased Dishevelled levels, attenuated Wnt/β-catenin signaling, and blocked progression through the G1–S cell-cycle transition. In mouse xenograft and allograft models, inducible PLEKHA4 knockdown attenuated tumor growth in BRAF- and NRAS-mutant melanomas and exhibited an additive effect with the clinically used inhibitor encorafenib in a BRAF-mutant model. As an E3 ubiquitin ligase regulator with both lipid- and protein-binding partners, PLEKHA4 presents several opportunities for targeting with small molecules. Our work identifies PLEKHA4 as a promising drug target for melanoma and clarifies a controversial role for Wnt/β-catenin signaling in the control of melanoma proliferation. Significance: This study establishes that melanoma cell proliferation requires the protein PLEKHA4 to promote pathologic Wnt signaling for proliferation, highlighting PLEKHA4 inhibition as a new avenue for the development of targeted therapies.

Purpose: The clinical standard treatment for patients with malignant pleural mesothelioma (MPM) includes a cisplatin-based chemotherapy, leading to reduction of tumor size in only a minority of patients. Predicting response to chemotherapy in patients with MPM by using a genetic marker would, therefore, enable patient stratification. Experimental Design: In this retrospective biomarker study, eligible patients had resectable MPM, measurable disease, and available primary MPM tissue. All patients underwent first-line treatment with cisplatin and pemetrexed, followed by surgery. Thorough molecular analysis was performed (whole-exome and targeted deep sequencing, and copy-number analyses), and also mechanistic in vitro data (viability assays, Western blots, and immunoprecipitation) using mesothelioma cell lines with and without siRNA-mediated BRCA1-associated protein 1 (BAP1) knockdown were provided. Results: In a training cohort of patients with MPM (n = 28), mutations or deletions of BAP1 each predicted resistance to chemotherapy in patients with primary MPM. The negative predictive value of BAP1 loss in patients with MPM was confirmed by amplicon sequencing and copy-number array technology in an independent test cohort (n = 39). Preliminary mechanistic studies using siRNA-based knockdown of BAP1 in MPM cell culture models along with immunoprecipitation assays confirmed chemoresistance in vitro, possibly through inhibition of apoptosis and transcriptional regulation of the BAP1/HCF1/E2F1 axis. Conclusions: Alterations in BAP1 in MPM were a negative predictor for response to chemotherapy and could possibly be used as a companion biomarker for treatment decision.

Purpose: Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance. Experimental Design: We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer. Results: ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased. Conclusions: Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

Castration-resistant prostate cancer can be treated with the anti-androgen enzalutamide, but responses and duration of response are variable. To identify genes that support enzalutamide resistance, we performed a short hairpin RNA (shRNA) screen in the bone-homing, castration-resistant prostate cancer cell line, C4-2B. We identified eleven genes (TFAP2C, CAD, SPDEF, EIF6, GABRG2, CDC37, PSMD12, COL5A2, AR, MAP3K11, andACAT1), whose loss resulted in decreased cell survival in response to enzalutamide. To validate our screen, we performed transient knockdowns in C4-2B and 22Rv1 cells and evaluated cell survival in response to enzalutamide. Through these studies, we validated three genes (ACAT1, MAP3K11, andPSMD12) as supporters of enzalutamide resistancein vitro. AlthoughACAT1expression is lower in metastatic castration-resistant prostate cancer samples versus primary prostate cancer samples, knockdown ofACAT1was sufficient to reduce cell survival in C4-2B and 22Rv1 cells.MAP3K11expression increases with Gleason grade, and the highest expression is observed in metastatic castration-resistant disease. Knockdown ofMAP3K11reduced cell survival and pharmacologic inhibition of MAP3K11 with CEP-1347 in combination with enzalutamide resulted in a dramatic increase in cell death. This was associated with decreased phosphorylation of AR-Serine650, which is required for maximal AR activation. Finally, whilePSMD12expression did not change during disease progression, knockdown ofPSMD12resulted in decreased AR and AR splice variant expression, likely contributing to the C4-2B and 22Rv1 decrease in cell survival. Our study has therefore identified at least three new supporters of enzalutamide resistance in castration-resistant prostate cancer cellsin vitro.Financial supportThe authors would like to acknowledge funding from the Joe C. Davis Foundation (to RJM), the Vanderbilt Institute for Clinical and Translational Research (VICTR, to YY, PEC, and RJM). The Vanderbilt Institute for Clinical and Translational Research (VICTR) is funded by the National Center for Advancing Translational Sciences (NCATS) Clinical Translational Science Award (CTSA) Program, Award Number 5UL1TR002243. The content of this manuscript solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We would also like to acknowledge the Case Research Institute, a joint venture between University Hospitals and Case Western Reserve University, start-up funds (to MMG), and the Cell and Molecular Biology Training Program (T32 GM 008056 to SEK).

