Quantitative and Qualitative Analysis of Blood-based Liquid Biopsies to Inform Clinical Decision-making in Prostate Cancer.

CONTEXT: Genomic stratification can impact prostate cancer (PC) care through diagnostic, prognostic, and predictive biomarkers that aid in clinical decision-making. The temporal and spatial genomic heterogeneity of PC together with the challenges of acquiring metastatic tissue biopsies hinder implementation of tissue-based molecular profiling in routine clinical practice. Blood-based liquid biopsies are an attractive, minimally invasive alternative. OBJECTIVE: To review the clinical value of blood-based liquid biopsy assays in PC and identify potential applications to accelerate the development of precision medicine. EVIDENCE ACQUISITION: A systematic review of PubMed/MEDLINE was performed to identify relevant literature on blood-based circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and extracellular vesicles (EVs) in PC. EVIDENCE SYNTHESIS: Liquid biopsy has emerged as a practical tool to profile tumor dynamics over time, elucidating features that evolve (genome, epigenome, transcriptome, and proteome) with tumor progression. Liquid biopsy tests encompass analysis of DNA, RNA, and proteins that can be detected in CTCs, ctDNA, or EVs. Blood-based liquid biopsies have demonstrated promise in the context of localized tumors (diagnostic signatures, risk stratification, and disease monitoring) and advanced disease (response/resistance biomarkers and prognostic markers). CONCLUSIONS: Liquid biopsies have value as a source of prognostic, predictive, and response biomarkers in PC. Most clinical applications have been developed in the advanced metastatic setting, where CTC and ctDNA yields are significantly higher. However, standardization of assays and analytical/clinical validation is necessary prior to clinical implementation. PATIENT SUMMARY: Traces of tumors can be isolated from blood samples from patients with prostate cancer either as whole cells or as DNA fragments. These traces provide information on tumor features. These minimally invasive tests can guide diagnosis and treatment selection.

Advanced Prostate Cancer with ATM Loss: PARP and ATR Inhibitors.

BACKGROUND: Deleterious ATM alterations are found in metastatic prostate cancer (PC); PARP inhibition has antitumour activity against this subset, but only some ATM loss PCs respond. OBJECTIVE: To characterise ATM-deficient lethal PC and to study synthetic lethal therapeutic strategies for this subset. DESIGN, SETTING, AND PARTICIPANTS: We studied advanced PC biopsies using validated immunohistochemical (IHC) and next-generation sequencing (NGS) assays. In vitro cell line models modified using CRISPR-Cas9 to impair ATM function were generated and used in drug-sensitivity and functional assays, with validation in a patient-derived model. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: ATM expression by IHC was correlated with clinical outcome using Kaplan-Meier curves and log-rank test; sensitivity to different drug combinations was assessed in the preclinical models. RESULTS AND LIMITATIONS: Overall, we detected ATM IHC loss in 68/631 (11%) PC patients in at least one biopsy, with synchronous and metachronous intrapatient heterogeneity; 46/71 (65%) biopsies with ATM loss had ATM mutations or deletions by NGS. ATM IHC loss was not associated with worse outcome from advanced disease, but ATM loss was associated with increased genomic instability (NtAI:number of subchromosomal regions with allelic imbalance extending to the telomere, p = 0.005; large-scale transitions, p = 0.05). In vitro, ATM loss PC models were sensitive to ATR inhibition, but had variable sensitivity to PARP inhibition; superior antitumour activity was seen with combined PARP and ATR inhibition in these models. CONCLUSIONS: ATM loss in PC is not always detected by targeted NGS, associates with genomic instability, and is most sensitive to combined ATR and PARP inhibition. PATIENT SUMMARY: Of aggressive prostate cancers, 10% lose the DNA repair gene ATM; this loss may identify a distinct prostate cancer subtype that is most sensitive to the combination of oral drugs targeting PARP and ATR.

Analysis of copy number alterations reveals the lncRNA ALAL-1 as a regulator of lung cancer immune evasion.

Cancer is characterized by genomic instability leading to deletion or amplification of oncogenes or tumor suppressors. However, most of the altered regions are devoid of known cancer drivers. Here, we identify lncRNAs frequently lost or amplified in cancer. Among them, we found amplified lncRNA associated with lung cancer-1 (ALAL-1) as frequently amplified in lung adenocarcinomas. ALAL-1 is also overexpressed in additional tumor types, such as lung squamous carcinoma. The RNA product of ALAL-1 is able to promote the proliferation and tumorigenicity of lung cancer cells. ALAL-1 is a TNFα- and NF-κB-induced cytoplasmic lncRNA that specifically interacts with SART3, regulating the subcellular localization of the protein deubiquitinase USP4 and, in turn, its function in the cell. Interestingly, ALAL-1 expression inversely correlates with the immune infiltration of lung squamous tumors, while tumors with ALAL-1 amplification show lower infiltration of several types of immune cells. We have thus unveiled a pro-oncogenic lncRNA that mediates cancer immune evasion, pointing to a new target for immune potentiation.

