Integration of Cell-Free DNA End Motifs and Fragment Lengths Can Identify Active Genes in Liquid Biopsies.

Multiple studies have shown that cell-free DNA (cfDNA) from cancer patients differ in both fragment length and fragment end motif (FEM) from healthy individuals, yet there is a lack of understanding of how the two factors combined are associated with cancer and gene transcription. In this study, we conducted cfDNA fragmentomics evaluations using plasma from lung cancer patients (n = 12) and healthy individuals (n = 7). A personal gene expression profile was established from plasma using H3K36me3 cell-free chromatin immunoprecipitation sequencing (cfChIP-seq). The genes with the highest expression displayed an enrichment of short cfDNA fragments (median = 19.99%, IQR: 16.94-27.13%, p < 0.0001) compared to the genes with low expression. Furthermore, distinct GC-rich FEMs were enriched after cfChIP. Combining the frequency of short cfDNA fragments with the presence of distinct FEMs resulted in an even further enrichment of the most expressed genes (median = 37.85%, IQR: 30.10-39.49%, p < 0.0001). An in vitro size selection of <150 bp cfDNA could isolate cfDNA representing active genes and the size-selection enrichment correlated with the cfChIP-seq enrichment (Spearman r range: 0.499-0.882, p < 0.0001). This study expands the knowledge regarding cfDNA fragmentomics and sheds new light on how gene activity is associated with both cfDNA fragment lengths and distinct FEMs.

RGMb impacts partial epithelial-mesenchymal transition and BMP2-Induced ID mRNA expression independent of PD-L2 in nonsmall cell lung cancer cells.

PD-1/PD-ligand-axis immunotherapy-mediated activation of T-cells for cancer cell elimination is a promising treatment of nonsmall cell lung cancer (NSCLC). However, the effect of immunotherapy on intracellular signaling pathways in cancer cells still needs further delineation. Repulsive Guidance Molecule b (RGMb), a regulator of Bone Morphogenetic Proteins (BMPs) signaling, interacts with the PD-ligand, PD-L2, at cancer cell membranes. Accordingly, a clarification of the functions of RGMb and its relation to PD-L2 might provide insight into NSCLC cell signaling responses to PD-1/PD-ligand-axis immunotherapy. In this study, the functions of RGMb and PD-L2 were examined using the two NSCLC cell lines HCC827 and A549. CRISPR/Cas9 was used to decrease the expression of RGMb and PD-L2, while lentiviral vectors were used to increase their expression. Downstream effects were examined by RT-qPCR and immunoassays. Ectopic expression of RGMb impacted BMP2-induced expression of ID1 and ID2 messenger RNA (mRNA) independently of PD-L2, while RGMb depletion by CRISPR/Cas9 did not affect the BMP2-mediated induction of ID1, ID2, and ID3 mRNA. However, depletion of RGMb resulted in a partial epithelial-mesenchymal transition (EMT) gene expression profile in HCC827 cells, which was not mimicked by PD-L2 depletion. The results show that RGMb is a coregulator of BMP signaling and hence, ID mRNA expression and that RGMb can control the EMT balance in NSCLC cells. However, RGMb appears to exert these functions independently of PD-L2, and accordingly, the PD-1/PD-ligand axis for immune surveillance in NSCLC cells.

PD-L1 and PD-L2 immune checkpoint protein induction by type III interferon in non-small cell lung cancer cells.