Metastatic dissemination remains a significant barrier to successful therapy for melanoma. Wnt5A is a potent driver of invasion in melanoma and is believed to be secreted from the tumor microenvironment (TME). Our data suggest that myeloid-derived suppressor cells (MDSC) in the TME are a major source of Wnt5A and are reliant upon Wnt5A for multiple actions. Knockdown of Wnt5A specifically in the myeloid cells demonstrated a clear decrease in Wnt5A expression within the TME in vivo as well as a decrease in intratumoral MDSC and regulatory T cell (Treg). Wnt5A knockdown also decreased the immunosuppressive nature of MDSC and decreased expression of TGFβ1 and arginase 1. In the presence of Wnt5A-depleted MDSC, tumor-infiltrating lymphocytes expressed decreased PD-1 and LAG3, suggesting a less exhausted phenotype. Myeloid-specific Wnt5A knockdown also led to decreased lung metastasis. Tumor-infiltrating MDSC from control animals showed a strong positive correlation with Treg, which was completely ablated in animals with Wnt5A-negative MDSC. Overall, our data suggest that while MDSC contribute to an immunosuppressive and less immunogenic environment, they exhibit an additional function as the major source of Wnt5A in the TME. Significance: These findings demonstrate that myeloid cells provide a major source of Wnt5A to facilitate metastatic potential in melanoma cells and rely on Wnt5A for their immunosuppressive function.

Ovarian clear cell carcinoma (OCCC) is a rare, chemo-resistant subtype of ovarian cancer. To identify novel therapeutic targets and combination therapies for OCCC, we subjected a set of patient-derived ovarian cancer cell lines to arrayed high-throughput siRNA and drug screening. The results indicated OCCC cells are vulnerable to knockdown of epigenetic gene targets such as bromodomain and extra-terminal domain (BET) proteins BRD2 and BRD3. Subsequent RNA interference assays, as well as BET inhibitor treatments, validated these BET proteins as potential therapeutic targets. Because development of resistance to single targeted agents is common, we next performed sensitizer drug screens to identify potential combination therapies with the BET inhibitor CPI0610. Several PI3K or AKT inhibitors were among the top drug combinations identified and subsequent work showed CPI0610 synergized with alpelisib or MK2206 by inducing p53-independent apoptosis. We further verified synergy between CPI0610 and PI3K–AKT pathway inhibitors alpelisib, MK2206, or ipatasertib in tumor organoids obtained directly from patients with OCCC. These findings indicate further preclinical evaluation of BET inhibitors, alone or in combination with PI3K-AKT inhibitors for OCCC, is warranted.

Circular RNAs (circRNA) are a new member of endogenously produced noncoding RNAs that have been characterized as key regulators of gene expression in a variety of malignances. However, the role of circRNA in oral squamous cell carcinoma (OSCC) remains largely unknown. In this study, we identified unique circRNA that regulate OSCC progression and metastasis and pave roads for future research in early diagnosis, prevention, and treatment of OSCC. Transcriptomic analyses identified a circRNA derived from IGHG locus (circIGHG) as significantly upregulated in OSCC and positively associated with poor prognosis of OSCC. circIGHG directly bound miR-142-5p and consequently elevated IGF2BP3 activity. Knockdown of circIGHG led to impaired expression of IGF2BP3 and attenuated aggressiveness of OSCC cells. Epithelial–mesenchymal transition was the main mechanism through which circIGHG/IGF2BP3 promotes metastasis of OSCC. Overall, these results demonstrate that circIGHG plays a pivotal role in OSCC development and metastasis and has potential to serve as a biomarker and therapeutic target for early-stage diagnosis and treatment of OSCC. Significance: These findings broaden our insights regarding regulation of OSCC progression by circular RNA and serve as a reference for future clinical research in OSCC diagnosis and treatment.

Hypoxia induces thousands of mRNAs and miRNAs to mediate tumor malignancy. However, hypoxia-induced long noncoding RNA (lncRNA) transcriptome and their role in triple-negative breast cancer (TNBC) have not been defined. Here we identified hypoxia-induced lncRNA transcriptome in two human TNBC cell lines by whole transcriptome sequencing. AC093818.1 was one of 26 validated lncRNAs and abundantly expressed in TNBC in vitro and in vivo. 5′- and 3′-rapid amplification of cDNA ends assays revealed that the isoform 2 was a dominant AC093818.1 transcript in TNBC cells and thus referred to as lncIHAT (lncRNA induced by hypoxia and abundant in TNBC). Hypoxia-inducible factor 1 (HIF1) but not HIF2 bound to the hypoxia response element at the promoter of lncIHAT to activate its transcription in hypoxic TNBC cells. LncIHAT promoted TNBC cell survival in vitro and tumor growth and lung metastasis in mice. Mechanistically, lncIHAT was required for the expression of its proximal neighboring oncogenic genes PDK1 and ITGA6 in TNBC cells and tumors. Reexpression of PDK1 and ITGA6 rescued survival and growth of lncIHAT knockdown TNBC cells in vitro. Collectively, these findings uncovered lncIHAT as a new hypoxia-induced oncogenic cis-acting lncRNA in TNBC. Implications: This study systematically identified hypoxia-induced lncRNA transcriptome in TNBC and sheds light on multiple layers of regulatory mechanisms of gene expression under hypoxia.

