CircHIF1A regulated by FUS accelerates triple-negative breast cancer progression by modulating NFIB expression and translocation.

Emerging evidence has demonstrated that circular RNAs (circRNAs) play critical roles in the development and progression of human cancer. However, the biological functions and underlying mechanisms of circRNAs in triple-negative breast cancer (TNBC) remain to be investigated. In our present study, we found that the novel circRNA circHIF1A was significantly overexpressed in breast cancer tissues and that it was associated with metastasis, poor prognosis, and the TNBC subtype. Gain- and loss-of-function experiments were conducted to investigate the biological roles of circHIF1A in TNBC. Overexpression of circHIF1A significantly promoted TNBC growth and metastasis in vitro and in vivo, while knockdown of circHIF1A exerted the opposite effects. Mechanistically, circHIF1A modulated the expression and translocation of NFIB through posttranscriptional and posttranslational modifications, resulting in the activation of the AKT/STAT3 signaling pathway and inhibition of P21. The RNA binding protein FUS could regulate the biogenesis of circHIF1A by interacting with the flanking intron, and FUS was transcriptionally regulated by NFIB, thus forming the circHIF1A/NFIB/FUS positive feedback loop. Moreover, circHIF1A could be packaged into exosomes and was upregulated in the plasma of breast cancer patients. Our findings indicated that circHIF1A played a critical role in the growth and metastasis of TNBC via a positive feedback loop and that circHIF1A could be a promising biomarker for breast cancer diagnosis and a potential therapeutic target for TNBC treatment.

Increased nuclear factor I/B expression in prostate cancer correlates with AR expression.

BACKGROUND: Most prostate cancers express androgen receptor (AR), and our previous studies have focused on identifying transcription factors that modify AR function. We have shown that nuclear factor I/B (NFIB) regulates AR activity in androgen-dependent prostate cancer cells in vitro. However, the status of NFIB in prostate cancer was unknown. METHODS: We immunostained a tissue microarray including normal, hyperplastic, prostatic intraepithelial neoplasia, primary prostatic adenocarcinoma, and castration-resistant prostate cancer tissue samples for NFIB, AR, and synaptophysin, a marker of neuroendocrine differentiation. We interrogated publically available data sets in cBioPortal to correlate NFIB expression and AR and neuroendocrine prostate cancer (NEPCa) activity scores. We analyzed prostate cancer cell lines for NFIB expression via Western blot analysis and used nuclear and cytoplasmic fractionation to assess where NFIB is localized. We performed co-immunoprecipitation studies to determine if NFIB and AR interact. RESULTS: NFIB increased in the nucleus and cytoplasm of prostate cancer samples versus matched normal controls, independent of Gleason score. Similarly, cytoplasmic AR and synaptophysin increased in primary prostate cancer. We observed strong NFIB staining in primary small cell prostate cancer. The ratio of cytoplasmic-to-nuclear NFIB staining was predictive of earlier biochemical recurrence in prostate cancer, once adjusted for tumor margin status. Cytoplasmic AR was an independent predictor of biochemical recurrence. There was no statistically significant difference between NFIB and synaptophysin expression in primary and castration-resistant prostate cancer, but cytoplasmic AR expression was increased in castration-resistant samples. In primary prostate cancer, nuclear NFIB expression correlated with cytoplasmic NFIB and nuclear AR, while cytoplasmic NFIB correlated with synaptophysin, and nuclear and cytoplasmic AR. In castration-resistant prostate cancer samples, NFIB expression correlated positively with an AR activity score, and negatively with the NEPCa score. In prostate cancer cell lines, NFIB exists in several isoforms. We observed NFIB predominantly in the nuclear fraction of prostate cancer cells with increased cytoplasmic expression seen in castration-resistant cell lines. We observed an interaction between AR and NFIB through co-immunoprecipitation experiments. CONCLUSION: We have described the expression pattern of NFIB in primary and castration-resistant prostate cancer and its positive correlation with AR. We have also demonstrated AR interacts with NFIB.

