An ErbB2 splice variant lacking exon 16 drives lung carcinoma.

Lung cancer causes more deaths annually than any other malignancy. A subset of non-small cell lung cancer (NSCLC) is driven by amplification and overexpression or activating mutation of the receptor tyrosine kinase (RTK) ERBB2 In some contexts, notably breast cancer, alternative splicing of ERBB2 causes skipping of exon 16, leading to the expression of an oncogenic ERBB2 isoform (ERBB2ΔEx16) that forms constitutively active homodimers. However, the broader implications of ERBB2 alternative splicing in human cancers have not been explored. Here, we have used genomic and transcriptomic analysis to identify elevated ERBB2ΔEx16 expression in a subset of NSCLC cases, as well as splicing site mutations facilitating exon 16 skipping and deletions of exon 16 in a subset of these lung tumors and in a number of other carcinomas. Supporting the potential of ERBB2ΔEx16 as a lung cancer driver, its expression transformed immortalized lung epithelial cells while a transgenic model featuring inducible ERBB2ΔEx16 specifically in the lung epithelium rapidly developed lung adenocarcinomas following transgene induction. Collectively, these observations indicate that ERBB2ΔEx16 is a lung cancer oncogene with potential clinical importance for a proportion of patients.

Liquid Biopsy in Lung Cancer: Biomarkers for the Management of Recurrence and Metastasis.

Liquid biopsies have emerged as a promising tool for the detection of metastases as well as local and regional recurrence in lung cancer. Liquid biopsy tests involve analyzing a patient's blood, urine, or other body fluids for the detection of biomarkers, including circulating tumor cells or tumor-derived DNA/RNA that have been shed into the bloodstream. Studies have shown that liquid biopsies can detect lung cancer metastases with high accuracy and sensitivity, even before they are visible on imaging scans. Such tests are valuable for early intervention and personalized treatment, aiming to improve patient outcomes. Liquid biopsies are also minimally invasive compared to traditional tissue biopsies, which require the removal of a sample of the tumor for further analysis. This makes liquid biopsies a more convenient and less risky option for patients, particularly those who are not good candidates for invasive procedures due to other medical conditions. While liquid biopsies for lung cancer metastases and relapse are still being developed and validated, they hold great promise for improving the detection and treatment of this deadly disease. Herein, we summarize available and novel approaches to liquid biopsy tests for lung cancer metastases and recurrence detection and describe their applications in clinical practice.

Previously undescribed thyroid-specific miRNA sequences in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common thyroid malignancy, wherein diagnostic limitations and lack of accurate prognostic factors are important clinical challenges. In this study, we report the discovery of 234 novel miRNAs in non-neoplastic thyroid and PTC samples, obtained from publicly available small RNA sequencing datasets (TCGA and GEO). These sequences were observed to display similar molecular features compared to currently annotated miRNAs. These potentially novel miRNAs presented tissue-specificity and largely decreased expression in PTC compared to non-neoplastic samples. We showed that the disrupted novel miRNAs have diagnostic and prognostic potential, and were associated with BRAF mutation, a frequent alteration related to more aggressive PTC. In conclusion, our results expand the miRNA repertoire in thyroid tissues and highlight the potential biological role and clinical utility of previously unannotated miRNAs.

Large-scale discovery of previously undetected microRNAs specific to human liver.

MicroRNAs (miRNAs) are crucial regulators of gene expression in normal development and cellular homeostasis. While miRNA repositories contain thousands of unique sequences, they primarily contain molecules that are conserved across several tissues, largely excluding lineage and tissue-specific miRNAs. By analyzing small non-coding RNA sequencing data for abundance and secondary RNA structure, we discovered 103 miRNA candidates previously undescribed in liver tissue. While expression of some of these unannotated sequences is restricted to non-malignant tissue, downregulation of most of the sequences was detected in liver tumors, indicating their importance in the maintenance of liver homeostasis. Furthermore, target prediction revealed the involvement of the unannotated miRNA candidates in fatty-acid metabolism and tissue regeneration, which are key pathways in liver biology. Here, we provide a comprehensive analysis of the undiscovered liver miRNA transcriptome, providing new resources for a deeper exploration of organ-specific biology and disease.

