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Aggressive form of neuroblastoma (NB) is a malignant childhood cancer derived from granule neuron precursors and sympatho-adrenal lineage with N-MYC amplification. An insulinoma associated-1 (INSM1) transcription factor has emerged as a NB biomarker that plays critical role in facilitating tumor cell growth and transformation. N-myc activates INSM1 in NB was discovered. In order to elucidate the signaling pathways associated with INSM1 expression and NB tumor cell growth, we developed an INSM1 promoter-driven luciferase assay for new drug discovery. Promoter-driven luciferase assay demonstrated high Z' factor (>0.7). Performance measures using signal-to-noise ratio, signal window, and Z' factor confirmed this assay is a robust screening method. A panel of FDA-approved oncology drugs set (147 compounds) was subjected to the INSM1 promoter-driven luciferase assay. The positive-hit compounds were validated for effects on cell viability and INSM1 expression. Screening a FDA-approved oncology drugs set revealed multiple inhibitors against various signaling pathways via suppression of INSM1 promoter activity. The positive-hit compounds consist of 9 signaling pathway inhibitors negatively regulates INSM1 expression and cell viability in NB. These inhibitors target DNA/RNA/protein synthesis, tubulin assembly, and histone deacetylase signaling pathways. The outcome of this assay indicates that the newly identified pathways could be critical for NB growth and transformation. This technology and the positive-hits of multiple pathways represent a promising option of identifying re-purposed FDA-approved drugs valuable for NB treatment in the context of a NB-specific marker, INSM1., (Copyright © 2020. Published by Elsevier Inc.)

Insulinoma-associated-1 (IA-1 or INSM1) encodes a zinc-finger transcription factor, which was isolated from a human insulinoma subtraction library, with specific expression patterns, predominantly in developing neuroendocrine tissues and tumors. INSM1 is key in early pancreatic endocrine, sympatho-adrenal lineage, and pan-neurogenic precursor development. Insm1 gene ablation results in impairment of pancreatic β cells, catecholamine biosynthesis, and basal progenitor development during mammalian neocortex maturation. Recently, INSM1 has emerged as a superior, sensitive, and specific biomarker for neuroendocrine tumors. INSM1 regulates downstream target genes and exhibits extranuclear activities associated with multiple signaling pathways, including Sonic Hedgehog, PI3K/AKT, MEK/ERK 1/2 , ADK, p53, Wnt, histone acetylation, LSD1, cyclin D1, Ascl1, and N-myc. Novel strategies targeting INSM1-associated signaling pathways facilitate the suppression of neuroendocrine tumor growth. In addition, INSM1 promoter-driven reporter assay and/or suicide gene therapy are promising effective therapeutic approaches for targeted specific neuroendocrine tumor therapy. In this review, the current knowledge of the biological role of INSM1 as a neuroendocrine tumor biomarker is summarized, and novel strategies targeting multiple signaling pathways in the context of INSM1 expression in neuroendocrine tumors are further explored. IMPLICATIONS: Neuroendocrine transcription factor (INSM1) may serve as a neuroendocrine biomarker for the development of novel cancer therapeutics against neuroendocrine tumors., (©2019 American Association for Cancer Research.)