Glia-specific enhancers and chromatin structure regulate NFIA expression and glioma tumorigenesis.

Long-range enhancer interactions critically regulate gene expression, yet little is known about how their coordinated activities contribute to CNS development or how this may, in turn, relate to disease states. By examining the regulation of the transcription factor NFIA in the developing spinal cord, we identified long-range enhancers that recapitulate NFIA expression across glial and neuronal lineages in vivo. Complementary genetic studies found that Sox9-Brn2 and Isl1-Lhx3 regulate enhancer activity and NFIA expression in glial and neuronal populations. Chromatin conformation analysis revealed that these enhancers and transcription factors form distinct architectures within these lineages in the spinal cord. In glioma models, the glia-specific architecture is present in tumors, and these enhancers are required for NFIA expression and contribute to glioma formation. By delineating three-dimensional mechanisms of gene expression regulation, our studies identify lineage-specific chromatin architectures and associated enhancers that regulate cell fate and tumorigenesis in the CNS.

Predictors of Outcome in Adenoid Cystic Carcinoma of Salivary Glands: A Clinicopathologic Study With Correlation Between MYB Fusion and Protein Expression.

Adenoid cystic carcinoma (ACC) is the second most common salivary gland malignancy and it has a high rate of recurrences and a poor long-term prognosis. Our aim was to assess the prognostic factors in ACC and study MYB-NFIB fusion and MYB protein expression in a large retrospective cohort of 135 patients with a median follow-up of 6.3 years. The 5- and 10-year local recurrence-free survival (RFS) rate of 94% and 78%, 5- and 10-year distant metastasis survival rate of 77% and 58%, and 5- and 10-year RFS of 66% and 44%. The following features were identified as adverse prognostic factors of RFS on univariate analysis: large tumor size, solid growth pattern, increased mitoses, positive margin, American Joint Committee on Cancer clinical staging, high-grade transformation, vascular invasion, nuclear atypia, open chromatin, prominent nucleoli, and tumor necrosis. However, on multivariate analysis, only increased mitoses (≥5/10 high-power fields), any solid growth pattern, and advanced American Joint Committee on Cancer TNM staging were independent adverse predictors for RFS. MYB immunoexpression and MYB-NFIB translocation were common findings in ACC, occurring in 72% and 59% of the tested ACCs, respectively. The sensitivity and specificity of MYB immunohistochemistry in detecting MYB-NFIB fusion was relatively low at 78% sensitivity and 50% specificity. The high prevalence of alterations leading to high expression of the MYB transcription factor family suggests that targeted approaches developed to suppress the expression of these oncogenic transcription factors and/or the transcriptional activity of these proteins would be a rational therapeutic approach to investigate in ACC.

Differential neuronal and glial expression of nuclear factor I proteins in the cerebral cortex of adult mice.

The nuclear factor I (NFI) family of transcription factors plays an important role in the development of the cerebral cortex in humans and mice. Disruption of nuclear factor IA (NFIA), nuclear factor IB (NFIB), or nuclear factor IX (NFIX) results in abnormal development of the corpus callosum, lateral ventricles, and hippocampus. However, the expression or function of these genes has not been examined in detail in the adult brain, and the cell type-specific expression of NFIA, NFIB, and NFIX is currently unknown. Here, we demonstrate that the expression of each NFI protein shows a distinct laminar pattern in the adult mouse neocortex and that their cell type-specific expression differs depending on the family member. NFIA expression was more frequently observed in astrocytes and oligodendroglia, whereas NFIB expression was predominantly localized to astrocytes and neurons. NFIX expression was most commonly observed in neurons. The NFI proteins were equally distributed within microglia, and the ependymal cells lining the ventricles of the brain expressed all three proteins. In the hippocampus, the NFI proteins were expressed during all stages of neural stem cell differentiation in the dentate gyrus, with higher expression intensity in neuroblast cells as compared to quiescent stem cells and mature granule neurons. These findings suggest that the NFI proteins may play distinct roles in cell lineage specification or maintenance, and establish the basis for further investigation of their function in the adult brain and their emerging role in disease.