Developmental transcription factor NFIB is a putative target of oncofetal miRNAs and is associated with tumour aggressiveness in lung adenocarcinoma.

Genes involved in fetal lung development are thought to play crucial roles in the malignant transformation of adult lung cells. Consequently, the study of lung tumour biology in the context of lung development has the potential to reveal key developmentally relevant genes that play critical roles in lung cancer initiation/progression. Here, we describe for the first time a comprehensive characterization of miRNA expression in human fetal lung tissue, with subsequent identification of 37 miRNAs in non-small cell lung cancer (NSCLC) that recapitulate their fetal expression patterns. Nuclear factor I/B (NFIB), a transcription factor essential for lung development, was identified as a potential frequent target for these 'oncofetal' miRNAs. Concordantly, analysis of NFIB expression in multiple NSCLC independent cohorts revealed its recurrent underexpression (in ∼40-70% of tumours). Interrogation of NFIB copy number, methylation, and mutation status revealed that DNA level disruption of this gene is rare, and further supports the notion that oncofetal miRNAs are likely the primary mechanism responsible for NFIB underexpression in NSCLC. Reflecting its functional role in regulating lung differentiation, low expression of NFIB was significantly associated with biologically more aggressive subtypes and, ultimately, poorer survival in lung adenocarcinoma patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Piwi-interacting RNAs in cancer: emerging functions and clinical utility.

PIWI-interacting RNAs (piRNAs) are emerging players in cancer genomics. Originally described in the germline, there are over 20,000 piRNA genes in the human genome. In contrast to microRNAs, piRNAs interact with PIWI proteins, another member of the Argonaute family, and function primarily in the nucleus. There, they are involved in the epigenetic silencing of transposable elements in addition to the transcriptional regulation of genes. It has recently been demonstrated that piRNAs are also expressed across a variety of human somatic tissue types in a tissue-specific manner. An increasing number of studies have shown that aberrant piRNA expression is a signature feature across multiple tumour types; however, their specific tumorigenic functions remain unclear. In this article, we discuss the emerging functional roles of piRNAs in a variety of cancers, and highlight their potential clinical utilities.

Emerging roles of T helper 17 and regulatory T cells in lung cancer progression and metastasis.

Lung cancer is a leading cause of cancer-related deaths worldwide. Lung cancer risk factors, including smoking and exposure to environmental carcinogens, have been linked to chronic inflammation. An integral feature of inflammation is the activation, expansion and infiltration of diverse immune cell types, including CD4+ T cells. Within this T cell subset are immunosuppressive regulatory T (Treg) cells and pro-inflammatory T helper 17 (Th17) cells that act in a fine balance to regulate appropriate adaptive immune responses.In the context of lung cancer, evidence suggests that Tregs promote metastasis and metastatic tumor foci development. Additionally, Th17 cells have been shown to be an integral component of the inflammatory milieu in the tumor microenvironment, and potentially involved in promoting distinct lung tumor phenotypes. Studies have shown that the composition of Tregs and Th17 cells are altered in the tumor microenvironment, and that these two CD4+ T cell subsets play active roles in promoting lung cancer progression and metastasis.We review current knowledge on the influence of Treg and Th17 cells on lung cancer tumorigenesis, progression, metastasis and prognosis. Furthermore, we discuss the potential biological and clinical implications of the balance among Treg/Th17 cells in the context of the lung tumor microenvironment and highlight the potential prognostic function and relationship to metastasis in lung cancer.

An atlas of gastric PIWI-interacting RNA transcriptomes and their utility for identifying signatures of gastric cancer recurrence.

The poor survival and recurrence rate in gastric adenocarcinoma highlights the need for cancer gene discovery. Towards this end, we globally assessed the expression of an emerging class of small non-coding RNAs, called PIWI-interacting RNAs (piRNAs). We analysed the transcriptomes of 358 non-malignant stomach tissue and gastric adenocarcinoma samples, and found that nearly half of the expressed piRNAs were overexpressed in tumours. Our gastric piRNA atlas showed that most piRNAs were embedded in protein-coding sequences rather than known piRNA clusters. Furthermore, we identified a three-piRNA signature associated with recurrence-free survival. In this proof-of-principle study, we demonstrate the potential clinical utility of piRNAs in gastric cancer.

