Insulinoma-Associated Protein 1 Is a Crucial Regulator of Neuroendocrine Differentiation in Lung Cancer.

Insulinoma-associated protein 1 (INSM1) is expressed exclusively in embryonic developing neuroendocrine (NE) tissues. INSM1 gene expression is specific for small-cell lung cancer (SCLC), along with achaete-scute homolog-like 1 (ASCL1) and several NE molecules, such as chromogranin A, synaptophysin, and neural cell adhesion molecule 1. However, the underlying biological role of INSM1 in lung cancer remains largely unknown. We first showed that surgically resected SCLC samples specifically expressed INSM1. Forced expression of the INSM1 gene in adenocarcinoma cell lines (H358 and H1975) induced the expression of ASCL1, brain-2 (BRN2), chromogranin A, synaptophysin, and neural cell adhesion molecule 1; in contrast, knockdown of the INSM1 gene by siRNA in SCLC (H69 and H889) decreased their expression. However, forced/knockdown expression of ASCL1 and BRN2 did not affect INSM1 expression. A chromatin immunoprecipitation study revealed that INSM1 bound to the promoter region of the ASCL1 gene. A xenotransplantation assay using tet-on INSM1 gene-transfected adenocarcinoma cell lines demonstrated that INSM1 induced NE differentiation and growth inhibition. Furthermore, we found that INSM1 was not expressed in non-small-cell lung cancer and some SCLC cell lines expressing Notch1-Hes1. By forced/knockdown expression of Notch1 or Hes1 genes, we revealed that Notch1-Hes1 signaling suppressed INSM1, as well as ASCL1 and BRN2. INSM1, expressed exclusively in SCLC, is a crucial regulator of NE differentiation in SCLCs, and is regulated by the Notch1-Hes1 signaling pathway.

Analysis of Mll1 deficiency identifies neurogenic transcriptional modules and Brn4 as a factor for direct astrocyte-to-neuron reprogramming.

BACKGROUND: Mixed lineage leukemia-1 (Mll1) epigenetically regulates gene expression patterns that specify cellular identity in both embryonic development and adult stem cell populations. In the adult mouse brain, multipotent neural stem cells (NSCs) in the subventricular zone generate new neurons throughout life, and Mll1 is required for this postnatal neurogenesis but not for glial cell differentiation. Analysis of Mll1-dependent transcription may identify neurogenic genes useful for the direct reprogramming of astrocytes into neurons. OBJECTIVE: To identify Mll1-dependent transcriptional modules and to determine whether genes in the neurogenic modules can be used to directly reprogram astrocytes into neurons. METHODS: We performed gene coexpression module analysis on microarray data from differentiating wild-type and Mll1-deleted subventricular zone NSCs. Key developmental regulators belonging to the neurogenic modules were overexpressed in Mll1-deleted cells and cultured cortical astrocytes, and cell phenotypes were analyzed by immunocytochemistry and electrophysiology. RESULTS: Transcriptional modules that correspond to neurogenesis were identified in wild-type NSCs. Modules related to astrocytes and oligodendrocytes were enriched in Mll1-deleted NSCs, consistent with their gliogenic potential. Overexpression of genes selected from the neurogenic modules enhanced the production of neurons from Mll1-deleted cells, and overexpression of Brn4 (Pou3f4) in nonneurogenic cortical astroglia induced their transdifferentiation into electrophysiologically active neurons. CONCLUSION: Our results demonstrate that Mll1 is required for the expression of neurogenic but not gliogenic transcriptional modules in a multipotent NSC population and further indicate that specific Mll1-dependent genes may be useful for direct reprogramming strategies.

Neuronal transcription factors induce conversion of human glioma cells to neurons and inhibit tumorigenesis.