INTRODUCTION: Despite the clinical success of PD-1/PD-1-ligand immunotherapy in non-small cell lung cancer (NSCLC), the appearance of primary and acquired therapy resistance is a major challenge reflecting that the mechanisms regulating the expression of the PD-1-ligands PD-L1 and PD-L2 are not fully explored. Type I and II interferons (IFNs) induce PD-L1 and PD-L2 expression. Here, we examined if PD-L1 and PD-L2 expression also can be induced by type III IFN, IFN-λ, which is peculiarly important for airway epithelial surfaces. METHODS: In silico mRNA expression analysis of PD-L1 (CD274), PD-L2 (PDCD1LG2), and IFN- λ signaling signature genes in NSCLC tumors and cell lines was performed using RNA sequencing expression data from TCGA, OncoSG, and DepMap portals. IFN-λ-mediated induction of PD-L1 and PD-L2 expression in NSCLC cell lines was examined by real-time quantitative polymerase chain reaction and flow cytometry. RESULTS: IFNL genes encoding IFN- λ variants are expressed in the majority of NSCLC tumors and cell lines along with the IFNLR1 and IL10R2 genes encoding the IFN-λ receptor subunits. The expression of PD-L1 and PD-L2 mRNA is higher in NSCLC tumors with IFNL mRNA expression compared to tumors without IFNL expression. In the NSCLC cell line HCC827, stimulation with IFN-λ induced both an increase in PD-L1 and PD-L2 mRNA expression and cell surface abundance of the corresponding proteins. In the NSCLC cell line A427, displaying a low basal expression of PD-L1 and PD-L2 mRNA and corresponding proteins, stimulation with IFN-λ resulted in an induction of the former. CONCLUSION: The type III IFN, IFN- λ, is capable of inducing PD-L1 and PD-L2 expression, at least in some NSCLC cells, and this regulation will need acknowledgment in the development of new diagnostic procedures, such as gene expression signature profiles, to improve PD-1/PD-1-ligand immunotherapy in NSCLC.

Cell-free chromatin immunoprecipitation can determine tumor gene expression in lung cancer patients.

Cell-free DNA (cfDNA) in blood plasma can be bound to nucleosomes that contain post-translational modifications representing the epigenetic profile of the cell of origin. This includes histone H3 lysine 36 trimethylation (H3K36me3), a marker of active transcription. We hypothesised that cell-free chromatin immunoprecipitation (cfChIP) of H3K36me3-modified nucleosomes present in blood plasma can delineate tumour gene expression levels. H3K36me3 cfChIP followed by targeted NGS (cfChIP-seq) was performed on blood plasma samples from non-small-cell lung cancer (NSCLC) patients (NSCLC, n = 8), small-cell lung cancer (SCLC) patients (SCLC, n = 4) and healthy controls (n = 4). H3K36me3 cfChIP-seq demonstrated increased enrichment of mutated alleles compared with normal alleles in plasma from patients with known somatic cancer mutations. Additionally, genes identified to be differentially expressed in SCLC and NSCLC tumours had concordant H3K36me3 cfChIP enrichment profiles in NSCLC (sensitivity = 0.80) and SCLC blood plasma (sensitivity = 0.86). Findings here expand the utility of cfDNA in liquid biopsies to characterise treatment resistance, cancer subtyping and disease progression.

Genome-wide epigenetic and mRNA-expression profiling followed by CRISPR/Cas9-mediated gene-disruptions corroborate the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 axis in acquired EMT-associated EGFR TKI-resistance in NSCLC cells.