Microtubule affinity-regulating kinase 2 is associated with DNA damage response and cisplatin resistance in non-small cell lung cancer.

Microtubule affinity-regulating kinases (MARKs) are involved in several cellular functions but few studies have correlated MARK kinase expression with cancer, and none have explored their role in lung cancer. In this study, we identified MARK2 as frequently disrupted by DNA hypomethylation and copy gain, resulting in concordant overexpression in independent lung tumor cohorts and we demonstrate a role for MARK2 in lung tumor biology. Manipulation of MARK2 in lung cell lines revealed its involvement in cell viability and anchorage-independent growth. Analyses of both manipulated cell lines and clinical tumor specimens identified a potential role for MARK2 in cell cycle activation and DNA repair. Associations between MARK2 and the E2F, Myc/Max and NF-κB pathways were identified by luciferase assays and in-depth assessment of the NF-κB pathway suggests a negative association between MARK2 expression and NF-κB due to activation of non-canonical NF-κB signaling. Finally, we show that high MARK2 expression levels correlate with resistance to cisplatin, a standard first line chemotherapy for lung cancer. Collectively, our work supports a role for MARK2 in promoting malignant phenotypes of lung cancer and potentially modulating response to the DNA damaging chemotherapeutic, cisplatin.

Investigation of dosimetric characteristics of radiochromic film in response to alpha particles emitted from Americium-241.

BACKGROUND: In radiotherapy, it is essential to deliver prescribed doses to tumors while minimizing damage to surrounding healthy tissue. Accurate measurements of absorbed dose are required for this purpose. Gafchromic® external beam therapy (EBT) radiochromic films have been widely used in radiotherapy. While the dosimetric characteristics of the EBT3 model film have been extensively studied for photon and charged particle beams (protons, electrons, and carbon ions), little research has been done on α $\alpha$ -particle dosimetry. α $\alpha$ -emitting radionuclides have gained popularity in cancer treatment due to their high linear energy transfer, short range in tissue, and ability to spare surrounding organs at risk, thereby delivering a more localized dose distribution to the tumor. Therefore, a dose-calibration film protocol for α $\alpha$ -particles is required. PURPOSE: This study aimed to develop a dose-calibration protocol for the α $\alpha$ -particle emitting radionuclide 241Am, using Monte Carlo (MC) simulations and measurements with unlaminated EBT3 films. METHODS: In this study, a MC-based user code was developed using the Geant4 simulation toolkit to model and simulate an 241Am source and an unlaminated EBT3 film. Two simulations were performed: one with voxelized geometries of the EBT3 active volume composition and the other using water. The dose rate was calculated within a region of interest in the voxelized geometries. Unlaminated EBT3 film pieces were irradiated with the 241Am source at various exposure times inside a black box. Film irradiations were compared to a 6-MV photon beam from a Varian TrueBeam machine. The simulated dose rate was used to convert the exposure times into absorbed doses to water, describing a radiochromic-film-based reference dosimetry protocol for α $\alpha$ -particles. The irradiated films were scanned and through an in-house Python script, the normalized pixel values from the green-color channel of scanned film images were analyzed. RESULTS: The 241Am energy spectra obtained from the simulations were in good agreement with IAEA and NIST databases, having differences < $<$ 0.516% for the emitted γ $\gamma$ -rays and produced characteristic x-rays and < $<$ 0.006% for the α $\alpha$ -particles. Due to the short range of α $\alpha$ -particles, there was no energy deposition in the voxels outside the active 241Am source region projected onto the film surface. Thus, the total dose rate within the voxels covering the source was 0.847 ± $\pm$ 0.003 Gy/min within the sensitive layer of the film (LiPCDA) and 0.847 ± $\pm$ 0.004 Gy/min in water, indicating that the active volume can be considered water equivalent for the 241Am beam quality. A novel approach was employed in α $\alpha$ -film dosimetry using an exponential fit for the green channel, which showed promising results by reducing the uncertainty in dose estimation within 5%. Although the statistical analysis did not reveal significant differences between the 6-MV photon beam and the α $\alpha$ calibration curves, the dose-response curves exhibited the expected behavior. CONCLUSIONS: The developed MC user code simulated the experimental setup for α $\alpha$ -dosimetry using radiochromic film with acceptable uncertainty. Unlaminated EBT3 film is suitable for the dosimetry of α $\alpha$ -radiation at low doses and can be used in conjunction with other unlaminated GafChromic® films for quality assurance and research purposes.

