Microproteins encoded by noncanonical ORFs are a major source of tumor-specific antigens in a liver cancer patient meta-cohort.

The expression of tumor-specific antigens during cancer progression can trigger an immune response against the tumor. Here, we investigate if microproteins encoded by noncanonical open reading frames (ncORFs) are a relevant source of tumor-specific antigens. We analyze RNA sequencing data from 117 hepatocellular carcinoma (HCC) tumors and matched healthy tissue together with ribosome profiling and immunopeptidomics data. Combining human leukocyte antigen-epitope binding predictions and experimental validation experiments, we conclude that around 40% of the tumor-specific antigens in HCC are likely to be derived from ncORFs, including two peptides that can trigger an immune response in humanized mice. We identify a subset of 33 tumor-specific long noncoding RNAs expressing novel cancer antigens shared by more than 10% of the HCC samples analyzed, which, when combined, cover a large proportion of the patients. The results of the study open avenues for extending the range of anticancer vaccines.

Relevance of mutation-derived neoantigens and non-classical antigens for anticancer therapies.

Efficacy of cancer immunotherapies relies on correct recognition of tumor antigens by lymphocytes, eliciting thus functional responses capable of eliminating tumor cells. Therefore, important efforts have been carried out in antigen identification, with the aim of understanding mechanisms of response to immunotherapy and to design safer and more efficient strategies. In addition to classical tumor-associated antigens identified during the last decades, implementation of next-generation sequencing methodologies is enabling the identification of neoantigens (neoAgs) arising from mutations, leading to the development of new neoAg-directed therapies. Moreover, there are numerous non-classical tumor antigens originated from other sources and identified by new methodologies. Here, we review the relevance of neoAgs in different immunotherapies and the results obtained by applying neoAg-based strategies. In addition, the different types of non-classical tumor antigens and the best approaches for their identification are described. This will help to increase the spectrum of targetable molecules useful in cancer immunotherapies.

Relationship between cholestasis and altered progesterone metabolism in the placenta-maternal liver tandem.

BACKGROUND: In intrahepatic cholestasis of pregnancy (ICP), there are elevated maternal serum levels of total bile acids, progesterone, and some sulfated metabolites, such as allopregnanolone sulfate, which inhibits canalicular function. AIM: To investigate the relationship between cholestasis and the expression of crucial enzymes involved in progesterone metabolism in the liver and placenta. METHODS: Obstructive cholestasis was induced by bile duct ligation (BDL). RT-qPCR (mRNA) and western blot (protein) were used to determine expression levels. Srd5a1 and Akr1c2 enzymatic activities were assayed by substrate disappearance (progesterone and 5α-dihydroprogesterone, respectively), measured by HPLC-MS/MS. RESULTS: BDL induced decreased Srd5a1 and Akr1c2 expression and activity in rat liver, whereas both enzymes were up-regulated in rat placenta. Regarding sulfotransferases, Sult2b1 was also moderately up-regulated in the liver. In placenta from ICP patients, SRD5A1 and AKR1C2 expression was elevated, whereas both genes were down-regulated in liver biopsies collected from patients with several liver diseases accompanied by cholestasis. SRD5A1 and AKR1C2 expression was not affected by incubating human hepatoma HepG2 cells with FXR agonists (chenodeoxycholic acid and GW4064). Knocking-out Fxr in mice did not reduce Srd5a1 and Akr1c14 expression, which was similarly down-regulated by BDL. CONCLUSION: SRD5A1 and AKR1C2 expression was markedly altered by cholestasis. This was enhanced in the placenta but decreased in the liver, which is not mediated by FXR. These results suggest that the excess of progesterone metabolites in the serum of ICP patients can involve both enhanced placental production and decreased hepatic clearance. The latter may also occur in other cholestatic conditions.

Engineering U1-Based Tetracycline-Inducible Riboswitches to Control Gene Expression in Mammals.

Synthetic riboswitches are promising regulatory devices due to their small size, lack of immunogenicity, and ability to fine-tune gene expression in the absence of exogenous trans-acting factors. Based on a gene inhibitory system developed at our lab, termed U1snRNP interference (U1i), we developed tetracycline (TC)-inducible riboswitches that modulate mRNA polyadenylation through selective U1 snRNP recruitment. First, we engineered different TC-U1i riboswitches, which repress gene expression unless TC is added, leading to inductions of gene expression of 3-to-4-fold. Second, we developed a technique called Systematic Evolution of Riboswitches by Exponential Enrichment (SEREX), to isolate riboswitches with enhanced U1 snRNP binding capacity and activity, achieving inducibilities of up to 8-fold. Interestingly, by multiplexing riboswitches we increased inductions up to 37-fold. Finally, we demonstrated that U1i-based riboswitches are dose-dependent and reversible and can regulate the expression of reporter and endogenous genes in culture cells and mouse models, resulting in attractive systems for gene therapy applications. Our work probes SEREX as a much-needed technology for the in vitro identification of riboswitches capable of regulating gene expression in vivo.