The NF-E2–related factor 2 (referred to as NRF2) transcription factor binds antioxidant responsive elements within the promoters of cytoprotective genes to induce their expression. Next-generation sequencing studies in lung cancer have shown a significant number of activating mutations within the NRF2 signaling pathway. Mutations in components of the SWI/SNF chromatin-remodeling complex, a general regulator of transcription using either BRG1 or BRM as the catalytic subunit, also frequently occur in lung cancers. Importantly, low BRG1 expression levels in primary human NSCLC correlated with increased NRF2-target gene expression. Here, we show that loss of SWI/SNF complex function activated a subset of NRF2-mediated transcriptional targets. Using a series of isogenic NSCLC lines with reduced or depleted BRG1 and/or BRM expression, we observed significantly increased expression of the NRF2-target genes HMOX1 and GSTM4. In contrast, expression of the NRF2 target genes NQO1 and GCLM modestly increased following BRM reduction. Chromatin immunoprecipitation showed that BRG1 knockdown led to increased NRF2 binding at its respective ARE sites in the HMOX1 promoter but not in NQO1 and GCLM. Our data demonstrate that loss of BRG1 or BRM in lung cancer results in activation of the NRF2/KEAP1 pathway and HMOX1 expression. Therefore, we provide an additional molecular explanation for why patients harboring BRG1 or BRM mutations show poor prognoses. A better understanding of this mechanism may yield novel insights into the design of targeted treatment modalities. Implications: Our study identifies a novel mechanism for how mutations in the SMARCA4 gene may drive progression of human lung adenocarcinomas.

The IL6 family of cytokines, including IL6 and leukemia-inhibitory factor (LIF), are induced during inflammation and are also expressed in many types of cancer where they play an important role in tumor development. IL6 family cytokines mainly activate the JAK–STAT3 pathway via the coreceptor, gp130, and IL6 is known to activate the Src family kinase (SFK)–Yes-associated protein (YAP) pathway. The current study investigated the role of autocrine LIF in human esophageal squamous cell carcinoma (ESCC) that highly expresses LIF. LIF knockdown had various effects on cancer cells, including profound changes in gene expression, suppression of cell proliferation, migration/invasion and sphere formation, and induction of apoptosis. Similar to IL6, LIF activated the SFK–YAP pathway as well as the JAK–STAT3 pathway. LIF-induced YAP activation was more important for cancer cell proliferation than LIF-induced STAT3 activation, and concomitant YAP and STAT3 activation completely compensated for the role of LIF in human ESCC growth. We also confirmed that SFK activation and LIF expression were correlated with YAP activation in human ESCC clinical samples. Furthermore, simultaneous inhibition of the SFK–YAP and JAK–STAT3 pathways in human ESCC cells was more effective at suppressing cell proliferation than single inhibition, and autocrine LIF signaling promoted human ESCC growth in vivo. Therefore, the LIF–SFK–YAP axis may represent a new therapeutic target for human ESCC. Implications: Autocrine LIF signaling promotes human ESCC progression via SFK-dependent YAP activation and is a new potential target of treatment for human ESCC.

Triple-negative breast cancers contain a spectrum of epithelial and mesenchymal phenotypes. SUM-229PE cells represent a model for this heterogeneity, maintaining both epithelial and mesenchymal subpopulations that are genomically similar but distinct in gene expression profiles. We identified differential regions of open chromatin in epithelial and mesenchymal cells that were strongly correlated with regions of H3K27ac. Motif analysis of these regions identified consensus sequences for transcription factors that regulate cell identity. Treatment with the MEK inhibitor trametinib induced enhancer remodeling that is associated with transcriptional regulation of genes in epithelial and mesenchymal cells. Motif analysis of enhancer peaks downregulated in response to chronic treatment with trametinib identified AP-1 motif enrichment in both epithelial and mesenchymal subpopulations. Chromatin immunoprecipitation sequencing (ChIP-seq) of JUNB identified subpopulation-specific localization, which was significantly enriched at regions of open chromatin. These results indicate that cell identity controls localization of transcription factors and chromatin-modifying enzymes to enhancers for differential control of gene expression. We identified increased H3K27ac at an enhancer region proximal to CXCR7, a G-protein–coupled receptor that increased 15-fold in expression in the epithelial subpopulation during chronic treatment. RNAi knockdown of CXCR7 inhibited proliferation in trametinib-resistant cells. Thus, adaptive resistance to chronic trametinib treatment contributes to proliferation in the presence of the drug. Acquired amplification of KRAS following trametinib dose escalation further contributed to POS cell proliferation. Adaptive followed by acquired gene expression changes contributed to proliferation in trametinib-resistant cells, suggesting inhibition of early transcriptional reprogramming could prevent resistance and the bypass of targeted therapy. Implications: We defined the differential responses to trametinib in subpopulations of a clinically relevant in vitro model of TNBC, and identified both adaptive and acquired elements that contribute to the emergence of drug resistance mediated by increased expression of CXCR7 and amplification of KRAS.