Frequent concerted genetic mechanisms disrupt multiple components of the NRF2 inhibitor KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex in thyroid cancer.

BACKGROUND: Reactive oxygen species contribute to normal thyroid function. The NRF2 oxidative response pathway is frequently and constitutively activated in multiple tumor types, including papillary thyroid carcinoma (PTC). Genetic mechanisms underlying NRF2 pathway activation in PTC are not fully understood. Thus, we aimed to determine whether inactivating patterns of DNA-level alterations affect genes encoding for individual NRF2 inhibitor complex components (CUL3/KEAP1/RBX1) occur in PTC. FINDINGS: Combined patterns of epi/genetic alterations for KEAP1/CUL3/RBX1 E3 ubiquitin-ligase complex components were simultaneously interrogated for a panel of 310 PTC cases and 40 adjacent non-malignant tissues. Data were obtained from The Cancer Genome Atlas project. Enrichment of NRF2 pathway activation was assessed by gene-set enrichment analysis using transcriptome data. Our analyses revealed that PTC sustain a strikingly high frequency (80.6%) of disruption to multiple component genes of the NRF2 inhibitor complex. Hypermethylation is the predominant inactivating mechanism primarily affecting KEAP1 (70.6%) and CUL3 (20%), while copy number loss mostly affects RBX1 (16.8%). Concordantly, NRF2-associated gene expression signatures are positively and significantly enriched in PTC. CONCLUSIONS: The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is almost ubiquitously affected by multiple DNA-level mechanisms and downstream NRF2 pathway targets are activated in PTC. Given the importance of this pathway to normal thyroid function as well as to cancer; targeted inhibition of NRF2 regulators may impact strategies for therapeutic intervention involving this pathway.

Molecular features in arsenic-induced lung tumors.

Arsenic is a well-known human carcinogen, which potentially affects ~160 million people worldwide via exposure to unsafe levels in drinking water. Lungs are one of the main target organs for arsenic-related carcinogenesis. These tumors exhibit particular features, such as squamous cell-type specificity and high incidence among never smokers. Arsenic-induced malignant transformation is mainly related to the biotransformation process intended for the metabolic clearing of the carcinogen, which results in specific genetic and epigenetic alterations that ultimately affect key pathways in lung carcinogenesis. Based on this, lung tumors induced by arsenic exposure could be considered an additional subtype of lung cancer, especially in the case of never-smokers, where arsenic is a known etiological agent. In this article, we review the current knowledge on the various mechanisms of arsenic carcinogenicity and the specific roles of this metalloid in signaling pathways leading to lung cancer.

Sequence-Based Platforms for Discovering Biomarkers in Liquid Biopsy of Non-Small-Cell Lung Cancer.

Lung cancer detection and monitoring are hampered by a lack of sensitive biomarkers, which results in diagnosis at late stages and difficulty in tracking response to treatment. Recent developments have established liquid biopsies as promising non-invasive methods for detecting biomarkers in lung cancer patients. With concurrent advances in high-throughput sequencing technologies and bioinformatics tools, new approaches for biomarker discovery have emerged. In this article, we survey established and emerging biomarker discovery methods using nucleic acid materials derived from bodily fluids in the context of lung cancer. We introduce nucleic acid biomarkers extracted from liquid biopsies and outline biological sources and methods of isolation. We discuss next-generation sequencing (NGS) platforms commonly used to identify novel biomarkers and describe how these have been applied to liquid biopsy. We highlight emerging biomarker discovery methods, including applications of long-read sequencing, fragmentomics, whole-genome amplification methods for single-cell analysis, and whole-genome methylation assays. Finally, we discuss advanced bioinformatics tools, describing methods for processing NGS data, as well as recently developed software tailored for liquid biopsy biomarker detection, which holds promise for early diagnosis of lung cancer.