Recent findings have demonstrated that the overexpression of lineage-specific transcription factors induces cell fate changes among diverse cell types. For example, neurons can be generated from mouse and human fibroblasts. It is well known that neurons are terminally differentiated cells that do not divide. Therefore, we consider how to induce glioma cells to become neurons by introducing transcription factors. Here, we describe the efficient generation of induced neuronal (iN) cells from glioma cells by the infection with three transcription factors: Ascl1, Brn2 and Ngn2 (ABN). iN cells expressed multiple neuronal markers and fired action potentials, similar to the properties of authentic neurons. Importantly, the proliferation of glioma cells following ABN overexpression was dramatically inhibited in both in vitro and in vivo experiments. In addition, iN cells that originated from human glioma cells did not continue to grow when they were sorted and cultured in vitro. The strategies by which glioma cells are induced to become neurons may be used to clinically study methods for inhibiting tumor growth.

The forkhead transcription factor FoxB1 regulates the dorsal-ventral and anterior-posterior patterning of the ectoderm during early Xenopus embryogenesis.

The formation of the dorsal-ventral (DV) and anterior-posterior (AP) axes, fundamental to the body plan of animals, is regulated by several groups of polypeptide growth factors including the TGF-ß, FGF, and Wnt families. In order to ensure the establishment of the body plan, the processes of DV and AP axis formation must be linked and coordinately regulated. However, the molecular mechanisms responsible for these interactions remain unclear. Here, we demonstrate that the forkhead box transcription factor FoxB1, which is upregulated by the neuralizing factor Oct-25, plays an important role in the formation of the DV and AP axes. Overexpression of FoxB1 promoted neural induction and inhibited BMP-dependent epidermal differentiation in ectodermal explants, thereby regulating the DV patterning of the ectoderm. In addition, FoxB1 was also found to promote the formation of posterior neural tissue in both ectodermal explants and whole embryos, suggesting its involvement in embryonic AP patterning. Using knockdown analysis, we found that FoxB1 is required for the formation of posterior neural tissues, acting in concert with the Wnt and FGF pathways. Consistent with this, FoxB1 suppressed the formation of anterior structures via a process requiring the function of XWnt-8 and eFGF. Interestingly, while downregulation of FoxB1 had little effect on neural induction, we found that it functionally interacted with its upstream factor Oct-25 and plays a supportive role in the induction and/or maintenance of neural tissue. Our results suggest that FoxB1 is part of a mechanism that fine-tunes, and leads to the coordinated formation of, the DV and AP axes during early development.

Induced stem cell neoplasia in a cnidarian by ectopic expression of a POU domain transcription factor.

The evolutionary origin of stem cell pluripotency is an unresolved question. In mammals, pluripotency is limited to early embryos and is induced and maintained by a small number of key transcription factors, of which the POU domain protein Oct4 is considered central. Clonal invertebrates, by contrast, possess pluripotent stem cells throughout their life, but the molecular mechanisms that control their pluripotency are poorly defined. To address this problem, we analyzed the expression pattern and function of Polynem (Pln), a POU domain gene from the marine cnidarian Hydractinia echinata. We show that Pln is expressed in the embryo and adult stem cells of the animal and that ectopic expression in epithelial cells induces stem cell neoplasms and loss of epithelial tissue. Neoplasm cells downregulated the transgene but expressed the endogenous Pln gene and also Nanos, Vasa, Piwi and Myc, which are all known cnidarian stem cell markers. Retinoic acid treatment caused downregulation of Pln and the differentiation of neoplasm cells to neurosensory and epithelial cells. Pln downregulation by RNAi led to differentiation. Collectively, our results suggest an ancient role of POU proteins as key regulators of animal stem cells.

Brn2 is a transcription factor regulating keratinocyte differentiation with a possible role in the pathogenesis of lichen planus.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. In this study, we investigated the role of the POU domain-containing transcription factor Brn2 in keratinocyte differentiation. Immunohistochemical analysis showed that Brn2 is expressed primarily in the upper granular layer. Consistent with its epidermal localization, Brn2 expression was highly induced at 14 days after calcium treatment of cultured normal human epidermal keratinocytes. When Brn2 was overexpressed by adenoviral transduction, Brn2 led to increased expression of the differentiation-related genes involucrin, filaggrin, and loricrin in addition to inhibition of their proliferation. Chromatin immunoprecipitation demonstrated that Brn2 bound to the promoter regions of these differentiation-related genes. We injected the purified Brn2 adenovirus into rat skin, which led to a thickened epidermis with increased amounts of differentiation related markers. The histopathologic features of adenovirus-Brn2 injected skin tissues looked similar to the features of lichen planus, a human skin disease showing chronic inflammation and well-differentiated epidermal changes. Moreover, Brn2 is shown to be expressed in almost all cell nuclei of the thickened epidermis of lichen planus, and Brn2 also attracts T lymphocytes. Our results demonstrate that Brn2 is probably a transcriptional factor playing an important role in keratinocyte differentiation and probably also in the pathogenesis of lichen planus lesions.

