Significance of NKX2-1 as a biomarker for clinical prognosis, immune infiltration, and drug therapy in lung squamous cell carcinoma.

Background: This study was performed to determine the biological processes in which NKX2-1 is involved and thus its role in the development of lung squamous cell carcinoma (LUSC) toward improving the prognosis and treatment of LUSC. Methods: Raw RNA sequencing (RNA-seq) data of LUSC from The Cancer Genome Atlas (TCGA) were used in bioinformatics analysis to characterize NKX2-1 expression levels in tumor and normal tissues. Survival analysis of Kaplan-Meier curve, the time-dependent receiver operating characteristic (ROC) curve, and a nomogram were used to analyze the prognosis value of NKX2-1 for LUSC in terms of overall survival (OS) and progression-free survival (PFS). Then, differentially expressed genes (DEGs) were identified, and Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA) were used to clarify the biological mechanisms potentially involved in the development of LUSC. Moreover, the correlation between the NKX2-1 expression level and tumor mutation burden (TMB), tumor microenvironment (TME), and immune cell infiltration revealed that NKX2-1 participates in the development of LUSC. Finally, we studied the effects of NKX2-1 on drug therapy. To validate the protein and gene expression levels of NKX2-1 in LUSC, we employed immunohistochemistry(IHC) datasets, The Gene Expression Omnibus (GEO) database, and qRT-PCR analysis. Results: NKX2-1 expression levels were significantly lower in LUSC than in normal lung tissue. It significantly differed in gender, stage and N classification. The survival analysis revealed that high expression of NKX2-1 had shorter OS and PFS in LUSC. The multivariate Cox regression hazard model showed the NKX2-1 expression as an independent prognostic factor. Then, the nomogram predicted LUSC prognosis. There are 51 upregulated DEGs and 49 downregulated DEGs in the NKX2-1 high-level groups. GO, KEGG and GSEA analysis revealed that DEGs were enriched in cell cycle and DNA replication.The TME results show that NKX2-1 expression was positively associated with mast cells resting, neutrophils, monocytes, T cells CD4 memory resting, and M2 macrophages but negatively associated with M1 macrophages. The TMB correlated negatively with NKX2-1 expression. The pharmacotherapy had great sensitivity in the NKX2-1 low-level group, the immunotherapy is no significant difference in the NKX2-1 low-level and high-level groups. The analysis of GEO data demonstrated concurrence with TCGA results. IHC revealed NKX2-1 protein expression in tumor tissues of both LUAD and LUSC. Meanwhile qRT-PCR analysis indicated a significantly lower NKX2-1 expression level in LUSC compared to LUAD. These qRT-PCR findings were consistent with co-expression analysis of NKX2-1. Conclusion: We conclude that NKX2-1 is a potential biomarker for prognosis and treatment LUSC. A new insights of NKX2-1 in LUSC is still needed further research.

CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair.

Cell plasticity theoretically extends to all possible cell types, but naturally decreases as cells differentiate, whereas injury-repair re-engages the developmental plasticity. Here we show that the lung alveolar type 2 (AT2)-specific transcription factor (TF), CEBPA, restricts AT2 cell plasticity in the mouse lung. AT2 cells undergo transcriptional and epigenetic maturation postnatally. Without CEBPA, both neonatal and mature AT2 cells reduce the AT2 program, but only the former reactivate the SOX9 progenitor program. Sendai virus infection bestows mature AT2 cells with neonatal plasticity where Cebpa mutant, but not wild type, AT2 cells express SOX9, as well as more readily proliferate and form KRT8/CLDN4+ transitional cells. CEBPA promotes the AT2 program by recruiting the lung lineage TF NKX2-1. The temporal change in CEBPA-dependent plasticity reflects AT2 cell developmental history. The ontogeny of AT2 cell plasticity and its transcriptional and epigenetic mechanisms have implications in lung regeneration and cancer.

High risk of lung cancer in surfactant-related gene variant carriers.