APLF and long non-coding RNA NIHCOLE promote stable DNA synapsis in non-homologous end joining.

The synapsis of DNA ends is a critical step for the repair of double-strand breaks by non-homologous end joining (NHEJ). This is performed by a multicomponent protein complex assembled around Ku70-Ku80 heterodimers and regulated by accessory factors, including long non-coding RNAs, through poorly understood mechanisms. Here, we use magnetic tweezers to investigate the contributions of core NHEJ proteins and APLF and lncRNA NIHCOLE to DNA synapsis. APLF stabilizes DNA end bridging and, together with Ku70-Ku80, establishes a minimal complex that supports DNA synapsis for several minutes under piconewton forces. We find the C-terminal acidic region of APLF to be critical for bridging. NIHCOLE increases the dwell time of the synapses by Ku70-Ku80 and APLF. This effect is further enhanced by a small and structured RNA domain within NIHCOLE. We propose a model where Ku70-Ku80 can simultaneously bind DNA, APLF, and structured RNAs to promote the stable joining of DNA ends.

ISR8/IRF1-AS1 Is Relevant for IFNα and NF-κB Responses.

The study of the interferon (IFN) α-induced cell transcriptome has shown altered expression of several long non-coding RNAs (lncRNAs). ISR8/IRF1-AS1 (IFN stimulated RNA 8), located close to IFN regulatory factor 1 (IRF1) coding gene, transcribes a lncRNA induced at early times after IFNα treatment or IRF1 or NF-κB activation. Depletion or overexpression of ISR8 RNA does not lead to detected deregulation of the IFN response. Surprisingly, disruption of ISR8 locus with CRISPR-Cas9 genome editing results in cells that fail to induce several key ISGs and pro-inflammatory cytokines after a trigger with IFNα or overexpression of IRF1 or the NF-κB subunit RELA. This suggests that the ISR8 locus may play a relevant role in IFNα and NF-κB pathways. Interestingly, IFNα, IRFs and NF-κB-responding luciferase reporters are normally induced in ISR8-disrupted cells when expressed from a plasmid but not when integrated into the genome. Therefore, IFNα and NF-κB pathways are functional to induce the expression of exogenous episomic transcripts but fail to activate transcription from genomic promoters. Transcription from these promoters is not restored with silencing inhibitors, by decreasing the levels of several negative regulators or by overexpression of inducers. Transcriptome analyses indicate that ISR8-disrupted cells have a drastic increase in the levels of negative regulators such as XIST and Zinc finger proteins. Our results agree with ISR8 loci being an enhancer region that is fundamental for proper antiviral and proinflammatory responses. These results are relevant because several SNPs located in the ISR8 region are associated with chronic inflammatory and autoimmune diseases including Crohn's disease, inflammatory bowel disease, ulcerative colitis or asthma.

U1A is a positive regulator of the expression of heterologous and cellular genes involved in cell proliferation and migration.

Here, we show that direct recruitment of U1A to target transcripts can increase gene expression. This is a new regulatory role, in addition to previous knowledge showing that U1A decreases the levels of U1A mRNA and other specific targets. In fact, genome-wide, U1A more often increases rather than represses gene expression and many U1A-upregulated transcripts are directly bound by U1A according to individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) studies. Interestingly, U1A-mediated positive regulation can be transferred to a heterologous system for biotechnological purposes. Finally, U1A-bound genes are enriched for those involved in cell cycle and adhesion. In agreement with this, higher U1A mRNA expression associates with lower disease-free survival and overall survival in many cancer types, and U1A mRNA levels positively correlate with those of some oncogenes involved in cell proliferation. Accordingly, U1A depletion leads to decreased expression of these genes and the migration-related gene CCN2/CTGF, which shows the strongest regulation by U1A. A decrease in U1A causes a strong drop in CCN2 expression and CTGF secretion and defects in the expression of CTGF EMT targets, cell migration, and proliferation. These results support U1A as a putative therapeutic target for cancer treatment. In addition, U1A-binding sequences should be considered in biotechnological applications.

DNA Methylation Regulates a Set of Long Non-Coding RNAs Compromising Hepatic Identity during Hepatocarcinogenesis.