Insulinoma-associated-1 (INSM1) is a key protein functioning as a transcriptional repressor in neuroendocrine differentiation and is activated by N-Myc in human neuroblastoma (NB). INSM1 modulates the phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-glycogen synthase kinase 3β (GSK3β) signaling pathway through a positive-feedback loop, resulting in N-Myc stabilization. Accordingly, INSM1 has emerged as a critical player closely associated with N-Myc in facilitating NB cell growth. Here, an INSM1 promoter-driven luciferase-based screen revealed that the compound 5'-iodotubercidin suppresses adenosine kinase (ADK), an energy pathway enzyme, and also INSM1 expression and NB tumor growth. Next, we sought to dissect how the ADK pathway contributes to NB tumor cell growth in the context of INSM1 expression. We also found that 5'-iodotubercidin inhibits INSM1 expression and induces an intra- and extracellular adenosine imbalance. The adenosine imbalance, which triggers adenosine receptor-3 signaling that decreases cAMP levels and AKT phosphorylation and enhances GSK3β activity. We further observed that GSK3β then phosphorylates β-catenin and promotes the cytoplasmic proteasomal degradation pathway. 5'-Iodotubercidin treatment and INSM1 inhibition suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) activity and the AKT signaling pathways required for NB cell proliferation. The 5'-iodotubercidin treatment also suppressed β-catenin, lymphoid enhancer-binding factor 1 (LEF-1), cyclin D1, N-Myc, and INSM1 levels, ultimately leading to apoptosis via caspase-3 and p53 activation. The identification of the signaling pathways that control the proliferation of aggressive NB reported here suggests new options for combination treatments of NB patients., (© 2019 Chen et al.)

Neuroendocrine (NE) lung tumors account for 20% of total lung cancer cases and represent a subset of aggressive tumors with metastatic potential. High-risk NE lung cancer patients display disseminated disease, N-myc expression/amplification, and poorly differentiated tumors. In this study, we investigate the molecular mechanisms underlying a zinc-finger transcription factor, INSM1 in NE lung cancer. Our study revealed that INSM1 crosstalk with the Shh-PI3K/AKT-N-myc/Ascl1-MEK/ERK 1/2 transcriptional network in NE lung cancer. The INSM1 expression pattern and functional data demonstrated that INSM1 is not only critical for NE differentiation, but also served as a NE tumor-specific marker in small cell lung carcinoma (SCLC). The Shh signaling pathway activates INSM1 expression through N-myc and Ascl1 in aggressive SCLC. The E2-box in the INSM1 promoter is the direct target recognized by N-myc and Ascl1 transcription factors. N-myc or Ascl1 activates endogenous INSM1 expression in lung cancer cells. INSM1 functions as a key player in NE lung cancer via Shh signaling that crosstalk with PI3K/AKT and MEK/ERK 1/2 pathway to enhance N-myc stability in NE lung cancer. We investigate the negative effects of Shh inhibitor and knockdown of INSM1 in NE lung cancer cells. The combination of different Shh signaling pathway inhibitors targeting INSM1 and N-myc inhibits lung cancer cell growth and could be used as a new treatment option for SCLC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Insulinomas are the most common type of neuroendocrine (NE) pancreatic islet tumors. Patients with insulinomas may develop complications associated with hyperinsulinemia. To increase the treatment options for insulinoma patients, we have tested a conditionally replicating adenovirus that has been engineered in such a way that it can specifically express therapeutic genes in NE tumors., Methods: We used a promoter-specific adenoviral vector delivery system that is regulated by an INSM1 (insulinoma-associated-1) promoter, which is silent in normal adult tissues but active in developing NE cells and tumors. Through a series of modifications, using an insulator (HS4) and neuron-restrictive silencer elements (NRSEs), an oncolytic adenoviral vector was generated that retains tumor specificity and drives the expression of a mutated adenovirus E1A gene (Δ24E1A) and the herpes simplex virus thymidine kinase (HSV-tk) gene. The efficacy of this vector was tested in insulinoma-derived MIN, RIN, βTC-1 and pancreatic (Panc-1) cells using in vitro cell survival and in vivo tumor growth assays., Results: Using in vitro insulinoma-derived cell lines and an in vivo subcutaneous mouse tumor model we found that the INSM1 promoter-driven viruses were able to replicate specifically in INSM1-positive cells. INSM1-specific HSV-tk expression in combination with ganciclovir treatment resulted in dose-dependent tumor cell killing, leaving INSM1-negative cells unharmed. When we combined the INSM1-promoter driven HSV-tk with Δ24E1A and INSM1p-HSV-tk (K5) viruses, we found that the co-infected insulinoma-derived cells expressed higher levels of HSV-tk and exhibited more efficient tumor suppression than cells infected with INSM1p-HSV-tk virus alone., Conclusions: INSM1 promoter-driven conditionally replicating adenoviruses may serve as a new tool for the treatment of insulinoma and may provide clinicians with additional options to combat this disease.