Background: Epithelial-mesenchymal-transition (EMT) is an epigenetic-based mechanism contributing to the acquired treatment resistance against receptor tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cells harboring epidermal growth factor receptor (EGFR)-mutations. Delineating the exact epigenetic and gene-expression alterations in EMT-associated EGFR TKI-resistance (EMT-E-TKI-R) is vital for improved diagnosis and treatment of NSCLC patients. Methods: We characterized genome-wide changes in mRNA-expression, DNA-methylation and the histone-modification H3K36me3 in EGFR-mutated NSCLC HCC827 cells in result of acquired EMT-E-TKI-R. CRISPR/Cas9 was used to functional examine key findings from the omics analyses. Results: Acquired EMT-E-TKI-R was analyzed with three omics approaches. RNA-sequencing identified 2,233 and 1,972 up- and down-regulated genes, respectively, and among these were established EMT-markers. DNA-methylation EPIC array analyses identified 14,163 and 7,999 hyper- and hypo-methylated, respectively, differential methylated positions of which several were present in EMT-markers. Finally, H3K36me3 chromatin immunoprecipitation (ChIP)-sequencing detected 2,873 and 3,836 genes with enrichment and depletion, respectively, and among these were established EMT-markers. Correlation analyses showed that EMT-E-TKI-R mRNA-expression changes correlated better with H3K36me3 changes than with DNA-methylation changes. Moreover, the omics data supported the involvement of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 signaling axis for acquired EMT-E-TKI-R. CRISPR/Cas9-mediated analyses corroborated the importance of ZEB1 in acquired EMT-E-TKI-R, MIR200C and MIR141 to be in an EMT-E-TKI-R-associated auto-regulatory loop with ZEB1, and FGFR1 to mediate cell survival in EMT-E-TKI-R. Conclusions: The current study describes the synchronous genome-wide changes in mRNA-expression, DNA-methylation, and H3K36me3 in NSCLC EMT-E-TKI-R. The omics approaches revealed potential novel diagnostic markers and treatment targets. Besides, the study consolidates the functional impact of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1-signaling axis in NSCLC EMT-E-TKI-R.

An Extended PD-L2 Cytoplasmic Domain Results From Alternative Splicing in NSCLC Cells.

Antibody-based immunotherapy targeting the interaction between programmed cell death 1 (PD-1) and its ligand PD-L1 has shown impressive clinical outcomes in various cancer types, including nonsmall cell lung cancer (NSCLC). However, regulatory mechanisms in this immune checkpoint pathway still needs clarification. PD-L2 is structurally homologous to PD-L1 and is a second PD-1 ligand. Alternative mRNA splicing from the CD274 and PDCD1LG2 genes holds the potential to generate PD-L1 and PD-L2 isoforms, respectively, with novel functionality in regulation of the PD-1 immune checkpoint pathway. Here, we describe alternative splicing in NSCLC cells potentially generating eight different PD-L2 isoforms from the PDCD1LG2 gene. Extension of exon 6 by four nucleotides is the most prominent alternative splicing event and results in PD-L2 isoform V with a cytoplasmic domain containing a 10 amino acid extension. On average 13% of the PDCD1LG2 transcripts in NSCLC cell lines and 22% of the transcripts in NSCLC tumor biopsies encode PD-L2 isoform V. PD-L2 isoform V localizes to the cell surface membrane but less efficiently than the canonical PD-L2 isoform I. The cytoplasmic domains of PD-1 ligands can affect immune checkpoint pathways by conferring membrane localization and protein stability and thereby represent alternative targets for immunotherapy. In addition, cytoplasmic domains are involved in intracellular signalling cascades in cancer cells. The presented observations of different cytoplasmic domains of PD-L2 will be important in the future delineation of the PD-1 immune checkpoint pathway.

EGFR transcription in non-small-cell lung cancer tumours can be revealed in ctDNA by cell-free chromatin immunoprecipitation (cfChIP).

Determination of tumour-specific transcription based on liquid biopsies possesses a large diagnostic and prognostic potential in non-small cell lung cancer (NSCLC). Cell-free DNA (cfDNA) packed in nucleosomes mirrors the histone modification profiles present in the cells of origin. H3 lysine 36 trimethylation (H3K36me3)-modified nucleosomes are associated with active genes, and therefore, cell-free chromatin immunoprecipitation (cfChIP) of H3K36me3-associated cfDNA has the potential to delineate whether transcription of a particular gene is occurring in the cells from which its cfDNA originates. We hypothesized that cfChIP can delineate transcriptional status of genes harbouring somatic cancer mutations and analysed the recurrently observed EGFR-L858R mutation as an example. In representative NSCLC cell lines, the relationship between wild-type (WT) and mutated EGFR transcriptional activity and mRNA expression levels was analysed using H3K36me3 ChIP and EGFR mRNA reverse transcription quantitative PCR (RT-qPCR), respectively. The ChIP analysis showed that both WT and mutated EGFR are transcribed and that mRNA is similarly expressed per EGFR copy. Based on this observation, we proceeded with EGFR cfChIP using blood plasma from NSCLC patients harbouring the EGFR-L858R mutation. EGFR-WT fragments can originate from both nontumour cells with no or low EGFR transcription and tumour cells with active EGFR transcription, whereas EGFR-L858R fragments must specifically originate from tumour cells. H3K36me3 cfChIP followed by droplet digital PCR (ddPCR) revealed significantly higher enrichment of EGFR-L858R compared to EGFR-WT fragments. This is in alignment with EGFR-L858R being actively transcribed in the NSCLC tumour cells. This study is proof-of-principle that cfChIP can be used to identify tumour-specific transcriptional activity of mutated alleles, which can expand the utility of liquid biopsy-based cfDNA analyses to enhance tumour diagnostics and therapeutics.