Arsenic, asbestos and radon: emerging players in lung tumorigenesis.

The cause of lung cancer is generally attributed to tobacco smoking. However lung cancer in never smokers accounts for 10 to 25% of all lung cancer cases. Arsenic, asbestos and radon are three prominent non-tobacco carcinogens strongly associated with lung cancer. Exposure to these agents can lead to genetic and epigenetic alterations in tumor genomes, impacting genes and pathways involved in lung cancer development. Moreover, these agents not only exhibit unique mechanisms in causing genomic alterations, but also exert deleterious effects through common mechanisms, such as oxidative stress, commonly associated with carcinogenesis. This article provides a comprehensive review of arsenic, asbestos, and radon induced molecular mechanisms responsible for the generation of genetic and epigenetic alterations in lung cancer. A better understanding of the mode of action of these carcinogens will facilitate the prevention and management of lung cancer related to such environmental hazards.

Genetic and Epigenetic Mechanisms Deregulate the CRL2pVHL Complex in Hepatocellular Carcinoma.

Dysregulation of ubiquitin-proteasome pathway genes through copy number alteration, promoter hypomethylation, and miRNA deregulation is involved in cancer development and progression. Further characterizing alterations in these genes may uncover novel drug targets across a range of diseases in which druggable alterations are uncommon, including hepatocellular carcinoma (HCC). We analyzed 377 HCC and 59 adjacent non-malignant liver tissue samples, focusing on alterations to component genes of the widely studied CRL2pVHL E3 ubiquitin ligase complex. mRNA upregulation of the component genes was common, and was correlated with DNA hypomethylation and copy number increase, but many tumours displayed overexpression that was not explained by either mechanism. Interestingly, we found 66 miRNAs, including 39 previously unannotated miRNAs, that were downregulated in HCC and predicted to target one or more CRL2pVHL components. Several miRNAs, including hsa-miR-101-3p and hsa-miR-139-5p, were negatively correlated with multiple component genes, suggesting that miRNA deregulation may contribute to CRL2pVHL overexpression. Combining miRNA and mRNA expression, DNA copy number, and methylation status into one multidimensional survival analysis, we found a significant association between greater numbers of alterations and poorer overall survival for multiple component genes. While the intricacies of CRL2pVHL complex gene regulation require additional research, it is evident that multiple causes for the deregulation of these genes must be considered in HCC, including non-traditional mechanisms.

Health Effects Associated With Pre- and Perinatal Exposure to Arsenic.

Inorganic arsenic is a well-established human carcinogen, able to induce genetic and epigenetic alterations. More than 200 million people worldwide are exposed to arsenic concentrations in drinking water exceeding the recommended WHO threshold (10µg/l). Additionally, chronic exposure to levels below this threshold is known to result in long-term health effects in humans. The arsenic-related health effects in humans are associated with its biotransformation process, whereby the resulting metabolites can induce molecular damage that accumulates over time. The effects derived from these alterations include genomic instability associated with oxidative damage, alteration of gene expression (including coding and non-coding RNAs), global and localized epigenetic reprogramming, and histone posttranslational modifications. These alterations directly affect molecular pathways involved in the onset and progression of many conditions that can arise even decades after the exposure occurs. Importantly, arsenic metabolites generated during its biotransformation can also pass through the placental barrier, resulting in fetal exposure to this carcinogen at similar levels to those of the mother. As such, more immediate effects of the arsenic-induced molecular damage can be observed as detrimental effects on fetal development, pregnancy, and birth outcomes. In this review, we focus on the genetic and epigenetic damage associated with exposure to low levels of arsenic, particularly those affecting early developmental stages. We also present how these alterations occurring during early life can impact the development of certain diseases in adult life.