Accurate detection of neuroendocrine (NE) tumors is critically important for better prognosis and treatment outcomes in patients. To demonstrate the efficacy of using an adenoviral vector for the detection of NE tumors, we have constructed a pair of adenoviral vectors which, in combination, can conditionally replicate and release Gaussia luciferase into the circulation after infecting the NE tumors. The expression of these two vectors is regulated upstream by an INSM1-promoter (insulinoma-associated-1) that is specifically active in NE tumors and developing NE tissues, but silenced in normal adult tissues. In order to retain the tumor-specificity of the INSM1 promoter, we have modified the promoter using the core insulator sequence from the chicken β-globin HS4 insulator and the neuronal restrictive silencing element (NRSE). This modified INSM1-promoter can retain NE tumor specificity in an adenoviral construct while driving a mutated adenovirus E1A gene (∆24E1A), the Metridia, or Gaussia luciferase gene. The in vitro cell line and mouse xenograft human tumor studies revealed the NE specificity of the INSM1-promoter in NE lung cancer, neuroblastoma, medulloblastoma, retinoblastoma, and insulinoma. When we combined the INSM1-promoter driven Gaussia luciferase with ∆24E1A, the co-infected NE tumor secreted higher levels of Gaussia luciferase as compared to the INSM1p-Gaussia virus alone. In a mouse subcutaneous xenograft tumor model, the combination viruses secreted detectable level of Gaussia luciferase after infecting an INSM1-positive NE lung tumor for ≥12 days. Therefore, the INSM1-promoter specific conditional replicating adenovirus represents a sensitive diagnostic tool to aid clinicians in the detection of NE tumors.

Insulinoma associated-1 (IA-1/INSM1) gene is exclusively expressed during early embryonic development, but has been found to be re-expressed at high levels in neuroendocrine tumors including neuroblastoma. Using over-expression and knockdown experiments in neuroblastoma cells, we showed that INSM1 is critical for cell proliferation, BME-coated invasion, and soft agar colony formation. Here, we identified INSM1 as a novel target gene activated by N-myc in N-myc amplified neuroblastoma cells. The Sonic hedgehog signaling pathway induced INSM1 by increasing N-myc expression. INSM1 activated PI3K/AKT/GSK3β pathways to suppress N-myc phosphorylation (Thr-58) and inhibited degradation of N-myc. Inversely, N-myc protein bound to the E2-box region of the INSM1 promoter and activated INSM1 expression. The invasion assay and the xenograft nude mouse tumor model revealed that the INSM1 factor facilitated growth and oncogenesis of neuroblastoma. The current data supports our hypothesis that a positive-feedback loop of sonic hedgehog signaling induced INSM1 through N-myc and INSM1 enhanced N-myc stability contributing to the transformation of human neuroblastoma.

In this study, the functional role of INSM1 is examined with an AR42J acinar cell model for trans-differentiation into insulin-positive cells. Islet transcription factors (ITFs: INSM1, Pdx-1, and NeuroD1) are over-expressed in AR42J cells using adenoviral vectors. Addition of Ad-INSM1 alone or the combination of three ITFs to the AR42J cells triggers cellular trans-differentiation. Ectopic expression of INSM1 directly induces insulin, Pax6, and Nkx6.1 expression, whereas Pdx-1 and NeuroD1 were slightly suppressed by INSM1. Addition of Pdx-1 and NeuroD1 with INSM1 further enhances endocrine trans-differentiation by increasing both the numbers and intensity of the insulin-positive cells with simultaneous activation of ITFs, Ngn3 and MafA. INSM1 expression alone partially inhibits dexamethasone-induced exocrine amylase expression. The combination of the three ITFs completely inhibits amylase expression and concomitantly induces greater acinar cell trans-differentiation into endocrine cells. Also, addition of the three ITFs promotes EGF and TGFβ receptors expression. Stimulation by the three ITFs along with the EGF/TGFβ growth factors strongly promotes insulin gene expression. The combination of the three ITFs and EGF/TGFβ growth factors with the primary cultured pancreatic acini also facilitates exocrine to endocrine cell differentiation. Taken together, both the AR42J cell line and the primary cultured mouse acinar cells support INSM1 induced acini trans-differentiation model., (Copyright © 2011 Wiley Periodicals, Inc.)