Epithelial-to-mesenchymal transition is a resistance mechanism to sequential MET-TKI treatment of MET-amplified EGFR-TKI resistant non-small cell lung cancer cells.

BACKGROUND: Tyrosine kinase inhibitor (TKI) resistance is a major obstacle in treatment of non-small cell lung cancer (NSCLC). MET amplification drives resistance to EGFR-TKIs in 5-20% of initially sensitive. EGFR: mutated NSCLC patients, and combined treatment with EGFR-TKIs and MET-TKIs can overcome this resistance. Yet, inevitably MET-TKI resistance will also occur. Hence, knowledge on development of this sequential resistance is important for identifying the proper next step in treatment. METHODS: To investigate sequential resistance to MET-TKI treatment, we established a two-step TKI resistance model in EGFR-mutated HCC827 cells with MET amplification-mediated erlotinib resistance. These cells were subsequently treated with increasing doses of the MET-TKIs capmatinib or crizotinib in combination with erlotinib to establish resistance. RESULTS: In all the MET-TKI resistant cell lines, we systematically observed epithelial-to-mesenchymal transition (EMT) evident by decreased expression of E-cadherin and increased expression of vimentin and ZEB1. Furthermore, FGFR1 expression was increased in all MET-TKI resistant cell lines and four out of the six resistant cell lines had increased sensitivity to FGFR inhibition, indicating FGFR1-mediated bypass signaling. CONCLUSIONS: EMT is common in the development of sequential EGFR-TKI and MET-TKI resistance in NSCLC cells. Our findings contribute to the evidence of EMT as a common TKI resistance mechanism.

Cell-free Chromatin Immunoprecipitation (cfChIP) from blood plasma can determine gene-expression in tumors from non-small-cell lung cancer patients.

OBJECTIVES: Lung cancer is the leading cause of cancer related death worldwide. Accurate molecular diagnostics from a tumor biopsy is paramount for correct diagnosis, treatment strategy, and prediction of outcome. However, a tumor biopsy can be misleading due to tumor heterogeneity and consecutive biopsies are rarely achievable. Importantly, tumor-specific genetic information concerning mutations and translocations, can also be obtained from liquid biopsies, e.g. blood plasma, containing cell-free DNA (cfDNA) with both systemic and tumor origin. Tumor-specific gene-expression information can also be determined from liquid biopsies using cfDNA methylation and cell-free RNA analyses. However, supplementary methodologies that can determine gene-expression patterns in lung tumors from liquid biopsies could also have diagnostic impact. MATERIALS AND METHODS: We here present the method cell-free chromatin Immunoprecipitation (cfChIP), which for genes having high expression specifically in the tumor, can determine such gene-expression from blood plasma. In cfChIP cell-free nucleosomes modified with histone H3 lysine 36 tri-methylation (H3K36me3), a mark quantitatively correlated with the transcription of the underlying gene, are isolated, and associated cfDNA quantified. RESULTS: We demonstrate that cfChIP from lung cancer patient blood plasma can successfully quantify the level of H3K36me3 associated with circulating cell-free nucleosomes and thereby quantify the transcriptional level of genes associated with these nucleosomes. Moreover, as a proof-of-principle we show that in blood plasma from 14 lung cancer patients, H3K36me3 cfChIP can replicate the expected higher expression of KRT6 in lung squamous cell carcinoma relative to adenocarcinoma. CONCLUSION: This work shows that for genes with a high expression specifically in tumor, cfChIP can determine this gene-expression pattern from blood plasma. cfChIP is a method that determine gene-expression at the transcriptional level and accordingly can supplement cfDNA methylation and cell-free RNA analyses.