BACKGROUND: Several rare surfactant-related gene (SRG) variants associated with interstitial lung disease are suspected to be associated with lung cancer, but data are missing. We aimed to study the epidemiology and phenotype of lung cancer in an international cohort of SRG variant carriers. METHODS: We conducted a cross-sectional study of all adults with SRG variants in the OrphaLung network and compared lung cancer risk with telomere-related gene (TRG) variant carriers. RESULTS: We identified 99 SRG adult variant carriers (SFTPA1 (n=18), SFTPA2 (n=31), SFTPC (n=24), ABCA3 (n=14) and NKX2-1 (n=12)), including 20 (20.2%) with lung cancer (SFTPA1 (n=7), SFTPA2 (n=8), SFTPC (n=3), NKX2-1 (n=2) and ABCA3 (n=0)). Among SRG variant carriers, the odds of lung cancer was associated with age (OR 1.04, 95% CI 1.01-1.08), smoking (OR 20.7, 95% CI 6.60-76.2) and SFTPA1/SFTPA2 variants (OR 3.97, 95% CI 1.39-13.2). Adenocarcinoma was the only histological type reported, with programmed death ligand-1 expression ≥1% in tumour cells in three samples. Cancer staging was localised (I/II) in eight (40%) individuals, locally advanced (III) in two (10%) and metastatic (IV) in 10 (50%). We found no somatic variant eligible for targeted therapy. Seven cancers were surgically removed, 10 received systemic therapy, and three received the best supportive care according to their stage and performance status. The median overall survival was 24 months, with stage I/II cancers showing better survival. We identified 233 TRG variant carriers. The comparative risk (subdistribution hazard ratio) for lung cancer in SRG patients versus TRG patients was 18.1 (95% CI 7.1-44.7). CONCLUSIONS: The high risk of lung cancer among SRG variant carriers suggests specific screening and diagnostic and therapeutic challenges. The benefit of regular computed tomography scan follow-up should be evaluated.

TTF-1 status in early-stage lung adenocarcinoma is an independent predictor of relapse and survival superior to tumor grading.

OBJECTIVES: Thyroid transcription factor 1 (TTF-1) is a well-established independent prognostic factor in lung adenocarcinoma (LUAD), irrespective of stage. This study aims to determine if TTF-1's prognostic impact is solely based on histomorphological differentiation (tumor grading) or if it independently relates to a biologically more aggressive phenotype. We analyzed a large bi-centric LUAD cohort to accurately assess TTF-1's prognostic value in relation to tumor grade. PATIENTS AND METHODS: We studied 447 patients with resected LUAD from major German lung cancer centers (Berlin and Cologne), correlating TTF-1 status and grading with clinical, pathologic, and molecular data, alongside patient outcomes. TTF-1's impact was evaluated through univariate and multivariate Cox regression. Causal graph analysis was used to identify and account for potential confounders, improving the statistical estimation of TTF-1's predictive power for clinical outcomes. RESULTS: Univariate analysis revealed TTF-1 positivity associated with significantly longer disease-free survival (DFS) (median log HR -0.83; p = 0.018). Higher tumor grade showed a non-significant association with shorter DFS (median log HR 0.30; p = 0,62 for G1 to G2 and 0.68; p = 0,34 for G2 to G3). In multivariate analysis, TTF-1 positivity resulted in a significantly longer DFS (median log HR -0.65; p = 0.05) independent of all other parameters, including grading. Adjusting for potential confounders as indicated by the causal graph confirmed the superiority of TTF-1 over tumor grading in prognostics power. CONCLUSIONS: TTF-1 status predicts relapse and survival in LUAD independently of tumor grading. The prognostic power of tumor grading is limited to TTF-1-positive patients, and the effect size of TTF-1 surpasses that of tumor grading. We recommend including TTF1 status as a prognostic factor in the diagnostic guidelines of LUAD.

Expression landscapes in non-small cell lung cancer shaped by the thyroid transcription factor 1.