CD1d-expressing cells present lipid Ag to CD1d-restricted NKT cells, which play an important role in immune regulation and tumor rejection. Lymphoid enhancer-binding factor-1 (LEF-1) is one of the regulators of the Wnt signaling pathway, which is a powerful regulator in cellular growth, differentiation, and transformation. There is little evidence connecting Wnt signaling to CD1d expression. In this study, we have identified LEF-1 as a regulator of the expression of the gene encoding the human CD1d molecule (CD1D). We found that LEF-1 binds specifically to the CD1D promoter. Overexpression of LEF-1 in K562 or Jurkat cells suppresses CD1D promoter activity and downregulates endogenous CD1D transcripts, whereas knockdown of LEF-1 using LEF-1-specific small interfering RNA increases CD1D transcripts in K562 and Jurkat cells but there are different levels of surface CD1d on these two cell types. Chromatin immunoprecipitation showed that the endogenous LEF-1 is situated at the CD1D promoter and interacts with histone deacetylase-1 to facilitate the transcriptional repressor activity. Knockdown of LEF-1 using small interfering RNA potentiates an acetylation state of histone H3/H4, supporting the notion that LEF-1 acts as a transcriptional repressor for the CD1D gene. Our finding links LEF-1 to CD1D and suggests a role of Wnt signaling in the regulation of the human CD1D gene.

Insulinoma-associated antigen-1 (INSM1/IA-1) is a unique zinc-finger transcription factor restrictedly expressed in pancreatic beta-cells during early pancreas development. INSM1 is transiently activated by the islet-specific endocrine factor neurogenin 3, and it subsequently regulates downstream target genes NeuroD1 and insulin during beta-cell maturation. Here, we examined how the INSM1 transcription factor contributes to endocrine cell differentiation using a defined serum-free medium-primed pancreatic duct cell model. We showed that ectopic expression of INSM1 can promote Panc-1 cell trans-differentiation. INSM1 up-regulates two islet transcription factors (ITFs), paired box 6 and homeodomain transcription factor 6.1, whereas other ITFs, including pancreatic duodenal homeobox-1 (Pdx-1), homeodomain transcription factor 2.2, NeuroD1, paired box 4, and neurogenin 3, were either down-regulated or absent. The result suggests that INSM1 is capable of regulating multiple ITFs and the insulin gene either directly or indirectly. When we overexpressed three ITFs, INSM1/Pdx-1/NeuroD1, in the Panc-1 differentiation model, higher insulin expression was observed in parallel with the activation of an additional ITF, neurogenin 3, signifying endocrine cell activation. Insulin expression from the three ITFs stimulation was readily detected by immunostaining and increased 40% as compared with the insulin-transferrin-selenium-LacZ control. Furthermore, we examined the differential chromatin acetylation patterns within the insulin promoter region using the chromatin immunoprecipitation assay. INSM1 alone can selectively enhance acetylation of histone H4, whereas NeuroD1 and Pdx-1 favor the acetylation of histone H3. Both H3 and H4 histone acetylations facilitate insulin gene expression. The consistent functional effect of INSM1, either with or without other ITFs, promotes pancreatic duct cell differentiation as well as induces Panc-1 cell cycle arrest.