The IFNL4 Gene Is a Noncanonical Interferon Gene with a Unique but Evolutionarily Conserved Regulation.

Interferon lambda 4 (IFN-λ4) is a recently identified enigmatic member of the interferon (IFN) lambda family. Genetic data suggest that the IFNL4 gene acts in a proviral and anti-inflammatory manner in patients. However, the protein is indistinguishable in vitro from the other members of the interferon lambda family. We have investigated the gene regulation of IFNL4 in detail and found that it differs radically from that of canonical antiviral interferons. Being induced by viral infection is a defining characteristic of interferons, but viral infection or overexpression of members of the interferon regulatory factor (IRF) family of transcription factors only leads to a minute induction of IFNL4 This behavior is evolutionarily conserved and can be reversed by inserting a functional IRF3 binding site into the IFNL4 promoter. Thus, the regulation of the IFNL4 gene is radically different and might explain some of the atypical phenotypes associated with the IFNL4 gene in humans.IMPORTANCE Recent genetic evidence has highlighted how the IFNL4 gene acts in a counterintuitive manner, as patients with a nonfunctional IFNL4 gene exhibit increased clearance of hepatitis C virus (HCV) but also increased liver inflammation. This suggests that the IFNL4 gene acts in a proviral and anti-inflammatory manner. These surprising but quite clear genetic data have prompted an extensive examination of the basic characteristics of the IFNL4 gene and its gene product, interferon lambda 4 (IFN-λ4). We have investigated the expression of the IFNL4 gene and found it to be poorly induced by viral infections. A thorough investigation of the IFNL4 promoter revealed a highly conserved and functional promoter, but also one that lacks the defining characteristic of interferons (IFNs), i.e., the ability to be effectively induced by viral infections. We suggest that the unique function of the IFNL4 gene is related to its noncanonical transcriptional regulation.

PD-L1 and PD-L2 expression correlated genes in non-small-cell lung cancer.

BACKGROUND: Programmed cell death ligand-1 (PD-L1) and ligand-2 (PD-L2) interaction with programmed cell death protein-1 (PD-1) represent an immune-inhibiting checkpoint mediating immune evasion and is, accordingly, an important target for blockade-based immunotherapy in cancer. In non-small-cell lung cancer (NSCLC), improved understanding of PD-1 checkpoint blockade-responsive biology and identification of biomarkers for prediction of a clinical response to immunotherapy is warranted. Thus, in the present study, we systematically described PD-L1 and PD-L2 expression correlated genes in NSCLC. METHODS: We performed comparative retrospective analyses to identify PD-L1 and PD-L2 mRNA expression correlated genes in NSCLC. For this, we examined available datasets from the cancer cell line encyclopedia (CCLE) project lung non-small-cell (Lung_NSC) and the cancer genome atlas (TCGA) projects lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). RESULTS: Analysis of the CCLE dataset Lung_NSC identified expression correlation between PD-L1 and PD-L2. Moreover, we identified expression correlation between 489 genes and PD-L1, 191 genes and PD-L2, and 111 genes for both. PD-L1 and PD-L2 also expression correlated in TCGA datasets LUAD and LUSC. In LUAD, we identified expression correlation between 257 genes and PD-L1, 914 genes and PD-L2, and 211 genes for both. In LUSC, we identified expression correlation between 26 genes and PD-L1, 326 genes and PD-L2, and 13 genes for both. Only a few genes expression correlated with PD-L1 and PD-L2 across the CCLE and TCGA datasets. Expression of Interferon signaling-involved genes converged in particular with the expression correlated genes for PD-L1 in Lung_NSC, for PD-L2 in LUSC, and for both PD-L1 and PD-L2 in LUAD. In LUSC, PD-L1, and to a lesser extent PD-L2, expression correlated with chromosome 9p24 localized genes, indicating a chromosome 9p24 topologically associated domain as an important driver of in particular LUSC PD-L1 expression. Expression correlation analyses of the PD-L1 and PD-L2 receptors programmed cell death protein-1 (PD-1), Cluster of differentiation 80 (CD80), and Repulsive guidance molecule B (RGMB) showed that PD-1 and CD80 expression correlated with both PD-L1 and PD-L2 in LUAD. CD80 expression correlated with PD-L2 in LUSC. CONCLUSIONS: We present gene signatures associated with PD-L1 and PD-L2 mRNA expression in NSCLC which could possess importance in relation to understand PD-1 checkpoint blockade-responsive biology and development of gene signature based biomarkers for predicting clinical responses to immunotherapy.