Targeting DNA Repair Defects for Precision Medicine in Prostate Cancer.

PURPOSE OF REVIEW: Genomic studies of localized and metastatic prostate cancer have identified a high prevalence of clinically actionable alterations including mutations in DNA repair genes. In this manuscript, we review the current knowledge on DNA repair defects in prostate cancer and provide an overview of how these alterations can be targeted towards a personalized prostate cancer management. RECENT FINDINGS: Twenty to 25% of metastatic prostate cancers harbor defects in DNA repair genes, most commonly in the homologous recombination genes. These defects confer increased sensitivity to platinum chemotherapy or poly (ADP-ribose) polymerase (PARP) inhibitors. Recent trials also support a synergistic effect of combining these therapies with androgen receptor-targeting agents. Identification of mismatch-repair defects could result in defining a prostate cancer population who may benefit from immune checkpoint inhibitors. These data have implications for family testing and early diagnosis, as many of these mutations are linked to inherited risk of prostate cancer. The DNA damage repair pathways are clinically relevant in prostate cancer, being a target for precision medicine; combination with standard-of-care androgen receptor (AR)-targeting agents may be synergistic.

Germline and Somatic Defects in DNA Repair Pathways in Prostate Cancer.

Recent studies have provided a better understanding of the molecular underpinnings of prostate cancer. Alterations in genes encoding for proteins involved in the different pathways in charge of preserving genomic integrity and repairing DNA damage are common in prostate cancer, particularly in late-stage disease. Generally, these alterations would confer a survival advantage for tumors, resulting in a more aggressive phenotype. However, DNA repair defects can also represent a vulnerability for tumors that can be exploited therapeutically, offering the possibility of precision medicine strategies. Moreover, many of these mutations are linked to hereditary risk for cancers; hence, identification of DNA repair mutations could also be relevant for cancer prevention and screening in healthy individuals, including relatives of prostate cancer patients. In this chapter, we summarize current knowledge about the prevalence of different DNA repair gene alterations across different stages of prostate cancer and review the clinical relevance of such events in terms of prognosis and treatment stratification.

The human lncRNA LINC-PINT inhibits tumor cell invasion through a highly conserved sequence element.

BACKGROUND: It is now obvious that the majority of cellular transcripts do not code for proteins, and a significant subset of them are long non-coding RNAs (lncRNAs). Many lncRNAs show aberrant expression in cancer, and some of them have been linked to cell transformation. However, the underlying mechanisms remain poorly understood and it is unknown how the sequences of lncRNA dictate their function. RESULTS: Here we characterize the function of the p53-regulated human lncRNA LINC-PINT in cancer. We find that LINC-PINT is downregulated in multiple types of cancer and acts as a tumor suppressor lncRNA by reducing the invasive phenotype of cancer cells. A cross-species analysis identifies a highly conserved sequence element in LINC-PINT that is essential for its function. This sequence mediates a specific interaction with PRC2, necessary for the LINC-PINT-dependent repression of a pro-invasion signature of genes regulated by the transcription factor EGR1. CONCLUSIONS: Our findings support a conserved functional co-dependence between LINC-PINT and PRC2 and lead us to propose a new mechanism where the lncRNA regulates the availability of free PRC2 at the proximity of co-regulated genomic loci.

A Long Noncoding RNA Regulates Sister Chromatid Cohesion.

Long noncoding RNAs (lncRNAs) are involved in diverse cellular processes through multiple mechanisms. Here, we describe a previously uncharacterized human lncRNA, CONCR (cohesion regulator noncoding RNA), that is transcriptionally activated by MYC and is upregulated in multiple cancer types. The expression of CONCR is cell cycle regulated, and it is required for cell-cycle progression and DNA replication. Moreover, cells depleted of CONCR show severe defects in sister chromatid cohesion, suggesting an essential role for CONCR in cohesion establishment during cell division. CONCR interacts with and regulates the activity of DDX11, a DNA-dependent ATPase and helicase involved in DNA replication and sister chromatid cohesion. These findings unveil a direct role for an lncRNA in the establishment of sister chromatid cohesion by modulating DDX11 enzymatic activity.

Genome-wide analysis of the human p53 transcriptional network unveils a lncRNA tumour suppressor signature.