The INSM1 gene encodes a developmentally regulated zinc finger transcription factor. INSM1 expression is normally absent in adult tissues, but is reactivated in neuroendocrine tumor cells. In the present study, we analyzed the therapeutic potential of an adenoviral INSM1 promoter-driven herpes simplex virus thymidine kinase (HSV-tk) construct in primitive neuroectodermal tumors (PNETs). We constructed an adenoviral INSM1 promoter-driven HSV-tk gene for therapy in PNETs. The PNET-specific adeno-INSM1 promoter HSV-tk construct was tested both in vitro and in vivo in a nude mouse tumor model. Northern blot analysis and transient transfection of an INSM1 promoter-driven luciferase reporter gene indicated that the INSM1 promoter was active in neuroblastoma (IMR-32), retinoblastoma (Y79), and medulloblastoma (D283 Med) cells, but not in glioblastoma (U-87 MG) cells. After Ad-INSM1p-HSV-tk infection, the levels of HSV-tk protein expression were consistent with INSM1 promoter activities. Furthermore, in vitro multiplicity of infection and ganciclovir (GCV) sensitivity studies indicated that the INSM1 promoter could mediate specific expression of the HSV-tk gene and selective killing of INSM1-positive PNETs. In vivo intratumoral adenoviral delivery demonstrated that the INSM1 promoter could direct HSV-tk gene expression in a nude mouse tumor model and effectively repressed tumor growth in response to GCV treatment. Taken together, our data show that the INSM1 promoter is specific and effective for targeted cancer gene therapy in PNETs.

Zinc-finger transcription factors are DNA-binding proteins that are implicated in many diverse biological functions. INSM1 (formerly IA-1) contains five zinc-finger motifs and functions as a transcription factor. INSM1 protein structure is highly conserved in homologues of different species. It is predominantly expressed in developing neuroendocrine tissues and the nervous system in mammals. INSM1 represents an important player in early embryonic neurogenesis. In pancreatic endocrine cell differentiation, Ngn3 first activates INSM1 and subsequently NeuroD/beta2. Conversely, INSM1 exerts a feedback mechanism to suppress NeuroD/beta2 and its own gene expression. INSM1 gene ablation in the mouse results in the impairment of pancreatic endocrine cell maturation. Further, deletion of INSM1 severely impairs catecholamine biosynthesis and secretion from the adrenal gland that results in early embryonic lethality. Genetically, INSM1 acts as a downstream factor of Mash 1 and Phox2b in the differentiation of the sympatho-adrenal lineage. In the developing neocortex, mouse embryos lacking INSM1 expression contain half the number of basal progenitors and show a reduction in cortical plate radial thickness. Cell signaling studies reveal that INSM1 contributes to the induction of cell cycle arrest/exit necessary to facilitate cellular differentiation. INSM1 is highly expressed in tumors of neuroendocrine origin. Hence, its promoter could serve as a tumor-specific promoter that drives a specific targeted cancer gene therapy for the treatment of neuroendocrine tumors. Taken together, all of these features of INSM1 strongly support its role as an important regulator during neuroendocrine differentiation.

INSM1 is a zinc finger transcription factor that plays an important role in pancreatic beta-cell development. To further evaluate its role in cell fate determination, we investigated INSM1 effects on cell cycle function. The cyclin box of cyclin D1 is essential for INSM1 binding. Competitive pull-down and co-immunoprecipitation revealed that INSM1 binding to cyclin D1 interrupts its association with CDK4 and induces hypophosphorylation of the retinoblastoma protein. An inducible Tet-on system was established in Cos-7 and Panc-1 cells. Using serum starvation, we synchronized the cell cycle and subsequently induced cell cycle progression by serum stimulation. Comparison of the INSM1 induction group with the noninduced control group, INSM1 ectopic expression causes cell cycle arrest, whereas the INSM1-mediated cell cycle arrest could be reversed by cyclin D1 and CDK4 overexpression. The proline-rich N-terminal portion of INSM1 is required for cyclin D1 binding. Mutation of proline residues abolished cyclin D1 binding and also diminished its ability to induce cell cycle arrest. Cellular proliferation of Panc-1 cells was inhibited by INSM1 overexpression demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, soft agar colony formation, as well as tumor growth in a nude mouse model. Taken together, we provide evidence to support that INSM1 binds to cyclin D1, interrupts cell cycle signaling, and inhibits cellular proliferation.