Cause-and-Effect relationship between FGFR1 expression and epithelial-mesenchymal transition in EGFR-mutated non-small cell lung cancer cells.

OBJECTIVES: Increased FGFR1 expression is associated with resistance to tyrosine kinase inhibitors (TKIs) in EGFR-mutated NSCLC cells and often concomitant with epithelial to mesenchymal transition (EMT). However, the cause-and-effect relationship between increased FGFR1 expression and EMT in the genetic background of EGFR-mutated non-small cell lung cancer (NSCLC) cells is not clear. Previous studies have specifically addressed the relationship between EMT and increased FGFR1 expression in the context of simultaneous TKI-mediated blocking of EGFR-signaling. Here, in the context of EGFR-mutated NSCLC cells with active EGFR-signaling, we have examined whether increased FGFR1 expression drives EMT or is an EMT passenger event. MATERIALS AND METHODS: For cause-and-effect analyses between EMT and FGFR1 expression, including expression of alternative spliced FGFR1 isoforms, we used CRISPR-dCAS9-SAM-mediated induction of the endogenous FGFR1 and ZEB1 genes, as well as biochemical EMT-induction, in PC9 and HCC827 NSCLC cell lines harboring activating EGFR-mutations. RESULTS: We find that FGFR1 expression correlates with a ZEB1-associated EMT gene expression profile in NSCLC cells. In experiments using NSCLC cell lines harboring activating EGFR-mutations we show that CRISPR-dCAS9-SAM-mediated induction of FGFR1 expression is neither driving an increase in ZEB1 expression nor EMT characteristics. However, CRISPR-dCAS9-SAM-mediated induction of ZEB1 expression drives EMT characteristics and an increase in FGFR1 expression. Biochemical induction of EMT also drives an increase in FGFR1 expression. CONCLUSION: From our findings concerning the cause-and-effect relationship in the genetic background of EGFR-mutated NSCLC cells, we conclude that an increase in ZEB1 expression is a driver of EMT resulting in concomitant increased FGFR1 expression, whereas an increase in FGFR1 expression is insufficient to drive concomitant EMT.

Up-Regulated FGFR1 Expression as a Mediator of Intrinsic TKI Resistance in EGFR-Mutated NSCLC.

Non-small cell lung carcinoma patients with epidermal growth factor receptor (EGFR) mutations are offered EGFR tyrosine kinase inhibitors (TKI) as first line treatment, but 20-40% of these patients do not respond. High expression of alternative receptor tyrosine kinases, such as Fibroblast growth factor receptor 1 (FGFR1), potentially mediates intrinsic EGFR TKI resistance. To study this in molecular detail, we used CRISPR-dCas9 Synergistic Activation Mediator (SAM) for up-regulation of FGFR1 in physiological relevant levels in the EGFR mutated NSCLC cell lines HCC827 and PC9 thereby generating HCC827gFGFR1 and PC9gFGFR1. The sensitivity to the TKI erlotinib was investigated in vitro and in a BALBc nu/nu mouse xenograft model. FGFR1 up-regulation decreased TKI-sensitivity in both NSCLC cell lines in the presence of the ligand fibroblast growth factor 2 (FGF2). Xenografts were established with PC9gFGFR1 cells and it was demonstrated that there was no significant difference in tumor size between TKI- and vehicle-treated PC9gFGFR1 tumors. This supports decreased TKI-sensitivity in NSCLC cells with FGFR1 up-regulation. Our study points to FGFR1 signaling being an intrinsic resistance mechanism abolishing TKI response in EGFR mutated NSCLC.