BACKGROUND: Long noncoding RNAs (lncRNAs) are emerging as key players in cancer, including hepatocellular carcinoma (HCC). Here we identify the mechanism implicated in the HCC inhibition of a set of lncRNAs, and their contribution to the process of hepatocarcinogenesis. METHODS AND RESULTS: The top-ranked 35 lncRNAs downregulated in HCC (Top35 LNDH) were validated in several human HCC cohorts. We demonstrate that their inhibition is associated with promoter hypermethylation in HCC compared to control tissue, and in HCC human cell lines compared to primary hepatocytes. Moreover, demethylating treatment of HCC human cell lines induced the expression of these lncRNAs. The Top35 LNDH were preferentially expressed in the adult healthy liver compared to other tissues and fetal liver and were induced in well-differentiated HepaRG cells. Remarkably, their knockdown compromised the expression of other hepato-specific genes. Finally, the expression of the Top35 LNDH positively correlates with the grade of tumor differentiation and, more importantly, with a better patient prognosis. CONCLUSIONS: Our results demonstrate that the selected Top35 LNDH are not only part of the genes that compose the hepatic differentiated signature but participate in its establishment. Moreover, their downregulation through DNA methylation occurs during the process of hepatocarcinogenesis compromising hepatocellular differentiation and HCC patients' prognosis.

Long Noncoding RNA NIHCOLE Promotes Ligation Efficiency of DNA Double-Strand Breaks in Hepatocellular Carcinoma.

Long noncoding RNAs (lncRNA) are emerging as key players in cancer as parts of poorly understood molecular mechanisms. Here, we investigated lncRNAs that play a role in hepatocellular carcinoma (HCC) and identified NIHCOLE, a novel lncRNA induced in HCC with oncogenic potential and a role in the ligation efficiency of DNA double-stranded breaks (DSB). NIHCOLE expression was associated with poor prognosis and survival of HCC patients. Depletion of NIHCOLE from HCC cells led to impaired proliferation and increased apoptosis. NIHCOLE deficiency led to accumulation of DNA damage due to a specific decrease in the activity of the nonhomologous end-joining (NHEJ) pathway of DSB repair. DNA damage induction in NIHCOLE-depleted cells further decreased HCC cell growth. NIHCOLE was associated with DSB markers and recruited several molecules of the Ku70/Ku80 heterodimer. Further, NIHCOLE putative structural domains supported stable multimeric complexes formed by several NHEJ factors including Ku70/80, APLF, XRCC4, and DNA ligase IV. NHEJ reconstitution assays showed that NIHCOLE promoted the ligation efficiency of blunt-ended DSBs. Collectively, these data show that NIHCOLE serves as a scaffold and facilitator of NHEJ machinery and confers an advantage to HCC cells, which could be exploited as a targetable vulnerability. SIGNIFICANCE: This study characterizes the role of lncRNA NIHCOLE in DNA repair and cellular fitness in HCC, thus implicating it as a therapeutic target.See related commentary by Barcena-Varela and Lujambio, p. 4899.

The Landscape of lncRNAs in Hepatocellular Carcinoma: A Translational Perspective.

LncRNAs are emerging as relevant regulators of multiple cellular processes involved in cell physiology as well as in the development and progression of human diseases, most notably, cancer. Hepatocellular carcinoma (HCC) is a prominent cause of cancer-related death worldwide due to the high prevalence of causative factors, usual cirrhotic status of the tumor-harboring livers and the suboptimal benefit of locoregional and systemic therapies. Despite huge progress in the molecular characterization of HCC, no oncogenic loop addiction has been identified and most genetic alterations remain non-druggable, underscoring the importance of advancing research in novel approaches for HCC treatment. In this context, long non-coding RNAs (lncRNAs) appear as potentially useful targets as they often exhibit high tumor- and tissue-specific expression and many studies have reported an outstanding dysregulation of lncRNAs in HCC. However, there is a limited perspective of the potential role that deregulated lncRNAs may play in HCC progression and aggressiveness or the mechanisms and therapeutic implications behind such effects. In this review, we offer a clarifying landscape of current efforts to evaluate lncRNA potential as therapeutic targets in HCC using evidence from preclinical models as well as from recent studies on novel oncogenic pathways that show lncRNA-dependency.

Long Noncoding RNA EGOT Responds to Stress Signals to Regulate Cell Inflammation and Growth.