TTF-1-expressing non-small cell lung cancer (NSCLC) is one of the most prevalent lung cancer types worldwide. However, theparadoxical activity of TTF-1 in both lung carcinogenesis and tumor suppression is believed to be context-dependentwhich calls for a deeper understanding about the pathological expression of TTF-1. In addition, the expression circuitry of TTF-1-target genes in NSCLC has not been well examined which necessitates to revisit the involvement of TTF-1- in a multitude of oncologic pathways. We used RNA-seq and clinical data of patients from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), including ChIP-seq data from different NSCLC cell lines, and mapped the proteome of NSCLC tumor. Our analysis showed significant variability in TTF-1 expression among NSCLC,and further clarified that this variability is orchestrated at the transcriptional level. We also found that high TTF-1 expression could negatively influence the survival outcomes of stage 1 LUAD which may be attributed to growth factor receptor-driven activation of mitogenic and angiogenic pathways. Mechanistically, TTF-1 may also control the genes associated with pathways involved in acquired TKI drug resistance or response to immune checkpoint inhibitors. Lastly, proteome-based biomarker discovery in stage 1 LUAD showed that TTF-1 positivity is potentially associated with the upregulation of several oncogenes which includes interferon proteins, MUC1, STAT3, and EIF2AK2. Collectively, this study highlights the potential involvement of TTF-1 in cell proliferation, immune evasion, and angiogenesis in early-stage NSCLC.

Transcriptome analysis reveals the essential role of NK2 homeobox 1/thyroid transcription factor 1 (NKX2-1/TTF-1) in gastric adenocarcinoma of fundic-gland type.

BACKGROUND: Gastric adenocarcinoma of fundic-gland type (GA-FG) is a gastric malignancy with little relation to Helicobacter pylori. Clinical characteristics of GA-FG have been established, but molecular mechanisms leading to tumorigenesis have not yet been elucidated. METHODS: We subjected three GA-FG tumors-normal mucosa pairs to microarray analysis. Network analysis was performed for the top 30 up-regulated gene transcripts, followed by immunohistochemical staining to confirm the gene expression analysis results. AGS and NUGC4 cells were transfected with the gene-encoding NK2 homeobox 1/thyroid transcription factor 1 (NKX2-1/TTF-1) to evaluate transcriptional changes in its target genes. RESULTS: Comprehensive gene expression analysis identified 1410 up-regulated and 1395 down-regulated gene probes with ≥ two-fold difference in expression. Among the top 30 up-regulated genes in GA-FG, we identified transcription factor NKX2-1/TTF-1, a master regulator of lung/thyroid differentiation, together with surfactant protein B (SFTPB), SFTPC, and secretoglobin family 3A member 2(SCGB3A2), which are regulated by NKX2-1/TTF-1. Immunohistochemical analysis of 16 GA-FG specimens demonstrated significantly higher NKX2-1/TTF-1 and SFTPB levels, as compared to that in adjacent normal mucosa (P < 0.05), while SCGB3A2 levels did not differ (P = 0.341). Transduction of NKX2-1/TTF-1 into AGS and NUGC4 cells induced transactivation of SFTPB and SFTPC, indicating that NKX2-1/TTF-1 can function as normally in gastric cells as it can in the lung cells. CONCLUSIONS: Our first transcriptome analysis of GA-FG indicates significant expression of NKX2-1/TTF1 in GA-FG. Immunohistochemistry and cell biology show ectopic expression and normal transactivation ability of NKX2-1/TTF-1, suggesting that it plays an essential role in GA-FG development.

Optimized thyroid transcription factor-1 core promoter-driven microRNA-7 expression effectively inhibits the growth of human non-small-cell lung cancer cells.