TNF-α-mediated microRNA-136 induces differentiation of myeloid cells by targeting NFIA.

Immune cell-lineage specification and function are influenced by progenitor origin and environmental factors. The mechanism of differentiation of immune cells, such as neutrophils, monocytes, and myeloid-derived suppressor cells, in inflammatory environments has not been elucidated completely. In this study, we have identified human microRNA-136 as a positive regulator of the differentiation of granulocytes and monocytes. Ectopic microRNA-136 induced cells to express higher levels of CD11b, CD14, and C/EBPε, secrete more cytokines, and synthesize higher levels of reactive oxygen species and H(2)O(2). microRNA-136 was shown to target and degrade multiple differentiation-associated molecules, such as the transcription factor NFIA, which induced the release of another microRNA, microRNA-223, with the ability to promote CD11b expression. Furthermore, microRNA-136 expression was remarkably increased by TNF-α, which activated NF-κB to bind to the DNA-promoter region controlling microRNA-136 expression. Additionally, TNF-α may alter NFIA expression through its modulation of microRNA-136 expression. Thus, TNF-α-mediated microRNA-136 may play a critical role in the generation and differentiation of inflammatory immune cells.

PAX6 does not regulate Nfia and Nfib expression during neocortical development.

The Nuclear factor I (NFI) family of transcription factors regulates proliferation and differentiation throughout the developing central nervous system. In the developing telencephalon of humans and mice, reduced Nfi expression is associated with agenesis of the corpus callosum and other neurodevelopmental defects. Currently, little is known about how Nfi expression is regulated during early telencephalic development. PAX6, a transcription factor important for telencephalic development, has been proposed as an upstream regulator of Nfi expression in the neocortex. Here we demonstrate that, in the developing neocortex of mice, NFIA and NFIB are endogenously expressed in gradients with high caudo-medial to low rostro-lateral expression and are most highly expressed in the cortical plate. We found that this expression pattern deviates from that of PAX6, suggesting that PAX6 does not drive Nfi expression. This is supported by in vitro reporter assays showing that PAX6 over-expression does not regulate Nfi promoter activity. Similarly, we also found that in the Pax6 Small Eye mutant, no changes in Nfi mRNA or protein expression are observed in the neocortical ventricular zone where PAX6 and the NFIs are expressed. Together these data demonstrate that in mice, PAX6 is not a transcriptional activator of Nfi expression during neocortical development.

p53-induced microRNA-1246 inhibits the cell growth of human hepatocellular carcinoma cells by targeting NFIB.

In recent years, miR-1246 has been identified as a transcriptional target of p53 in Down syndrome and may provide a new p53-miR-1246-DYRK1A-NFAT pathway in cancer. The present study aimed to explore the role of miR-1246 in the tumorigenesis of human hepatocellular carcinoma (HCC). We found that wild-type p53 regulated the expression of miR-1246 in HCC cell lines, and alteration of miR-1246 modulated cell proliferation, colony formation ability and apoptosis. The nuclear factor I/B (NFIB), an oncogene, was identified as a direct target gene of miR-1246 using a fluorescent reporter assay. Overexpression of NFIB abolished the regulation of cell apoptosis caused by miR-1246 in HepG2 cells. This finding suggests that miR-1246 is regulated by p53 and suppresses the growth of human HCC by targeting NFIB. Here, we propose a new p53-miR-1246-NFIB pathway in HCC.

miR-365 promotes cutaneous squamous cell carcinoma (CSCC) through targeting nuclear factor I/B (NFIB).