In this study, an insulinoma-associated antigen-1 (INSM1)-binding site in the proximal promoter sequence of the insulin gene was identified. The co-transfection of INSM1 with rat insulin I/II promoter-driven reporter genes exhibited a 40-50% inhibitory effect on the reporter activity. Mutational experiments were performed by introducing a substitution, GG to AT, into the INSM1 core binding site of the rat insulin I/II promoters. The mutated insulin promoter exhibited a three- to 20-fold increase in the promoter activity over the wild-type promoter in several insulinoma cell lines. Moreover, INSM1 overexpression exhibited no inhibitory effect on the mutated insulin promoter. Chromatin immunoprecipitation assays using beta TC-1, mouse fetal pancreas, and Ad-INSM1-transduced human islets demonstrated that INSM1 occupies the endogenous insulin promoter sequence containing the INSM1-binding site in vivo. The binding of the INSM1 to the insulin promoter could suppress approximately 50% of insulin message in human islets. The mechanism for transcriptional repression of the insulin gene by INSM1 is mediated through the recruitment of cyclin D1 and histone deacetylase-3 to the insulin promoter. Anti-INSM1 or anti-cyclin D1 morpholino treatment of fetal mouse pancreas enhances the insulin promoter activity. These data strongly support the view that INSM1 is a new zinc-finger transcription factor that modulates insulin gene transcription during early pancreas development.

INSM1 is a downstream target gene of neurogenin 3 (ngn3). A promoter construct containing the -426/+40bp region transiently co-transfected into NIH-3T3 cells with a ngn3 expression plasmid resulted in a 12-fold increase in promoter activity. The ngn3/E47 heterodimer selectively binds and activates the E-box3 of the INSM1 promoter. The endogenous ngn3 and CREB-binding protein (CBP) co-activator occupy the INSM1 promoter, resulting in hyper-acetylation of histone H3/H4 chromatin in a human neuroblastoma cell line, IMR-32. Additionally, adenoviral ngn3 can induce endogenous INSM-1 expression in pancreatic ductal carcinoma-1 cells through the recruitment of CBP to the INSM1 promoter and increase the acetylation of the INSM1 promoter region.

INSM1/IA-1 (insulinoma-associated 1) is a developmentally regulated zinc-finger transcription factor, exclusively expressed in the foetal pancreas and nervous system, and in tumours of neuroendocrine origin. We have identified an INSM1 binding site in the neuroD/beta2 promoter and demonstrated transcriptional repressor activity of INSM1 by transient transfection assay. A chromatin immunoprecipitation assay confirmed that in vivo INSM1 is situated on the promoter region of the neuroD/beta2 gene. In an attempt to elucidate the molecular mechanism of transcriptional repression by the INSM1 gene, cyclin D1 was identified as an interacting protein by using a 45-day-old human foetal brain cDNA library and a yeast two-hybrid screen. The physical association between INSM1 and cyclin D1 was confirmed by in vitro and in vivo pull-down assay. Cyclin D1 co-operates with INSM1 and suppresses neuroD/beta2 promoter activity. Co-immunoprecipitation of INSM1, cyclin D1 and HDACs (histone deacetylases) in mammalian cells revealed that INSM1 interacts with HDAC-1 and -3 and that this interaction is mediated through cyclin D1. Overexpression of cyclin D1 and HDAC-3 significantly enhanced the transcriptional repression activity of INSM1 on the neuroD/beta2 promoter. A further chromatin immunoprecipitation assay confirmed that HDAC-3 occupies this same region of the neuroD/beta2 promoter, by forming a transcription complex with INSM1. Thus we conclude that INSM1 recruits cyclin D1 and HDACs, which confer transcriptional repressor activity.