Targeted gene therapy has become a promising approach for lung cancer treatment. In our previous work, we reported that the targeted expression of microRNA-7 (miR-7) operated by thyroid transcription factor-1 (TTF-1) promoter inhibited the growth of human lung cancer cells in vitro and in vivo; however, the intervention efficiency needed to be further improved. In this study, we identified the core promoter of TTF-1 (from -1299 bp to -871 bp) by 5' deletion assay and screened out the putative transcription factors nuclear factor-1 (NF-1) and activator protein-1 (AP-1). Further analysis revealed that the expression level of NF-1, but not AP-1, was positively connected with the activation of TTF-1 core promoter in human non-small-cell lung cancer (NSCLC) cells. Moreover, the silencing of NF-1 could reduce the expression level of miR-7 operated by TTF-1 core promoter. Of note, we optimized four distinct sequences to form additional NF-1-binding sites (TGGCA) in the sequence of TTF-1 core promoter (termed as optTTF-1 promoter), and verified the binding efficiency of NF-1 on the optTTF-1 promoter by electrophoretic mobility shift assay (EMSA). As expected, the optTTF-1 promoter could more effectively drive miR-7 expression and inhibit the growth of human NSCLC cells in vitro, accompanied by a reduced transduction of NADH dehydrogenase (ubiquinone) 1α subcomplex 4 (NDUFA4)/protein kinase B (Akt) pathway. Consistently, optTTF-1 promoter-driven miR-7 expression could also effectively abrogate the growth and metastasis of tumor cells in a murine xenograft model of human NSCLC. Finally, no significant changes were detected in the biological indicators or the histology of some important tissues and organs, including heart, liver, and spleen. On the whole, our study revealed that the optimized TTF-1 promoter could more effectively operate miR-7 to influence the growth of human NSCLC cells, providing a new basis for the development of microRNA-based targeting gene therapy against clinical lung cancer.

Transcriptional Circuitry of NKX2-1 and SOX1 Defines an Unrecognized Lineage Subtype of Small-Cell Lung Cancer.

Rationale: The current molecular classification of small-cell lung cancer (SCLC) on the basis of the expression of four lineage transcription factors still leaves its major subtype SCLC-A as a heterogeneous group, necessitating more precise characterization of lineage subclasses. Objectives: To refine the current SCLC classification with epigenomic profiles and to identify features of the redefined SCLC subtypes. Methods: We performed unsupervised clustering of epigenomic profiles on 25 SCLC cell lines. Functional significance of NKX2-1 (NK2 homeobox 1) was evaluated by cell growth, apoptosis, and xenograft using clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-associated protein 9)-mediated deletion. NKX2-1-specific cistromic profiles were determined using chromatin immunoprecipitation followed by sequencing, and its functional transcriptional partners were determined using coimmunoprecipitation followed by mass spectrometry. Rb1flox/flox; Trp53flox/flox and Rb1flox/flox; Trp53flox/flox; Nkx2-1flox/flox mouse models were engineered to explore the function of Nkx2-1 in SCLC tumorigenesis. Epigenomic landscapes of six human SCLC specimens and 20 tumors from two mouse models were characterized. Measurements and Main Results: We identified two epigenomic subclusters of the major SCLC-A subtype: SCLC-Aα and SCLC-Aσ. SCLC-Aα was characterized by the presence of a super-enhancer at the NKX2-1 locus, which was observed in human SCLC specimens and a murine SCLC model. We found that NKX2-1, a dual lung and neural lineage factor, is uniquely relevant in SCLC-Aα. In addition, we found that maintenance of this neural identity in SCLC-Aα is mediated by collaborative transcriptional activity with another neuronal transcriptional factor, SOX1 (SRY-box transcription factor 1). Conclusions: We comprehensively describe additional epigenomic heterogeneity of the major SCLC-A subtype and define the SCLC-Aα subtype by the core regulatory circuitry of NKX2-1 and SOX1 super-enhancers and their functional collaborations to maintain neuronal linage state.

Persisting embryonal infundibular recess in a case of TITF-1 gene mutation.

The thyroid transcription factor 1 (TITF-1) gene plays an important role in the development of the ventral forebrain, thyroid and lungs. Mutations of this gene are known to cause benign hereditary chorea (BHC) and can cause the full spectrum of abnormalities seen in the brain-thyroid-lung syndrome. Abnormalities of the ventral forebrain on imaging have been variably documented in the literature. Multiple previous reports describe a cystic pituitary mass, as well as duplication of the pituitary stalk and communication between an intrasellar cyst and the third ventricle. The initial MRI performed in our case was interpreted as an intrasellar cyst, but the high-resolution MRI performed later was able to resolve this as a persisting embryonal infundibular recess (PEIR), rather than the cystic pituitary mass which has previously been described. This case illustrates the role of the TITF-1 gene in the development of the pituitary and hypothalamus.