Aberrant expression of microRNAs plays vital roles in tumor development and progression. As transcription factors (TFs) are the critical components of signaling cascades, specific targeting effects of microRNAs to specific TFs may determine the role of microRNAs in different cancers. In this study, we identified Nuclear Factor I/B (NFIB) as one of the targets of miR-365 which was previously verified as an onco-miR in cutaneous squamous cell carcinoma (CSCC). Down-regulation of NFIB was a general feature in both CSCC cell lines and tumors from patients which show drastically up-regulated miR-365 expression levels. The siRNA-based knockdown of NFIB mimic the carcinogenic transformation of normal cells by ectopically expression of miR-365 which indicates depletion of NFIB is necessary for miR-365 to exert its pro-carcinogenic function. NFIB may represent a functional barrier targeted by miR-365 to the development of CSCC. Further studies also discovered a conserved feedback regulatory circuitry formed by NFIB and miR-365 in CSCC development which may be potentially utilized as therapeutic target to improve the clinical CSCC treatment.

Temporal regulation of nuclear factor one occupancy by calcineurin/NFAT governs a voltage-sensitive developmental switch in late maturing neurons.

Dendrite and synapse development are critical for establishing appropriate neuronal circuits, and disrupted timing of these events can alter neural connectivity. Using microarrays, we have identified a nuclear factor I (NFI)-regulated temporal switch program linked to dendrite formation in developing mouse cerebellar granule neurons (CGNs). NFI function was required for upregulation of many synapse-related genes as well as downregulation of genes expressed in immature CGNs. Chromatin immunoprecipitation analysis revealed that a central feature of this program was temporally regulated NFI occupancy of late-expressed gene promoters. Developing CGNs undergo a hyperpolarizing shift in membrane potential, and depolarization inhibits their dendritic and synaptic maturation via activation of calcineurin (CaN) (Okazawa et al., 2009). Maintaining immature CGNs in a depolarized state blocked NFI temporal occupancy of late-expressed genes and the NFI switch program via activation of the CaN/nuclear factor of activated T-cells, cytoplasmic (NFATc) pathway and promotion of late-gene occupancy by NFATc4, and these mechanisms inhibited dendritogenesis. Conversely, inhibition of the CaN/NFATc pathway in CGNs maturing under physiological nondepolarizing conditions upregulated the NFI switch program, NFI temporal occupancy, and dendrite formation. NFATc4 occupied the promoters of late-expressed NFI program genes in immature mouse cerebellum, and its binding was temporally downregulated with development. Further, NFI temporal binding and switch gene expression were upregulated in the developing cerebellum of Nfatc4 (-/-) mice. These findings define a novel NFI switch and temporal occupancy program that forms a critical link between membrane potential/CaN and dendritic maturation in CGNs. CaN inhibits the program and NFI occupancy in immature CGNs by promoting NFATc4 binding to late-expressed genes. As maturing CGNs become more hyperpolarized, NFATc4 binding declines leading to onset of NFI temporal binding and the NFI switch program.

Molecular regulatory mechanism of root development.

TGF-beta signaling is known to function during tooth formation. The authors' study investigated the role of TGF-beta signaling during tooth root development and determined how the common mediator for TGF-beta signaling, Smad4, affected root formation in mice. Smod4 was specifically inactivated in all epidermal-derived tissues by using a two-component genetic system. The authors' findings show that when Smad4 expression is eliminated in the dental epithelium, there is lack of root formation and severe crown defects.

Enhanced expression of nuclear factor I/B in oxaliplatin-resistant human cancer cell lines.

Oxaliplatin is a third-generation platinum drug that has favorable activity in cisplatin-resistant cells. However, the molecular mechanisms underlying oxaliplatin resistance are not well understood. To investigate the molecular mechanisms involved, resistant cell lines were independently derived from colon cancer (DLD1) and bladder cancer (T24) cells. Oxaliplatin-resistant DLD1 OX1 and DLD1 OX2 cell lines were approximately 16.3-fold and 17.8-fold more resistant to oxaliplatin than the parent cell lines, respectively, and had 1.7- and 2.2-fold higher cross-resistance to cisplatin, respectively. Oxaliplatin-resistant T24 OX2 and T24 OX3 cell lines were approximately 5.0-fold more resistant to oxaliplatin than the parent cell line and had 1.9-fold higher cross-resistance to cisplatin. One hundred and fifty-eight genes commonly upregulated in both DLD1 OX1 and DLD1 OX2 were identified by microarray analysis. These genes were mainly involved in the function of transcriptional regulators (14.6%), metabolic molecules (14.6%), and transporters (9.5%). Of these, nuclear factor I/B (NFIB) was upregulated in all oxaliplatin-resistant cells. Downregulation of NFIB rendered cells sensitive to oxaliplatin, but not to cisplatin. Forced expression of NFIB induced resistance to oxaliplatin, but not to cisplatin. Taken together, these results suggest that NFIB is a novel and specific biomarker for oxaliplatin resistance in human cancers.