Amplification and overexpression of the MYC oncogene in tumor cells, including ovarian cancer cells, correlates with poor responses to chemotherapy. As MYC is not directly targetable, we have analyzed molecular pathways downstream of MYC to identify potential therapeutic targets. Here we report that ovarian cancer cells overexpressing glutaminase (GLS), a target of MYC and a key enzyme in glutaminolysis, are intrinsically resistant to platinum-based chemotherapy and are enriched with intracellular antioxidant glutathione. Deprivation of glutamine by glutamine-withdrawal, GLS knockdown, or exposure to the GLS inhibitor CB-839 resulted in robust induction of reactive oxygen species in high GLS-expressing but not in low GLS-expressing ovarian cancer cells. Treatment with CB-839 rendered GLShigh cells vulnerable to the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, and prolonged survival in tumor-bearing mice. These findings suggest consideration of applying a combined therapy of GLS inhibitor and PARP inhibitor to treat chemoresistant ovarian cancers, especially those with high GLS expression. Significance: Targeting glutaminase disturbs redox homeostasis and nucleotide synthesis and causes replication stress in cancer cells, representing an exploitable vulnerability for the development of effective therapeutics.

PTEN deletion or mutation occurs in 30% to 60% of patients with glioblastoma (GBM) and is associated with poor prognosis. Efficacious therapy for this subgroup of patients is currently lacking. To identify potential target(s) to selectively suppress PTEN-deficient GBM growth, we performed a three-step synthetic lethal screen on LN18 PTEN wild-type (WT) and knockout (KO) isogeneic GBM cell lines using a library containing 606 target-selective inhibitors. A MCL1 inhibitor UMI-77 identified in the screen exhibited excellent suppression on the proliferation, colony formation, 3D spheroid, and neurosphere formation of PTEN-deficient GBM cells. Mechanistically, loss of PTEN in GBM cells led to upregulation of MCL1 in posttranslational level via inhibition of GSK3β, and consequently confer cells resistance to apoptosis. Pharmacologic inhibition or knockdown of MCL1 blocked this PI3K–GSK3β–MCL1 axis and caused reduction of several antiapoptotic proteins, finally induced massive caspase-3 cleavage and apoptosis. In both subcutaneous and orthotopic GBM models, knockdown of MCL1 significantly impaired the in vivo growth of PTEN-deficient xenografts. Moreover, the combination of UMI-77 and temozolomide synergistically killed PTEN-deficient GBM cells. Collectively, our work identified MCL1 as a promising target for PTEN-deficient GBM. For future clinical investigations, priority should be given to the development of a selective MCL1 inhibitor with efficient brain delivery and minimal in vivo toxicity.

Elevated expression of EZH2, the enzymatic subunit of polycomb repressive complex 2 (PRC2), often occurs in cancer. EZH2 expression results in the silencing of genes that suppress tumor formation and metastasis through trimethylation of histone H3 at lysine 27 (H3K27me3) at their promoters. However, inhibitors of EZH2 enzymatic activity have not shown the expected efficacy against cancer in clinical trials, suggesting a need for other strategies to address EZH2 overexpression. Here, we show that SUMOylation, a posttranslational modification characterized by covalent attachment of small ubiquitin-like modifier (SUMO) proteins to a lysine (Lys) residue on target proteins, enhances EZH2 transcription. Either knockdown of the SUMO-activating enzyme SAE2 or pharmacologic inhibition of SUMOylation resulted in decreased levels of EZH2 mRNA and protein as well as reduced H3K27me3 levels. SUMOylation regulated EZH2 expression by enhancing binding of the E2F1 transcriptional activator to the EZH2 promoter. Inhibition of SUMOylation not only resulted in reduced EZH2 mRNA and protein levels but also increased expression of genes silenced by EZH2, such as E-cadherin, which suppresses epithelial–mesenchymal transition and metastasis. In more than 6,500 patient tumor samples across different cancer types, expression of UBA2 and EZH2 was positively correlated. Taken together, our findings suggest that inhibition of SUMOylation may serve as a potential strategy to address EZH2 overexpression and improve current cancer therapeutic approaches. Significance: These findings provide important biological insights into the mechanism of EZH2 overexpression in cancers and suggest that inhibiting SUMOylation may improve current cancer therapeutic approaches.