Despite the inarguable relevance of p53 in cancer, genome-wide studies relating endogenous p53 activity to the expression of lncRNAs in human cells are still missing. Here, by integrating RNA-seq with p53 ChIP-seq analyses of a human cancer cell line under DNA damage, we define a high-confidence set of 18 lncRNAs that are p53 transcriptional targets. We demonstrate that two of the p53-regulated lncRNAs are required for the efficient binding of p53 to some of its target genes, modulating the p53 transcriptional network and contributing to apoptosis induction by DNA damage. We also show that the expression of p53-lncRNAs is lowered in colorectal cancer samples, constituting a tumour suppressor signature with high diagnostic power. Thus, p53-regulated lncRNAs establish a positive regulatory feedback loop that enhances p53 tumour suppressor activity. Furthermore, the signature defined by p53-regulated lncRNAs supports their potential use in the clinic as biomarkers and therapeutic targets.

FAL1ing inside an amplicon.

Frequently amplified regions of the cancer genome contain well-known oncogenes. In this issue of Cancer Cell, Hu and colleagues discover that FAL1, a long noncoding RNA is encoded in one of these regions. FAL1 acts as an oncogene by stabilizing BMI1, which results in the repression of CDKN1A expression.

Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2.

BACKGROUND: The p53 transcription factor is located at the core of a complex wiring of signaling pathways that are critical for the preservation of cellular homeostasis. Only recently it has become clear that p53 regulates the expression of several long intergenic noncoding RNAs (lincRNAs). However, relatively little is known about the role that lincRNAs play in this pathway. RESULTS: Here we characterize a lincRNA named Pint (p53 induced noncoding transcript). We show that Pint is aubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, Pint promotes cell proliferation and survival by regulating the expression of genes of the TGF-b, MAPK and p53 pathways. Pint is a nuclear lincRNA that directly interacts with the Polycomb repressive complex 2 (PRC2), and is required for PRC2 targeting of specific genes for H3K27 tri-methylation and repression. Furthermore, Pint functional activity is highly dependent on PRC2 expression. We have also identified Pint human ortholog (PINT), which presents suggestive analogies with the murine lincRNA. PINT is similarly regulated by p53, and its expression significantly correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is downregulated in colon primary tumors, while its overexpression inhibits the proliferation of tumor cells, suggesting a possible role as tumor suppressor. CONCLUSIONS: Our results reveal a p53 autoregulatory negative mechanism where a lincRNA connects p53 activation with epigenetic silencing by PRC2. Additionally, we show analogies and differences between the murine and human orthologs, identifying a novel tumor suppressor candidate lincRNA.

CUP-1 is a novel protein involved in dietary cholesterol uptake in Caenorhabditis elegans.

Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (ChUP-1), [corrected] is involved in dietary cholesterol uptake in C. elegans. Animals lacking ChUP-1 [corrected] showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A ChUP-1-GFP [corrected] fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane "cholesterol recognition/interaction amino acid consensus" (CRAC) motif present in C. elegans ChUP-1. [corrected]. In-silico analysis identified two mammalian homologues of ChUP-1. [corrected]. Most interestingly, CRAC motifs are conserved in mammalian ChUP-1 [corrected] homologous. Our results suggest a role of ChUP-1 [corrected] in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals.

Expression profiling of synaptic microRNAs from the adult rat brain identifies regional differences and seizure-induced dynamic modulation.

In recent years, microRNAs or miRNAs have been proposed to target neuronal mRNAs localized near the synapse, exerting a pivotal role in modulating local protein synthesis, and presumably affecting adaptive mechanisms such as synaptic plasticity. In the present study we have characterized the distribution of miRNAs in five regions of the adult mammalian brain and compared the relative abundance between total fractions and purified synaptoneurosomes (SN), using three different methodologies. The results show selective enrichment or depletion of some miRNAs when comparing total versus SN fractions. These miRNAs were different for each brain region explored. Changes in distribution could not be attributed to simple diffusion or to a targeting sequence inside the miRNAs. In silico analysis suggest that the differences in distribution may be related to the preferential concentration of synaptically localized mRNA targeted by the miRNAs. These results favor a model of co-transport of the miRNA-mRNA complex to the synapse, although further studies are required to validate this hypothesis. Using an in vivo model for increasing excitatory activity in the cortex and the hippocampus indicates that the distribution of some miRNAs can be modulated by enhanced neuronal (epileptogenic) activity. All these results demonstrate the dynamic modulation in the local distribution of miRNAs from the adult brain, which may play key roles in controlling localized protein synthesis at the synapse.