IA-1 is a novel zinc finger transcription factor with a restricted tissue distribution in the embryonic nervous system and tumors of neuroendocrine origin. The 1.7-kilobase 5'-upstream DNA sequence of the human IA-1 gene directed transgene expression predominantly in the developing nervous system including forebrain, midbrain, hindbrain, spinal cord, retina, olfactory bulb, and cerebellum, which recapitulated the expression patterns of neuroendocrine tissues and childhood brain tumors. The IA-1 promoter deletion reporter gene constructs revealed that the sequence between -426 and -65 bp containing three putative E-boxes (approximately 361 bp) upstream of the transcription start site was sufficient to confer tissue-specific transcriptional activity. Further mutation analysis revealed that the proximal E-box (E3) closest to the start site is critical to confer transcriptional activity. Electrophoretic mobility shift assay and transient transfection studies demonstrated that the NeuroD1 and E47 heterodimer are the key transcription factors that regulate the proximal E-box of the IA-1 promoter. Therefore, we concluded that the IA-1 gene is developmentally expressed in the nervous system and the NeuroD1/E47 transcription factors up-regulate IA-1 gene expression through the proximal E-box element of the IA-1 promoter.

Introduction: IA-1, an insulinoma-associated cDNA-1, encodes a zinc-finger DNA-binding protein originally isolated from a human insulinoma subtraction library., Aim: To demonstrate the restriction of IA-1 gene expression in human fetal pancreata of different gestational stages and to determine whether the expression of IA-1 gene is associated with rat AR42J cell differentiation into insulin-positive cells., Methodology: To examine whether the IA-1 gene is associated with pancreatic endocrine cell differentiation, we used a rat pancreatic amphicrine cell line, AR42J, to investigate whether the expression of the IA-1 gene coincides with AR42J cells converting into either endocrine or exocrine lineage. We also examined a set of islet transcription factors that regulate key differentiation steps involved in activating the genes that confer the specialized functions of terminally differentiated pancreatic islet cells., Results: When the AR42J cells were converted into insulin-positive cells induced by GLP-1, insulinoma conditioned-medium, or both, we observed a significant elevated expression of mRNA for IA-1 and islet-specific transcription factors such as Pdx-1, NeuroD/beta2, and Nkx6.1. In contrast, dramatically decreased expression of mRNA for IA-1 and islet-specific transcription factors was displayed when AR42J cells were converted into the acinar-like phenotype by dexamethasone., Conclusions: IA-1 gene was shown to be developmentally regulated in fetal pancreatic cells, and its expression pattern is consistent with parallel changes in islet-specific transcription factors during the endocrine differentiation of AR42J cells.

A novel cDNA, insulinoma-associated antigen-1 (IA-1), containing five zinc-finger DNA-binding motifs, was isolated from a human insulinoma subtraction library. IA-1 expression is restricted to fetal but not adult pancreatic and brain tissues as well as tumors of neuroendocrine origin. Using various GAL4 DNA binding domain (DBD)/IA-1 fusion protein constructs, we demonstrated that IA-1 functions as a transcriptional repressor and that the region between amino acids 168 and 263 contains the majority of the repressor activity. Using a selected and amplified random oligonucleotide binding assay and bacterially expressed GST-IA-1DBD fusion protein (257-510 a.a.), we identified the consensus IA-1 binding sequence, TG/TC/TC/TT/AGGGGG/TCG/A. Further experiments showed that zinc-fingers 2 and 3 of IA-1 are sufficient to demonstrate transcriptional activity using an IA-1 consensus site containing a reporter construct. A database search with the consensus IA-1 binding sequence revealed target sites in a number of pancreas- and brain-specific genes consistent with its restricted expression pattern. The most significant matches were for the 5'-flanking regions of IA-1 and NeuroD/beta2 genes. Co-transfection of cells with either the full-length IA-1 or hEgr-1AD/IA-1DBD construct and IA-1 or NeuroD/beta2 promoter/CAT construct modulated CAT activity. These findings suggest that the IA-1 protein may be auto-regulated and play a role in pancreas and neuronal development, specifically in the regulation of the NeuroD/beta2 gene.