Sox9 is involved in the thyroid differentiation program and is regulated by crosstalk between TSH, TGFß and thyroid transcription factors.

While the signaling pathways and transcription factors involved in the differentiation of thyroid follicular cells, both in embryonic and adult life, are increasingly well understood, the underlying mechanisms and potential crosstalk between the thyroid transcription factors Nkx2.1, Foxe1 and Pax8 and inductive signals remain unclear. Here, we focused on the transcription factor Sox9, which is expressed in Nkx2.1-positive embryonic thyroid precursor cells and is maintained from embryonic development to adulthood, but its function and control are unknown. We show that two of the main signals regulating thyroid differentiation, TSH and TGFß, modulate Sox9 expression. Specifically, TSH stimulates the cAMP/PKA pathway to transcriptionally upregulate Sox9 mRNA and protein expression, a mechanism that is mediated by the binding of CREB to a CRE site within the Sox9 promoter. Contrastingly, TGFß signals through Smad proteins to inhibit TSH-induced Sox9 transcription. Our data also reveal that Sox9 transcription is regulated by the thyroid transcription factors, particularly Pax8. Interestingly, Sox9 significantly increased the transcriptional activation of Pax8 and Foxe1 promoters and, consequently, their expression, but had no effect on Nkx2.1. Our study establishes the involvement of Sox9 in thyroid follicular cell differentiation and broadens our understanding of transcription factor regulation of thyroid function.

SRGN-Triggered Aggressive and Immunosuppressive Phenotype in a Subset of TTF-1-Negative Lung Adenocarcinomas.

BACKGROUND: Approximately 20% of lung adenocarcinoma (LUAD) is negative for the lineage-specific oncogene Thyroid transcription factor 1 (TTF-1) and exhibits worse clinical outcome with a low frequency of actionable genomic alterations. To identify molecular features associated with TTF-1-negative LUAD, we compared the transcriptomic and proteomic profiles of LUAD cell lines. SRGN , a chondroitin sulfate proteoglycan Serglycin, was identified as a markedly overexpressed gene in TTF-1-negative LUAD. We therefore investigated the roles and regulation of SRGN in TTF-1-negative LUAD. METHODS: Proteomic and metabolomic analyses of 41 LUAD cell lines were done using mass spectrometry. The function of SRGN was investigated in 3 TTF-1-negative and 4 TTF-1-positive LUAD cell lines and in a syngeneic mouse model (n = 5 to 8 mice per group). Expression of SRGN was evaluated in 94 and 105 surgically resected LUAD tumor specimens using immunohistochemistry. All statistical tests were 2-sided. RESULTS: SRGN was markedly overexpressed at mRNA and protein levels in TTF-1-negative LUAD cell lines (P < .001 for both mRNA and protein levels). Expression of SRGN in LUAD tumor tissue was associated with poor outcome (hazard ratio = 4.22, 95% confidence interval = 1.12 to 15.86, likelihood ratio test, P = .03), and with higher expression of Programmed cell death 1 ligand 1 (PD-L1) in tumor cells and higher infiltration of Programmed cell death protein 1-positive lymphocytes. SRGN regulated expression of PD-L1 as well as proinflammatory cytokines, including Interleukin-6, Interleukin-8, and C-X-C motif chemokine 1 in LUAD cell lines; increased migratory and invasive properties of LUAD cells and fibroblasts; and enhanced angiogenesis. SRGN was induced by DNA demethylation resulting from Nicotinamide N-methyltransferase-mediated impairment of methionine metabolism. CONCLUSIONS: Our findings suggest that SRGN plays a pivotal role in tumor-stromal interaction and reprogramming into an aggressive and immunosuppressive tumor microenvironment in TTF-1-negative LUAD.

Transcription start site-level expression of thyroid transcription factor 1 isoforms in lung adenocarcinoma and its clinicopathological significance.