POU6F1 is the transcription factor that might be involved in cell proliferation of clear cell adenocarcinoma of the ovary.

Clear cell adenocarcinoma of the ovary often shows resistance to anticancer agents. We investigated new molecules to use when developing molecular-targeting therapy for clear cell adenocarcinoma of the ovary. RMG-I cells without invasive potential and RMG-V cells with invasive potential (derived from clear cell adenocarcinoma of the ovary) were subjected to complementary deoxyribonucleic acid microarray analysis. Caveolin-1, a molecule involved in cellular motility and invasion, showed differing expression between the two cell lines. An RNA interference experiment using the published siRNA for caveolin-1 was carried out. The results showed suppression of RMG-V cell infiltration by siRNA, but proliferation of the cancer cells was also suppressed. In other words, RMG-V cell infiltration may have been suppressed simply because cell proliferation was suppressed by RNA interference. These findings suggested that POU6F1 might be a transcription factor involved in the proliferation of ovarian cancer cells. Clear cell adenocarcinoma of the ovary shows little response to standard therapy. The results of the present study suggest that the transcription factor POU6F1 could be a new molecular target for treatment of this cancer.

SOX9 and SOX10 but not BRN2 are required for nestin expression in human melanoma cells.

Nestin is an intermediate filament protein and a marker of neuroectodermal stem cells indicating multipotentiality and regenerative capability. In melanoma tissues, nestin re-expression was correlated with tumor progression. Activation of the nestin neural enhancer was shown to be dependent on the binding of class III POU transcription factors, with brain-2 (BRN2) suggested to play a key role. We found both nestin and BRN2 mRNA in almost all of 13 analyzed melanoma cell lines of different progression stages, but expression levels did not correlate. Nestin protein was detected in 11 of 13 and BRN2 protein in 7 of 13 melanoma cell lines independent of progression stage. Downregulation of BRN2 by small-interfering RNA did not alter nestin expression in melanoma cells. However, POU proteins, such as BRN2, commonly cooperate with transcription factors of the Sry-box (SOX) family by binding to a nearby DNA site necessary for their action. SOX9 and SOX10 have been shown to be expressed in melanocyte precursors, with SOX10 downregulated upon differentiation. We now demonstrate SOX9 and SOX10 protein expression in melanoma tissues and cell lines. Downregulation of SOX9 and of SOX10 markedly decreased nestin levels in melanoma cells in a cooperative manner. Thus, SOX9 and SOX10 but not BRN2 seem to be required for nestin expression in human melanoma.

POU domain transcription factors: BRN2 as a regulator of melanocytic growth and tumourigenesis.

Several parallels between stem cell biology and tumour behaviour have been discovered in recent times. Such commonality is apparent in the unlimited capacity for cell division together with the lack of a differentiated phenotype in embryonic and adult stem cells, traits shared with tumour cells. Differentiation is a tightly regulated process that is mediated by the actions of multiple transcription factor families. The POU domain-containing family of transcription factors contains multiple mammalian members divided into six classes, which can be expressed broadly or in a cell-specific manner, and which are regulators of cell fate decisions of many different lineages. Target gene regulation can occur via a POU factor acting alone, or in combination with other POU proteins, ubiquitous co-activators or co-repressors, or other lineage restricted transcription factors. Aberrant levels of POU proteins have been found in several malignancies, including melanoma, connecting the otherwise developmentally restricted gene regulatory functions of POU transcription factors to the critical determinants of malignant transformation. Here, we focus on the role of the BRN2 (POU3F2/N-Oct-3) transcription factor in the melanocytic lineage where it may co-ordinate normal developmental cues that can be re-activated in melanoma. Recent studies have shown BRN2 to be responsive to MAPK pathway activation and to modulate the levels of MITF so as to suppress the differentiated melanocytic phenotype and to enhance tumour metastasis.