The R-spondin family plays important roles in embryonic development, including in humans. However, information on the relationship between R-spondin2 and hepatocellular carcinoma (HCC) is lacking. This study aimed was to explore the mechanisms of R-spondin2 action in the progression of HCC. By analyzing R-spondin2 expression levels in HCC tissues by IHC and database, we identified that HCC tissues had lower expression levels of R-spondin2, correlated with a poor prognosis. We also established R-spondin2–overexpressing and knockdown cell lines and measured their viabilities and invasion abilities in vitro and their oncogenic capacity in vivo. Human mRNA microarray analysis was performed to screen for mRNAs that were differentially expressed between R-spondin2–overexpressing and control HCC cells. Microarray and Western blot analyses showed significant changes in the MAPK signaling pathway after transfection. Furthermore, in vivo experiments indicated that R-spondin2 knockdown increased the tumorigenicity of HCC cells after subcutaneous implantation in mice. Altogether, our results indicate that the R-spondin2, which might be a novel tumor suppressor gene, were responsible for inhibiting the proliferation and invasion of HCC via the MAPK signaling pathway. Implications: R-spondin2 gene might be a novel tumor suppressor gene providing new clues to clarify the biological behavior of HCC and thus reduce patient mortality and prolong survival.

Although intravesical bacillus Calmette-Guérin (BCG) immunotherapy has been the gold standard for nonsurgical management of non–muscle-invasive bladder cancer, a considerable number of patients exhibit resistance to the adjuvant treatment with unexplained mechanisms. This study aimed to investigate whether and how androgen receptor (AR) signals modulate BCG cytotoxicity in bladder cancer. AR knockdown or overexpression in bladder cancer lines resulted in induction or reduction, respectively, in intracellular BCG quantity and its cytotoxic activity. Microarray screening identified Rab27b, a small GTPase known to mediate bacterial exocytosis, which was upregulated in BCG-resistant cells and downregulated in AR-shRNA cells. Knockdown of Rab27b, or its effector SYTL3, or overexpression of Rab27b also induced or reduced, respectively, BCG quantity and cytotoxicity. In addition, treatment with GW4869, which was previously shown to inhibit Rab27b-dependent secretion, induced them and reduced Rab27b expression in bladder cancer cells. Meanwhile, AR expression was upregulated in BCG-resistant lines, compared with respective controls. In a mouse orthotopic xenograft model, Rab27b/SYTL3 knockdown or GW4869 treatment enhanced the amount of BCG within tumors and its suppressive effect on tumor growth. Moreover, in non–muscle-invasive bladder cancer specimens from patients subsequently undergoing BCG therapy, positivity of AR/Rab27b expression was associated with significantly higher risks of tumor recurrence. AR activation thus correlates with resistance to BCG treatment, presumably via upregulating Rab27b expression. Mechanistically, it is suggested that BCG elimination from urothelial cells is induced by Rab27b/SYTL3-mediated exocytosis. Accordingly, Rab27b inactivation, potentially via antiandrogenic drugs and/or exocytosis inhibition are anticipated to sensitize the efficacy of BCG therapy, especially in patients with BCG-refractory AR/Rab27b-positive bladder cancer.

The majority of patients with prostate cancer treated with docetaxel develop resistance to it. To better understand the mechanism behind the acquisition of resistance, we conducted single-cell RNA-sequencing (scRNA-seq) of docetaxel-sensitive and -resistant variants of DU145 and PC3 prostate cancer cell lines. Overall, sensitive and resistant cells clustered separately. Differential gene expression analysis between resistant and sensitive cells revealed 182 differentially expressed genes common to both prostate cancer cell lines. A subset of these genes gave a gene expression profile in the resistant transcriptome-like–sensitive cells similar to the resistant cells. Exploration for functional gene pathways identified 218 common pathways between the two cell lines. Protein ubiquitination was the most differentially regulated pathway and was enriched in the resistant cells. Transcriptional regulator analysis identified 321 potential regulators across both cell lines. One of the top regulators identified was nuclear protein 1 (NUPR1). In contrast to the single-cell analysis, bulk analysis of the cells did not reveal NUPR1 as a promising candidate. Knockdown and overexpression of NUPR1 in the prostate cancer cells demonstrated that NUPR1 confers docetaxel resistance in both cell lines. Collectively, these data demonstrate the utility of scRNA-seq to identify regulators of drug resistance. Furthermore, NUPR1 was identified as a mediator of prostate cancer drug resistance, which provides the rationale to explore NUPR1 and its target genes for reversal of docetaxel resistance. Implications: Using single-cell sequencing of prostate cancer, we show that NUPR1 plays a role in docetaxel resistance.