Nuclear factor IA is expressed in astrocytomas and is associated with improved survival.

Nuclear factor IA (NFIA) is a transcription factor that specifies glial cell identity and promotes astrocyte differentiation during embryonic development. Its expression and function in gliomas are not known. Here, we examined NFIA protein expression in gliomas and its association with clinical outcome in pediatric malignant astrocytomas. We analyzed expression of NFIA by immunohistochemistry in 88 existing glioma specimens from Childrens Hospital Los Angeles and the University of Southern California. Association between NFIA expression and progression-free survival (PFS) was examined in high-grade astrocytomas for which clinical data were available (n = 23, all children). NFIA was highly expressed in astrocytomas of all grades, but only in a minority of cells in oligodendroglial tumors. NFIA was expressed on a higher percentage of tumor cells in low-grade astrocytomas (91 +/- 5% and 77 +/- 14% in World Health Organization [WHO] I and II, respectively) compared with high-grade astrocytomas (48 +/- 18% and 37 +/- 16% in WHO III and IV, respectively; P < .001, low- vs high-grade astrocytomas). There was a significant association between NFIA expression and PFS in children with astrocytoma WHO grade III or IV (Cox regression P = .019; logrank trend test for NFIA tertiles P = .0040 and NFIA quartiles P = .014). The association was not consistently significant in this small series of patients after adjustment was made for WHO grade III or IV. This is the first study to demonstrate expression of NFIA protein in astrocytomas and its association with grades of astrocytoma and PFS, suggesting that NFIA may play a role in astrocytoma biology.

Impaired micro-RNA pathways diminish osteoclast differentiation and function.

Micro-RNAs (miRNAs) are important in regulating cell fate determination because many of their target mRNA transcripts are engaged in cell proliferation, differentiation, and apoptosis. DGCR8, Dicer, and Ago2 are essential factors for miRNA homeostasis. Here we show that these three factors have critical roles in osteoclast differentiation and function. Gene silencing of DGCR8, Dicer, or Ago2 by small interfering RNA revealed global inhibition of osteoclast transcription factor expression and function, decreased osteoclastogenesis, and decreased bone resorption in vitro. In vivo, CD11b(+)-cre/Dicer-null mice had mild osteopetrosis caused by decreased osteoclast number and bone resorption. These results suggest that miRNAs play important roles in differentiation and function of osteoclasts in vitro and in vivo. We found a novel mechanism mediating these results in which PU.1, miRNA-223, NFI-A, and the macrophage colony-stimulating factor receptor (M-CSFR) are closely linked through a positive feedback loop. PU.1 stimulates miRNA-223 expression, and this up-regulation is implicated in stimulating differentiation and function of osteoclasts through negative regulation of NFI-A levels. Down-regulation of NFI-A levels is important for expression of the M-CSFR, which is critical for osteoclast differentiation and function. NFI-A overexpression decreased osteoclast formation and function with down-regulation of M-CSFR levels. Forced expression of the M-CSFR in M-CSF-dependent bone marrow macrophages from Dicer-deficient mice rescued osteoclast differentiation with up-regulation of PU.1 levels. Our studies provide new molecular mechanisms controlling osteoclast differentiation and function by the miRNA system and specifically by miRNA-223, which regulates NFI-A and the M-CSFR levels.