There are multiple transcription start sites (TSSs) in agreement with multiple transcript variants encoding different isoforms of NKX2-1/TTF-1 (thyroid transcription factor 1); however, the clinicopathological significance of each transcript isoform of NKX2-1/TTF-1 in lung adenocarcinoma (LAD) is unknown. Herein, TSS-level expression of NKX2-1/TTF-1 isoforms was evaluated in 71 LADs using bioinformatic analysis of cap analysis of gene expression (CAGE)-sequencing data, which provides genome-wide expression levels of the 5'-untranslated regions and the TSSs of different isoforms. Results of CAGE were further validated in 664 LADs using in situ hybridisation. Fourteen of 17 TSSs in NKX2-1/TTF-1 (80% of known TSSs in FANTOM5, an atlas of mammalian promoters) were identified in LADs, including TSSs 1-13 and 15; four isoforms of NKX2-1/TTF-1 transcripts (NKX2-1_001, NKX2-1_002, NKX2-1_004, and NKX2-1_005) were expressed in LADs, although NKX2-1_005 did not contain a homeodomain. Among those, six TSSs regulated NKX2-1_004 and NKX2-1_005, both of which contain exon 1. LADs with low expression of isoforms from TSS region 11 regulating exon 1 were significantly associated with poor prognosis in the CAGE data set. In the validation set, 62 tumours (9.3%) showed no expression of NKX2-1/TTF-1 exon 1; such tumours were significantly associated with older age, EGFR wild-type tumours, and poor prognosis. In contrast, 94 tumours, including 22 of 30 pulmonary invasive mucinous adenocarcinomas (IMAs) exhibited exon 1 expression without immunohistochemical TTF-1 protein expression. Furthermore, IMAs commonly exhibited higher exon 1 expression relative to that of exon 4/5, which contained a homeodomain in comparison with EGFR-mutated LADs. These transcriptome and clinicopathological results reveal that LAD use at least 80% of NKX2-1 TSSs and expression of the NKX2-1/TTF-1 transcript isoform without exon 1 (NKX2-1_004 and NKX2-1_005) defines a distinct subset of LAD characterised by aggressive behaviour in elder patients. Moreover, usage of alternative TSSs regions regulating NKX2-1_005 may occur in subsets of LADs.

CRISPRi-mediated functional analysis of NKX2-1-binding sites in the lung.

The transcription factor NKX2-1/TTF-1 is involved in lung pathophysiology, including breathing, innate defense and tumorigenesis. To understand the mechanism by which NKX2-1 regulates genes involved in such pathophysiology, we have previously performed ChIP-seq and identified genome-wide NKX2-1-binding sites, which revealed that NKX2-1 binds to not only proximal promoter regions but also multiple intra- and inter-genic regions of the genes regulated by NKX2-1. However, the roles of such regions, especially non-proximal ones, bound by NKX2-1 have not yet been determined. Here, using CRISPRi (CRISPR/dCas9-KRAB), we scrutinize the functional roles of 19 regions/sites bound by NKX2-1, which are located in genes involved in breathing and innate defense (SFTPB, LAMP3, SFTPA1, SFTPA2) and lung tumorigenesis (MYBPH, LMO3, CD274/PD-L1). Notably, the CRISPRi approach reveals that a portion of NKX2-1-binding sites are functionally indispensable while the rest are dispensable for the expression of the genes, indicating that functional roles of NKX2-1-binding sites are unequally yoked.

An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma.

Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E-driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2-1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies.