Prostate cancer chemoprevention by sulforaphane, which is a metabolic by-product of glucoraphanin found in broccoli, in preclinical models is associated with induction of both apoptosis and autophagy. However, the molecular mechanism underlying sulforaphane-mediated autophagy, which is protective against apoptotic cell death by this phytochemical, is still poorly understood. This study demonstrates a role for lysosome-associated membrane protein 2 (LAMP2) in sulforaphane-mediated autophagy and apoptosis. Western blotting revealed dose-dependent induction of LAMP2 protein after treatment with sulforaphane as well as its naturally occurring analogs in PC-3 and 22Rv1 human prostate cancer cell lines that was confirmed by microscopy (sulforaphane). The mRNA level of LAMP2 was also increased upon treatment with sulforaphane in both cell lines. Sulforaphane-mediated increase in the level of autophagy marker microtubule-associated protein light-chain 3B was augmented by RNAi of LAMP2 in PC-3 and 22Rv1 cells. Apoptosis induction by sulforaphane treatment was also increased significantly by knockdown of the LAMP2 protein in PC-3 and 22Rv1 cells. Augmentation of sulforaphane-mediated apoptosis by RNAi of LAMP2 was accompanied by induction and activation of proapoptotic protein Bak. Oral administration of sulforaphane to TRAMP mice also resulted in induction of LAMP2 protein expression. Targeted microarray in sulforaphane-treated PC-3 cells revealed induction of many autophagy-related genes (e.g., HSP90AA1, NRF2, etc.) and their expression positively correlated with that of LAMP2 in prostate cancer The Cancer Genome Atlas. In conclusion, this study reveals that induction of LAMP2 by sulforaphane inhibits its ability to induce apoptotic cell death at least in human prostate cancer cells.

Proneural-to-mesenchymal transition (PMT) is a common process in glioblastoma (GBM) progression that leads to increased radiotherapy resistance. However, the mechanism underlying PMT is poorly understood. Here, we found that tumor-associated macrophages triggered PMT in glioma stem cells (GSC) via small extracellular vesicles (sEV). sEVs from monocyte-derived macrophages transferred miR-27a-3p, miR-22-3p, and miR-221-3p to GSCs, and these miRNAs promoted several mesenchymal phenotypes in proneural (PN) GSCs by simultaneously targeting CHD7. We found that CHD7 played a critical role in the maintenance of the PN phenotype, and CHD7 knockdown significantly promoted PMT in GSCs via the RelB/P50 and p-STAT3 pathways. The induction of PMT by sEVs containing miR-27a-3p, miR-22-3p, and miR-221-3p in a xenograft nude mouse model exacerbated radiotherapy resistance and thus decreased the benefits of radiotherapy. Collectively, these findings identified macrophage-derived sEVs as key regulators of PMT in GSCs and demonstrated that CHD7 is a novel inhibitor of PMT.

Purpose: EGFR tyrosine kinase inhibitors (EGFR-TKI) benefit patients with advanced lung adenocarcinoma (ADC) harboring activating EGFR mutations. We aimed to identify biomarkers to monitor and predict the progression of patients receiving EGFR-TKIs via a comprehensive omic analysis. Experimental Design: We applied quantitative proteomics to generate the TKI resistance–associated pleural effusion (PE) proteome from patients with ADC with or without EGFR-TKI resistance. Candidates were selected from integrated genomic and proteomic datasets. The PE (n = 33) and serum (n = 329) levels of potential biomarkers were validated with ELISAs. Western blotting was applied to detect protein expression in tissues, PEs, and a cell line. Gene knockdown, TKI treatment, and proliferation assays were used to determine EGFR-TKI sensitivity. Progression-free survival (PFS) and overall survival (OS) were assessed to evaluate the prognostic values of the potential biomarkers. Results: Fifteen proteins were identified as potential biomarkers of EGFR-TKI resistance. Cadherin-3 (CDH3) was overexpressed in ADC tissues compared with normal tissues. CDH3 knockdown enhanced EGFR-TKI sensitivity in ADC cells. The PE level of soluble CDH3 (sCDH3) was increased in patients with resistance. The altered sCDH3 serum level reflected the efficacy of EGFR-TKI after 1 month of treatment (n = 43). Baseline sCDH3 was significantly associated with PFS and OS in patients with ADC after EGFR-TKI therapy (n = 76). Moreover, sCDH3 was positively associated with tumor stage in non–small cell lung cancer (n = 272). Conclusions: We provide useful marker candidates for drug resistance studies. sCDH3 is a survival predictor and real-time indicator of treatment efficacy in patients with ADC treated with EGFR-TKIs.

TAp63 is a p53 family member and potent tumor and metastasis suppressor. Here, we show that TAp63−/− mice exhibit an increased susceptibility to ultraviolet radiation–induced cutaneous squamous cell carcinoma (cuSCC). A human-to-mouse comparison of cuSCC tumors identified miR-30c-2* and miR-497 as underexpressed in TAp63-deficient cuSCC. Reintroduction of these miRNAs significantly inhibited the growth of cuSCC cell lines and tumors. Proteomic profiling of cells expressing either miRNA showed downregulation of cell-cycle progression and mitosis-associated proteins. A mouse to human and cross-platform comparison of RNA-sequencing and proteomics data identified a 7-gene signature, including AURKA, KIF18B, PKMYT1, and ORC1, which were overexpressed in cuSCC. Knockdown of these factors in cuSCC cell lines suppressed tumor cell proliferation and induced apoptosis. In addition, selective inhibition of AURKA suppressed cuSCC cell proliferation, induced apoptosis, and showed antitumor effects in vivo. Finally, treatment with miR-30c-2* or miR-497 miRNA mimics was highly effective in suppressing cuSCC growth in vivo. Our data establish TAp63 as an essential regulator of novel miRNAs that can be therapeutically targeted for potent suppression of cuSCC. Significance: This study provides preclinical evidence for the use of miR-30c-2*/miR-497 delivery and AURKA inhibition in the treatment of cuSCC, which currently has no FDA-approved targeted therapies. See related commentary by Parrales and Iwakuma, p. 2439