Brn-2 represses microphthalmia-associated transcription factor expression and marks a distinct subpopulation of microphthalmia-associated transcription factor-negative melanoma cells.

The origin of tumor heterogeneity is poorly understood, yet it represents a major barrier to effective therapy. In melanoma and in melanocyte development, the microphthalmia-associated transcription factor (Mitf) controls survival, differentiation, proliferation, and migration/metastasis. The Brn-2 (N-Oct-3, POU3F2) transcription factor also regulates melanoma proliferation and is up-regulated by BRAF and beta-catenin, two key melanoma-associated signaling molecules. Here, we show that Brn-2 also regulates invasiveness and directly represses Mitf expression. Remarkably, in melanoma biopsies, Mitf and Brn-2 each mark a distinct subpopulation of melanoma cells, providing a striking illustration of melanoma tumor heterogeneity with implications for melanoma therapy.

Cooperative function of Tbx1 and Brn4 in the periotic mesenchyme is necessary for cochlea formation.

The T-box transcription factor TBX1 has been identified as the major gene responsible for the etiology of velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS). Conductive hearing loss occurs in a majority of patients with this syndrome, while sensorineural deafness has also been reported in some cases. Mutations in POU3F4/BRN4, a POU domain transcription factor, cause DFN3, an X-linked nonsyndromic form of deafness characterized by mixed conductive and sensorineural hearing loss. Inactivation of the murine orthologues of these genes causes similar defects to those seen in humans and has provided excellent models for the study of inner ear development. Tbx1 and Brn4 are expressed in the mesenchymal cells surrounding the otic vesicle and have been shown to play roles in cochlear outgrowth. Furthermore, expression of Brn4 is reduced in Tbx1 null mutants, suggesting a possible genetic interaction between these genes. To test whether Tbx1 and Brn4 function in a common pathway, mice mutant for both genes were generated and analyzed for inner ear defects. Brn4-;Tbx1+/- mutants displayed a significant reduction in the number of turns of the cochlea compared to Brn4- or Tbx1+/- mice. In addition, Brn4-;Tbx1+/- mice displayed structural defects in the apical cochlea indicative of Mondini dysplasia found in patients with either VCFS/DGS or DFN3. These data establish a genetic interaction between Tbx1 and Brn4 relevant to human disease and indicate a function of these genes in signaling from the periotic mesenchyme to the otic vesicle to direct proper coiling of the cochlear duct.

Rb induces a proliferative arrest and curtails Brn-2 expression in retinoblastoma cells.

BACKGROUND: Retinoblastoma is caused by loss of the Rb protein in early retinal cells. Although numerous Rb functions have been identified, Rb effects that specifically relate to the suppression of retinoblastoma have not been defined. RESULTS: In this study, we examined the effects of restoring Rb to Y79 retinoblastoma cells, using novel retroviral and lentiviral vectors that co-express green fluorescent protein (GFP). The lentiviral vector permitted transduction with sufficient efficiency to perform biochemical analyses. Wild type Rb (RbWT) and to a lesser extent the low penetrance mutant Rb661W induced a G0/G1 arrest associated with induction of p27KIP1 and repression of cyclin E1 and cyclin E2. Microarray analyses revealed that in addition to down-regulating E2F-responsive genes, Rb repressed expression of Brn-2 (POU3F2), which is implicated as an important transcriptional regulator in retinal progenitor cells and other neuroendocrine cell types. The repression of Brn-2 was a specific Rb effect, as ectopic p27 induced a G0/G1 block, but enhanced, rather than repressed, Brn-2 expression. CONCLUSION: In addition to Rb effects that occur in many cell types, Rb regulates a gene that selectively governs the behavior of late retinal progenitors and related cells.