The cell has several mechanisms to sense and neutralize stress. Stress-related stimuli activate pathways that counteract danger, support cell survival, and activate the inflammatory response. We use human cells to show that these processes are modulated by EGOT, a long noncoding RNA highly induced by viral infection, whose inhibition results in increased levels of antiviral IFN-stimulated genes (ISGs) and decreased viral replication. We now show that EGOT is induced in response to cell stress, viral replication, or the presence of pathogen-associated molecular patterns via the PI3K/AKT, MAPKs, and NF-κB pathways, which lead to cell survival and inflammation. Transcriptome analysis and validation experiments show that EGOT modulates PI3K/AKT and NF-κB responses. On the one hand, EGOT inhibition decreases expression of PI3K/AKT-induced cellular receptors and cell proliferation. In fact, EGOT levels are increased in several tumors. On the other hand, EGOT inhibition results in decreased levels of key NF-κB target genes, including those required for inflammation and ISGs in those cells that build an antiviral response. Mechanistically, EGOT depletion decreases the levels of the key coactivator TBLR1, essential for transcription by NF-κB. In summary, EGOT is induced in response to stress and may function as a switch that represses ISG transcription until a proper antiviral or stress response is initiated. EGOT then helps PI3K/AKT, MAPKs, and NF-κB pathways to activate the antiviral response, cell inflammation, and growth. We believe that modulation of EGOT levels could be used as a therapy for the treatment of certain viral infections, immune diseases, and cancer.

Endogenous Retroelement Activation by Epigenetic Therapy Reverses the Warburg Effect and Elicits Mitochondrial-Mediated Cancer Cell Death.

For millions of years, endogenous retroelements have remained transcriptionally silent within mammalian genomes by epigenetic mechanisms. Modern anticancer therapies targeting the epigenetic machinery awaken retroelement expression, inducing antiviral responses that eliminate tumors through mechanisms not completely understood. Here, we find that massive binding of epigenetically activated retroelements by RIG-I and MDA5 viral sensors promotes ATP hydrolysis and depletes intracellular energy, driving tumor killing independently of immune signaling. Energy depletion boosts compensatory ATP production by switching glycolysis to mitochondrial oxidative phosphorylation, thereby reversing the Warburg effect. However, hyperfunctional succinate dehydrogenase in mitochondrial electron transport chain generates excessive oxidative stress that unleashes RIP1-mediated necroptosis. To maintain ATP generation, hyperactive mitochondrial membrane blocks intrinsic apoptosis by increasing BCL2 dependency. Accordingly, drugs targeting BCL2 family proteins and epigenetic inhibitors yield synergistic responses in multiple cancer types. Thus, epigenetic therapy kills cancer cells by rewiring mitochondrial metabolism upon retroelement activation, which primes mitochondria to apoptosis by BH3-mimetics. SIGNIFICANCE: The state of viral mimicry induced by epigenetic therapies in cancer cells remodels mitochondrial metabolism and drives caspase-independent tumor cell death, which sensitizes to BCL2 inhibitor drugs. This novel mechanism underlies clinical efficacy of hypomethylating agents and venetoclax in acute myeloid leukemia, suggesting similar combination therapies for other incurable cancers.This article is highlighted in the In This Issue feature, p. 995.

LncRNAs in the Type I Interferon Antiviral Response.

The proper functioning of the immune system requires a robust control over a delicate equilibrium between an ineffective response and immune overactivation. Poor responses to viral insults may lead to chronic or overwhelming infection, whereas unrestrained activation can cause autoimmune diseases and cancer. Control over the magnitude and duration of the antiviral immune response is exerted by a finely tuned positive or negative regulation at the DNA, RNA, and protein level of members of the type I interferon (IFN) signaling pathways and on the expression and activity of antiviral and proinflammatory factors. As summarized in this review, committed research during the last decade has shown that several of these processes are exquisitely regulated by long non-coding RNAs (lncRNAs), transcripts with poor coding capacity, but highly versatile functions. After infection, viruses, and the antiviral response they trigger, deregulate the expression of a subset of specific lncRNAs that function to promote or repress viral replication by inactivating or potentiating the antiviral response, respectively. These IFN-related lncRNAs are also highly tissue- and cell-type-specific, rendering them as promising biomarkers or therapeutic candidates to modulate specific stages of the antiviral immune response with fewer adverse effects.

The Mir181ab1 cluster promotes KRAS-driven oncogenesis and progression in lung and pancreas.

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.

Identification of Coding and Long Noncoding RNAs Differentially Expressed in Tumors and Preferentially Expressed in Healthy Tissues.