Purpose: ROS1 tyrosine kinase inhibitors (TKI) provide significant benefit in lung adenocarcinoma patients with ROS1 fusions. However, as observed with all targeted therapies, resistance arises. Detecting mechanisms of acquired resistance (AR) is crucial to finding novel therapies and improve patient outcomes. Experimental Design: ROS1 fusions were expressed in HBEC and NIH-3T3 cells either by cDNA overexpression (CD74/ROS1, SLC34A2/ROS1) or CRISPR-Cas9–mediated genomic engineering (EZR/ROS1). We reviewed targeted large-panel sequencing data (using the MSK-IMPACT assay) patients treated with ROS1 TKIs, and genetic alterations hypothesized to confer AR were modeled in these cell lines. Results: Eight of the 75 patients with a ROS1 fusion had a concurrent MAPK pathway alteration and this correlated with shorter overall survival. In addition, the induction of ROS1 fusions stimulated activation of MEK/ERK signaling with minimal effects on AKT signaling, suggesting the importance of the MAPK pathway in driving ROS1 fusion-positive cancers. Of 8 patients, 2 patients harbored novel in-frame deletions in MEK1 (MEK1delE41_L54) and MEKK1 (MEKK1delH907_C916) that were acquired after ROS1 TKIs, and 2 patients harbored NF1 loss-of-function mutations. Expression of MEK1del or MEKK1del, and knockdown of NF1 in ROS1 fusion-positive cells activated MEK/ERK signaling and conferred resistance to ROS1 TKIs. Combined targeting of ROS1 and MEK inhibited growth of cells expressing both ROS1 fusion and MEK1del. Conclusions: We demonstrate that downstream activation of the MAPK pathway can mediate of innate acquired resistance to ROS1 TKIs and that patients harboring ROS1 fusion and concurrent downstream MAPK pathway alterations have worse survival. Our findings suggest a treatment strategy to target both aberrations.

The detailed biological functions of circular RNA (circRNA) are largely unexplored. Using circRNA sequencing, we identified 169 differentially expressed circRNA in pancreatic ductal adenocarcinoma (PDAC) cells compared with nontumor human pancreatic ductal epithelial cells. Among them, circFOXK2 was validated with significant upregulation in PDAC cells and 63% of primary tumors (53 of 84). circFOXK2 promoted cell growth, migration, and invasion and was involved in cell-cycle progression and apoptosis. circFOXK2 contained multiple miRNA binding sites, functioning as a sponge for miR-942, which in turn promoted expression of ANK1, GDNF, and PAX6. A novel and highly specific circRNA-pulldown followed by mass spectrometry analysis identified 94 circFOXK2-interacting proteins, which were involved in cell adhesion, mRNA splicing, and structural molecule activity. Of these, circFOKX2 interactions with YBX1 and hnRNPK enhanced expression of oncogenes NUF2 and PDXK. Knockdown of circFOXK2 reduced binding of YBX1 and hnRNPK to NUF2 and PDXK, in turn decreasing their expression. Collectively, our findings demonstrate that circFOXK2 in complex with YBX1 and hnRNPK promotes expression of oncogenic proteins that contribute to PDAC progression. Significance: This study reveals a prominent role for the circRNA circFOXK2 in PDAC progression, suggesting that circFOXK2 might be a novel diagnostic marker for PDAC.

Endocrine therapy is standard treatment for estrogen receptor (ER)-positive breast cancer, yet long-term treatment often causes acquired resistance, which results in recurrence and metastasis. Recent studies have revealed that RNA-binding proteins (RBP) are involved in tumorigenesis. Here, we demonstrate that PSF/SFPQ is an RBP that potentially predicts poor prognosis of patients with ER-positive breast cancer by posttranscriptionally regulating ERα (ESR1) mRNA expression. Strong PSF immunoreactivity correlated with shorter overall survival in patients with ER-positive breast cancer. PSF was predominantly expressed in a model of tamoxifen-resistant breast cancer cells, and depletion of PSF attenuated proliferation of cultured cells and xenografted tumors. PSF expression was significantly associated with estrogen signaling. PSF siRNA downregulated ESR1 mRNA by inhibiting nuclear export of the RNA. Integrative analyses of microarray and RNA immunoprecipitation sequencing also identified SCFD2, TRA2B, and ASPM as targets of PSF. Among the PSF targets, SCFD2 was a poor prognostic indicator of breast cancer and SCFD2 knockdown significantly suppressed breast cancer cell proliferation. Collectively, this study shows that PSF plays a pathophysiologic role in ER-positive breast cancer by posttranscriptionally regulating expression of its target genes such as ESR1 and SCFD2. Overall, PSF and SCFD2 could be potential diagnostic and therapeutic targets for primary and hormone-refractory breast cancers. Significance: This study defines oncogenic roles of RNA-binding protein PSF, which exhibits posttranscriptional regulation in ER-positive breast cancer.