When cells become cancerous they grow uncontrollably and spread into surrounding healthy tissue. As the cancer progresses different genes are switched on and off which can cause tumor cells to change their identity and transition into other types of cell. How closely tumor cells resemble the healthy tissue they came from can influence how well the cancer responds to treatment. Many lung cancers have an identity similar to normal lung cells. However, some turn off a gene that codes for a protein called NKX2-1, which leads to a type of cancer called invasive mucinous adenocarcinoma (or IMA for short). Cells from this type of cancer develop an identity similar to mucous cells that line the surface of the stomach. But it was unclear how IMA tumor cells that developed from a mutation in the BRAF gene are affected by this loss in NKX2-1, and how transitioning to a different cell type impacts their response to treatment. To investigate this, Zewdu et al. studied lung cells from patients with IMA tumors driven by a mutation in BRAF and cells from mice that have been genetically engineered to have a similar form of cancer. This revealed that the NKX2-1 protein is needed to initiate the formation of cancer cells but is not required for the growth of already established BRAF-driven tumors. Further experiments showed that removing the gene for NKX2-1 made these cancer cells less responsive to drugs known as BRAF/MEK inhibitors that are commonly used to treat cancer. These drugs caused the IMA cancer cells to change their identity and become another type of stomach cell. This identity change was found to depend on two signaling pathways which cells use to communicate. This study provides some explanation of how IMA lung cancers that lack the gene for NKX2-1 resist treatment with BRAF/MEK inhibitors. It also shows new relationships between key genes in these cancers and systems for cell communication. These findings could lead to better therapies for lung cancer, particularly for patients whose tumor cells are deficient in NKX2-1 and therefore require specialized treatment.

Aberrant expression of thyroid transcription factor-1 in meningeal solitary fibrous tumor/hemangiopericytoma.

Meningeal solitary fibrous tumor (SFT) and hemangiopericytoma (HPC) were categorized as the same entity in the World Health Organization (WHO) 2016 classification of tumors of the central nervous system (CNS). Although NAB2-STAT6 fusion protein can be used to distinguish most of SFT/HPC from the other sarcomas, additional biomarkers were requested to separate meningeal SFT/HPC from meningioma and the molecular pathological difference between meningeal SFT/HPC and extra-CNS SFT/HPC still remains unclear. In this study, we evaluated the expression of TTF-1 in 67 meningeal SFT/HPC, 62 extra-CNS SFT/HPC and 201 meningiomas samples with immunohistochemistry (IHC) assays. The results showed that TTF-1 was detected in 23 of 67 (34.3%) meningeal SFT/HPC, 3 retroperitoneum SFT/HPC and none of meningiomas. Meanwhile, the copy number variation and mRNA expression of TTF-1 were measured by real-time quantitative PCR (qPCR) in meningeal SFT/HPC. These results demonstrated that TTF-1 protein expression level was significantly correlated with its transcription level, but independently related to the gene copy number variant. In conclusion, our study suggested that a large proportion of meningeal SFT/HPC was positive to TTF-1, while very few extra CNS SFT/HPC cases and no meningiomas were stained. So TTF-1 has value as an auxiliary diagnostic marker for meningeal SFT/HPC.

Novel insights into di­(2­ethylhexyl)phthalate activation: Implications for the hypothalamus­pituitary­thyroid axis.

Di (2­ethylhexyl) phthalate (DEHP), an environmental pollutant, is widely used as a plasticizer and causes serious pollution in the ecological environment. As previously reported, exposure to DEHP may cause thyroid dysfunction of the hypothalamic­pituitary­thyroid (HPT) axis. However, the underlying role of DEHP remains to be elucidated. The present study performed intragastrical administration of DEHP (150, 300 and 600 mg/kg) once a day for 90 consecutive days. DEHP­stimulated oxidative stress increased the thyroid follicular cavity diameter and caused thyrocyte oedema. Furthermore, DEHP exposure altered mRNA and protein levels. Thus, DEHP may perturb TH homeostasis by affecting biosynthesis, biotransformation, bio­transportation, receptor levels and metabolism through disruption of the HPT axis and activation of the thyroid­stimulating hormone (TSH)/TSH receptor signaling pathway. These results identified the formerly unappreciated endocrine­disrupting activities of phthalates and the molecular mechanisms of DEHP­induced thyrotoxicity.

An NKX2-1GFP and TP63tdTomato dual fluorescent reporter for the investigation of human lung basal cell biology.