The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets allow unprecedented gene expression analyses. Here, using these datasets, we performed pan-cancer and pan-tissue identification of coding and long noncoding RNA (lncRNA) transcripts differentially expressed in tumors and preferentially expressed in healthy tissues and/or tumors. Pan-cancer comparison of mRNAs and lncRNAs showed that lncRNAs were deregulated in a more tumor-specific manner. Given that lncRNAs are more tissue-specific than mRNAs, we identified healthy tissues that preferentially express lncRNAs upregulated in tumors and found that testis, brain, the digestive tract, and blood/spleen were the most prevalent. In addition, specific tumors also upregulate lncRNAs preferentially expressed in other tissues, generating a unique signature for each tumor type. Most tumors studied downregulated lncRNAs preferentially expressed in their tissue of origin, probably as a result of dedifferentiation. However, the same lncRNAs could be upregulated in other tumors, resulting in "bimorphic" transcripts. In hepatocellular carcinoma (HCC), the upregulated genes identified were expressed at higher levels in patients with worse prognosis. Some lncRNAs upregulated in HCC and preferentially expressed in healthy testis or brain were predicted to function as oncogenes and were significantly associated with higher tumor burden, and poor prognosis, suggesting their relevance in hepatocarcinogenesis and/or tumor evolution. Taken together, therapies targeting oncogenic lncRNAs should take into consideration the healthy tissue, where the lncRNAs are preferentially expressed, to predict and decrease unwanted secondary effects and increase potency. SIGNIFICANCE: Comprehensive analysis of coding and noncoding genes expressed in different tumors and normal tissues, which should be taken into account to predict side effects from potential coding and noncoding gene-targeting therapies.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/20/5167/F1.large.jpg.

Inhibition of a G9a/DNMT network triggers immune-mediated bladder cancer regression.

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances1,2. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer3,4. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients5-8. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1-/-) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.

The cirrhotic liver is depleted of docosahexaenoic acid (DHA), a key modulator of NF-κB and TGFß pathways in hepatic stellate cells.

Liver cirrhosis results from chronic hepatic damage and is characterized by derangement of the organ architecture with increased liver fibrogenesis and defective hepatocellular function. It frequently evolves into progressive hepatic insufficiency associated with high mortality unless liver transplantation is performed. We have hypothesized that the deficiency of critical nutrients such as essential omega-3 fatty acids might play a role in the progression of liver cirrhosis. Here we evaluated by LC-MS/MS the liver content of omega-3 docosahexaenoic fatty acid (DHA) in cirrhotic patients and investigated the effect of DHA in a murine model of liver injury and in the response of hepatic stellate cells (HSCs) (the main producers of collagen in the liver) to pro-fibrogenic stimuli. We found that cirrhotic livers exhibit a marked depletion of DHA and that this alteration correlates with the progression of the disease. Administration of DHA exerts potent anti-fibrogenic effects in an acute model of liver damage. Studies with HSCs show that DHA inhibits fibrogenesis more intensely than other omega-3 fatty acids. Data from expression arrays revealed that DHA blocks TGFß and NF-κB pathways. Mechanistically, DHA decreases late, but not early, SMAD3 nuclear accumulation and inhibits p65/RelA-S536 phosphorylation, which is required for HSC survival. Notably, DHA increases ADRP expression, leading to the formation of typical quiescence-associated perinuclear lipid droplets. In conclusion, a marked depletion of DHA is present in the liver of patients with advanced cirrhosis. DHA displays anti-fibrogenic activities on HSCs targeting NF-κB and TGFß pathways and inducing ADPR expression and quiescence in these cells.

Deregulation of linc-PINT in acute lymphoblastic leukemia is implicated in abnormal proliferation of leukemic cells.

Long Non-Coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. Several lncRNAs are involved in cell proliferation and are deregulated in several human tumors. Few lncRNAs have been described to play a role in Acute Lymphoblastic Leukemia (ALL). In this study, we carried out a genome wide lncRNA expression profiling in ALL samples and peripheral blood samples obtained from healthy donors. We detected 43 lncRNAs that were aberrantly expressed in ALL. Interestingly, among them, linc-PINT showed a significant downregulation in T and B-ALL. Re-expression of linc-PINT in ALL cells induced inhibition of leukemic cell growth that was associated with apoptosis induction and cell cycle arrest in G2/M phase. linc-PINT induced the transcription of HMOX1 which reduced the viability of ALL cells. Intriguingly, we observed that treatment with anti-tumoral epigenetic drugs like LBH-589 (Panobinostat) and Curcumin induced the expression of linc-PINT and HMOX1 in ALL. These results indicate that the downregulation of linc-PINT plays a relevant role in the pathogenesis of ALL, and linc-PINT re-expression may be one of the mechanisms exerted by epigenetic drugs to reduce cell proliferation in ALL.