TNF-related apoptosis-inducing ligand (TRAIL) and an agonistic antibody against the death-inducing TRAIL receptor 5, DR5, are thought to selectively induce tumor cell death and therefore, have gained attention as potential therapeutics currently under investigation in several clinical trials. However, some tumor cells are resistant to TRAIL/DR5–induced cell death, even though they express DR5. Previously, we reported that DR5 is transported into the nucleus by importin β1, and knockdown of importin β1 upregulates cell surface expression of DR5 resulting in increased TRAIL sensitivity in vitro. Here, we examined the impact of importin β1 knockdown on agonistic anti-human DR5 (hDR5) antibody therapy. Drug-inducible importin β1 knockdown sensitizes HeLa cells to TRAIL-induced cell death in vitro, and exerts an antitumor effect when combined with agonistic anti-hDR5 antibody administration in vivo. Therapeutic importin β1 knockdown, administered via the atelocollagen delivery system, as well as treatment with the importin β inhibitor, importazole, induced regression and/or eradication of two human TRAIL-resistant tumor cells when combined with agonistic anti-hDR5 antibody treatment. Thus, these findings suggest that the inhibition of importin β1 would be useful to improve the therapeutic effects of agonistic anti-hDR5 antibody against TRAIL-resistant cancers.

Long noncoding RNAs (lncRNA) are emerging as crucial regulators of cell biology. However, the role of lncRNAs in the development and function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) remains unclear. Here, we identified that the lncRNA F730016J06Rik (AK036396) was highly expressed in PMN-MDSCs and that lncRNA AK036396 knockdown promoted the maturation and decreased the suppressive function of PMN-MDSCs. Ficolin B (Fcnb), the expression of which could be assessed as a surrogate for PMN-MDSC development, was the predicted target gene of lncRNA AK036396 based on microarray results. LncRNA AK036396 knockdown attenuated Fcnb protein stability in a manner dependent on the ubiquitin-proteasome system. Moreover, Fcnb inhibition downregulated the suppressive function of PMN-MDSCs. In addition, the expression of human M-ficolin, which is an ortholog of mouse Fcnb, was increased and positively correlated with arginase1 (ARG1) expression. This suppressive molecule is released by MDSCs, and its production is commonly used to represent the suppressive activity of MDSCs in patients with lung cancer, suggesting clinical relevance for these findings. These results indicate that lncRNA AK036396 can inhibit maturation and accelerate immunosuppression of PMN-MDSCs by enhancing Fcnb protein stability.

Purpose: Long noncoding RNAs (lncRNA) have been observed in various cancer types. Our bioinformatic analysis of existing databases demonstrated overexpression of lncRNA THAP9-AS1 in pancreatic ductal adenocarcinoma (PDAC). We aimed to investigate the roles and mechanisms of THAP9-AS1 in PDAC. Experimental Design: The overexpression of THAP9-AS1 in samples of patients with pancreatic cancer was characterized and was associated with clinical outcomes. The nonprotein coding property of the THAP9-AS1 was verified. Various in vitro and in vivo experiments were performed to investigate the interaction between THAP9-AS1 and YAP signaling. Results: We demonstrated that lncRNA THAP9-AS1 is overexpressed in PDAC in multiple patient sample sets, which is significantly associated with poor outcome of patients with PDAC. THAP9-AS1 promotes PDAC cells growth both in vitro and in vivo. THAP9-AS1 exerts its effects via enhancing YAP signaling. Ectopic YAP expression overcame the effects of THAP9-AS1 knockdown. Inversely, YAP knockdown diminished the effects of THAP9-AS1 overexpression. THAP9-AS1 acts as a competing endogenous RNA for miR-484, leading to YAP upregulation. Moreover, THAP9-AS1 binds to YAP protein and inhibits the phosphorylation-mediated inactivation of YAP by LATS1. Reciprocally, YAP/TEAD1 complex promotes THAP9-AS1 transcription to form a feed-forward circuit. Importantly, THAP9-AS1 level positively correlates with YAP expression in PDAC tissues. YAP overexpression also predicts a poor outcome in patients with PDAC. Conclusions: Our findings indicate that THAP9-AS1 plays an important role in PDAC growth via enhancing YAP signaling, which in turn also modulates THAP9-AS1 transcription. THAP9-AS1/YAP axis may serve as a potential biomarker and therapeutic target for PDAC treatment.