Basal cells are multipotent stem cells responsible for the repair and regeneration of all the epithelial cell types present in the proximal lung. In mice, the elusive origins of basal cells and their contribution to lung development were recently revealed by high-resolution, lineage tracing studies. It however remains unclear if human basal cells originate and participate in lung development in a similar fashion, particularly with mounting evidence for significant species-specific differences in this process. To address this outstanding question, in the last several years differentiation protocols incorporating human pluripotent stem cells (hPSC) have been developed to produce human basal cells in vitro with varying efficiencies. To facilitate this endeavour, we introduced tdTomato into the human TP63 gene, whose expression specifically labels basal cells, in the background of a previously described hPSC line harbouring an NKX2-1GFP reporter allele. The functionality and specificity of the NKX2-1GFP;TP63tdTomato hPSC line was validated by directed differentiation into lung progenitors as well as more specialised lung epithelial subtypes using an organoid platform. This dual fluorescent reporter hPSC line will be useful for tracking, isolating and expanding basal cells from heterogenous differentiation cultures for further study.

Downregulation of TTF1 in the rat hypothalamic ARC or AVPV nucleus inhibits Kiss1 and GnRH expression, leading to puberty delay.

BACKGROUND: TTF1 is a transcription factor that is expressed in the hypothalamus after birth and plays crucial roles in pubertal development. TTF1 may regulate the expression of the Kiss1 gene, which may drive puberty onset in the hypothalamic arcuate (ARC) and anterior ventral paraventricular (AVPV) nuclei. METHODS: A dual-luciferase reporter assay was used to detect binding between TTF1 and the Kiss1 gene promoter. To investigate the effects of TTF1, we modified TTF1 expression in cell lines and in the ARC or AVPV nucleus of 21-day-old female rats via lentivirus infection. TTF1 and other puberty onset-related genes were detected by qRT-PCR and western blot analyses. RESULTS: The in vitro data indicated that TTF1 knockdown (KD) significantly reduced Kiss1 and GnRH expression. Overexpression (OE) of TTF1 promoted Kiss1 expression. In vivo, the expression of Kiss1 and GnRH decreased significantly in the rats with hypothalamic ARC- or AVPV-specific TTF1 KD. The TTF1-KD rats showed vaginal opening delay. H&E staining revealed that the corpus luteum was obviously reduced at the early puberty and adult stages in the rats with ARC- or AVPV-specific TTF1 KD. CONCLUSION: TTF1 bound to the promoter of the Kiss1 gene and enhanced its expression. For 21-day-old female rats, decreased TTF1 in the hypothalamic ARC or AVPV nucleus resulted in delayed vaginal opening and ovarian abnormalities. These observations suggested that TTF1 regulates puberty onset by promoting the expression of Kiss1 and plays an important role in gonad development.

MYB rearrangements and over-expression in T-cell acute lymphoblastic leukemia.

We investigated MYB rearrangements (MYB-R) and the levels of MYB expression, in 331 pediatric and adult patients with T-cell acute lymphoblastic leukemia (T-ALL). MYB-R were detected in 17 cases and consisted of MYB tandem duplication (tdup) (= 14) or T cell receptor beta locus (TRB)-MYB (= 3). As previously reported, TRB-MYB was found only in children (1.6%) while MYB tdup occurred in both age groups, although it was slightly more frequent in children (5.2% vs 2.8%). Shared features of MYB-R T-ALL were a non-early T-cell precursor (ETP) phenotype, a high incidence of NOTCH1/FBXW7 mutations (81%) and CDKN2AB deletions (70.5%). Moreover, they mainly belonged to HOXA (=8), NKX2-1/2-2/TLX1 (=4), and TLX3 (=3) homeobox-related subgroups. Overall, MYB-R cases had significantly higher levels of MYB expression than MYB wild type (MYB-wt) cases, although high levels of MYB were detected in ~ 30% of MYB-wt T-ALL. Consistent with the transcriptional regulatory networks, cases with high MYB expression were significantly enriched within the TAL/LMO subgroup (P = .017). Interestingly, analysis of paired diagnosis/remission samples demonstrated that a high MYB expression was restricted to the leukemic clone. Our study has indicated that different mechanisms underlie MYB deregulation in 30%-40% of T-ALL and highlighted that, MYB has potential as predictive/prognostic marker and/or target for tailored therapy.