Profiling the small non-coding RNA transcriptome of the human placenta.

Proper functioning of the human placenta is critical for maternal and fetal health. While microRNAs (miRNAs) are known to impact placental gene expression, the effects of other small non-coding RNAs (sncRNAs) on the placental transcriptome are not well-established, and are emerging topics in the study of environmental influence on fetal development and reproductive health. Here, we assembled a cohort of 30 placental chorionic villi samples of varying gestational ages (M ± SD = 23.7 ± 11.3 weeks) to delineate the human placental sncRNA transcriptome through small RNA sequence analysis. We observed expression of 1544 sncRNAs, which include 48 miRNAs previously unannotated in humans. Additionally, 18,003 miRNA variants (isomiRs) were identified from the 654 observed miRNA species. This characterization of the term and pre-term placental sncRNA transcriptomes provides data fundamental to future investigations of their regulatory functions in the human placenta, and the baseline expression pattern needed for identifying changes in response to environmental factors, or under disease conditions.

Human placental piwi-interacting RNA transcriptome is characterized by expression from the DLK1-DIO3 imprinted region.

The placenta is vital to embryonic development and requires a finely-tuned pattern of gene expression, achieved in part by its unique epigenetic landscape. Piwi-interacting RNAs (piRNAs) are a class of small-non-coding RNA with established roles as epigenetic regulators of gene expression, largely via methylation of targeted DNA sequences. The expression of piRNAs have mainly been described in germ cells, but a fraction have been shown to retain expression in adult somatic tissues. To aid in understanding the contribution of these regulators in the placenta, we provide the first description of the piRNA transcriptome in human placentas. We find 297 piRNAs to be preferentially expressed in the human placenta, a subset of which are expressed at higher levels relative to testes samples. We also observed a large proportion of placental piRNAs to be expressed from a single locus, as distinct from canonical cluster locations associated with transposable element silencing. Finally, we find that 15 of the highest-expressed placental piRNAs maps to the DLK1-DIO3 locus, suggesting a link to placental biology. Our findings suggest that piRNAs could contribute to the molecular networks defining placental function in humans, and a biological impact of piRNA expression beyond germ cells.

Aberrant Expression of Pseudogene-Derived lncRNAs as an Alternative Mechanism of Cancer Gene Regulation in Lung Adenocarcinoma.

Transcriptome sequencing has led to the widespread identification of long non-coding RNAs (lncRNAs). Subsequently, these genes have been shown to hold functional importance in human cellular biology, which can be exploited by tumors to drive the hallmarks of cancer. Due to the complex tertiary structure and unknown binding motifs of lncRNAs, there is a growing disparity between the number of lncRNAs identified and those that have been functionally characterized. As such, lncRNAs deregulated in cancer may represent critical components of cancer pathways that could serve as novel therapeutic intervention points. Pseudogenes are non-coding DNA sequences that are defunct relatives of their protein-coding parent genes but retain high sequence similarity. Interestingly, certain lncRNAs expressed from pseudogene loci have been shown to regulate the protein-coding parent genes of these pseudogenes in trans particularly because of this sequence complementarity. We hypothesize that this phenomenon occurs more broadly than previously realized, and that aberrant expression of lncRNAs overlapping pseudogene loci provides an alternative mechanism of cancer gene deregulation. Using RNA-sequencing data from two cohorts of lung adenocarcinoma, each paired with patient-matched non-malignant lung samples, we discovered 104 deregulated pseudogene-derived lncRNAs. Remarkably, many of these deregulated lncRNAs (i) were expressed from the loci of pseudogenes related to known cancer genes, (ii) had expression that significantly correlated with protein-coding parent gene expression, and (iii) had lncRNA protein-coding parent gene expression that was significantly associated with survival. Here, we uncover evidence to suggest the lncRNA-pseudogene-protein-coding gene axis as a prominent mechanism of cancer gene regulation in lung adenocarcinoma, and highlights the clinical utility of exploring the non-coding regions of the cancer transcriptome.