Simple method for assembly of CRISPR synergistic activation mediator gRNA expression array.

When studying complex interconnected regulatory networks, effective methods for simultaneously manipulating multiple genes expression are paramount. Previously, we have developed a simple method for generation of an all-in-one CRISPR gRNA expression array. We here present a Golden Gate Assembly-based system of synergistic activation mediator (SAM) compatible CRISPR/dCas9 gRNA expression array for the simultaneous activation of multiple genes. Using this system, we demonstrated the simultaneous activation of the transcription factors, TWIST, SNAIL, SLUG, and ZEB1 a human breast cancer cell line.

IGF1R depletion facilitates MET-amplification as mechanism of acquired resistance to erlotinib in HCC827 NSCLC cells.

EGFR-mutated non-small cell lung cancer patients experience relapse within 1-2 years of treatment with EGFR-inhibitors, such as erlotinib. Multiple resistance mechanisms have been identified including secondary EGFR-mutations, MET-amplification, and epithelial-mesenchymal transition (EMT). Previous studies have indicated a role of Insulin-like growth factor 1 receptor (IGF1R) in acquired resistance to EGFR-directed drugs as well as in EMT. In the present study, we have investigated the involvement of IGF1R in acquired high-dose erlotinib resistance in the EGFR-mutated lung adenocarcinoma cell line HCC827. We observed that IGF1R was upregulated in the immediate response to erlotinib and hyperactivated in erlotinib resistant HCC827 cells. Resistant cells additionally acquired features of EMT, whereas MET-amplification and secondary EGFR-mutations were absent. Using CRISPR/Cas9, we generated a HCC827(IGFR1-/-) cell line and subsequently investigated resistance development in response to high-dose erlotinib. Interestingly, HCC827(IGFR1-/-) cells were now observed to specifically amplify the MET gene. Additionally, we observed a reduced level of mesenchymal markers in HCC827(IGFR1-/-) indicating an intrinsic enhanced epithelial signature compared to HCC827 cells. In conclusion, our data show that IGF1R have an important role in defining selected resistance mechanisms in response to high doses of erlotinib.

The role of epithelial to mesenchymal transition in resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.

Inhibition of the epidermal growth factor receptor (EGFR) is an important strategy when treating non-small cell lung cancer (NSCLC) patients. However, intrinsic resistance or development of resistance during the course of treatment constitutes a major challenge. The knowledge on EGFR-directed tyrosine kinase inhibitors (TKIs) and their biological effect keeps increasing. Within the group of patients with EGFR mutations some benefit to a much higher degree than others, and for patients lacking EGFR mutations a subset experience an effect. Up to 70% of patients with EGFR mutations and 10-20% of patients without EGFR mutations initially respond to the EGFR-TKI erlotinib, but there is a severe absence of good prognostic markers. Despite initial effect, all patients acquire resistance to EGFR-TKIs. Multiple mechanisms have implications in resistance development, but much is still to be explored. Epithelial to mesenchymal transition (EMT) is a transcriptionally regulated phenotypic shift rendering cells more invasive and migratory. Within the EMT process lays a need for external or internal stimuli to give rise to changes in central signaling pathways. Expression of mesenchymal markers correlates to a bad prognosis and an inferior response to EGFR-TKIs in NSCLC due to the contribution to a resistant phenotype. A deeper understanding of the role of EMT in NSCLC and especially in EGFR-TKI resistance-development constitute one opportunity to improve the benefit of TKI treatment for the individual patient. Many scientific studies have linked the EMT process to EGFR-TKI resistance in NSCLC and our aim is to review the role of EMT in both intrinsic and acquired resistance to EGFR-TKIs.

Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.

Regulatory dissection of the CBX5 and hnRNPA1 bi-directional promoter in human breast cancer cells reveals novel transcript variants differentially associated with HP1α down-regulation in metastatic cells.

BACKGROUND: The three members of the human heterochromatin protein 1 (HP1) family of proteins, HP1α, HP1ß, and HPγ, are involved in chromatin packing and epigenetic gene regulation. HP1α is encoded from the CBX5 gene and is a suppressor of metastasis. CBX5 is down-regulated at the transcriptional and protein level in metastatic compared to non-metastatic breast cancer. CBX5 shares a bi-directional promoter structure with the hnRNPA1 gene. But whereas CBX5 expression is down-regulated in metastatic cells, hnRNAP1 expression is constant. Here, we address the regulation of CBX5 in human breast cancer. METHODS: Transient transfection and transposon mediated integration of dual-reporter mini-genes containing the bi-directional hnRNPA1 and CBX5 promoter was performed to investigate transcriptional regulation in breast cancer cell lines. Bioinformatics and functional analysis were performed to characterize transcriptional events specifically regulating CBX5 expression. TSA treatment and Chromatin Immunoprecipitation (ChIP) were performed to investigate the chromatin structure along CBX5 in breast cancer cells. Finally, expression of hnRNPA1 and CBX5 mRNA isoforms were measured by quantitative reverse transcriptase PCR (qRT-PCR) in breast cancer tissue samples. RESULTS: We demonstrate that an hnRNPA1 and CBX5 bi-directional core promoter fragment does not comprise intrinsic capacity for specific CBX5 down-regulation in metastatic cells. Characterization of transcriptional events in the 20 kb CBX5 intron 1 revealed existence of several novel CBX5 transcripts. Two of these encode consensus HP1α protein but used autonomous promoters in intron 1 by which HP1α expression could be de-coupled from the bi-directional promoter. In addition, another CBX5 transcriptional isoform, STET, was discovered. This transcript includes CBX5 exon 1 and part of intron 1 sequences but lacks inclusion of HP1α encoding exons. Inverse correlation between STET and HP1α coding CBX5 mRNA expression was observed in breast cancer cell lines and tissue samples from breast cancer patients. CONCLUSION: We find that HP1α is down-regulated in a mechanism involving CBX5 promoter downstream sequences and that regulation through alternative polyadenylation and splicing generates a transcript, STET, with potential importance in carcinogenesis.

EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions.

BACKGROUND: FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. RESULTS: To determine the gene regulatory functions of FUS and EWS at the level of chromatin, we have performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. CONCLUSIONS: The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.

Beyond the histone tale: HP1α deregulation in breast cancer epigenetics.

Heterochromatin protein 1α (HP1α) encoded from the CBX5-gene is an evolutionary conserved protein that binds histone H3 di- or tri-methylated at position lysine 9 (H3K9me2/3), a hallmark for heterochromatin, and has an essential role in forming higher order chromatin structures. HP1α has diverse functions in heterochromatin formation, gene regulation, and mitotic progression, and forms complex networks of gene, RNA, and protein interactions. Emerging evidence has shown that HP1α serves a unique biological role in breast cancer related processes and in particular for epigenetic control mechanisms involved in aberrant cell proliferation and metastasis. However, how HP1α deregulation plays dual mechanistic functions for cancer cell proliferation and metastasis suppression and the underlying cellular mechanisms are not yet comprehensively described. In this paper we provide an overview of the role of HP1α as a new sight of epigenetics in proliferation and metastasis of human breast cancer. This highlights the importance of addressing HP1α in breast cancer diagnostics and therapeutics.