IMP1 Expression Research Articles

Insulin-like Growth Factor II mRNA-Binding Protein 1 Regulates Pancreatic Cancer Cell Growth through the Surveillance of CDC25A mRNA.

A number of data indicate that the sources of different kinds of PDAC may be discovered at the transcription/transduction stage. RNA metabolism is manipulated at various steps by different RNA-binding proteins (RBPs), and the deregulation or irregular activity of RBPs is known to contribute to tumor promotion and progression. The insulin-like growth factor 2 mRNA-binding protein family (IMPs), and IMP1 in particular, has been linked with a poor prognosis in PDAC patients; however, little is known about its contribution in PDAC carcinogenesis. In this study, we investigated the function of IMP1 in PDAC. To evaluate IMP1 expression and correlation with PDAC prognosis, we utilized several public databases. Using a specific siRNA IMP1, we analyzed cell death and cell cycle progression in PDAC cell lines and 3D spheroids. The role of IMP1 was also evaluated in vivo in a Panc-1-derived tumor xenograft murine model. Public data suggest that PDAC patients with higher expression of IMP1 showed poor overall and progression-free survival. IMP1 silencing leads to reduced cell growth in PDAC cells and three-dimensional spheroids. Abrogation of IMP1 in PDAC cells showed lower levels of CDC25A, increased phosphorylation of the cyclin-dependent kinase (CDK)2, and accumulation of PDAC cells in the G1 phase. Immunoprecipitation experiments revealed that IMP1 binds CDC25A mRNA, thus controlling cell-cycle progression. Ultimately, we proved that suppression of IMP1 blocked in vivo growth of Panc-1 transferred into immunodeficient mice. Our results indicate that IMP1 drives the PDCA cell cycle and represents a novel strategy for overcoming PDCA cell proliferation.

Dystonia Linked to EIF4A2 Haploinsufficiency: A Disorder of Protein Translation Dysfunction.

Protein synthesis is a tightly controlled process, involving a host of translation-initiation factors and microRNA-associated repressors. Variants in the translational regulator EIF2AK2 were first linked to neurodevelopmental-delay phenotypes, followed by their implication in dystonia. Recently, de novo variants in EIF4A2, encoding eukaryotic translation initiation factor 4A isoform 2 (eIF4A2), have been described in pediatric cases with developmental delay and intellectual disability. We sought to characterize the role of EIF4A2 variants in dystonic conditions. We undertook an unbiased search for likely deleterious variants in mutation-constrained genes among 1100 families studied with dystonia. Independent cohorts were screened for EIF4A2 variants. Western blotting and immunocytochemical studies were performed in patient-derived fibroblasts. We report the discovery of a novel heterozygous EIF4A2 frameshift deletion (c.896_897del) in seven patients from two unrelated families. The disease was characterized by adolescence- to adulthood-onset dystonia with tremor. In patient-derived fibroblasts, eIF4A2 production amounted to only 50% of the normal quantity. Reduction of eIF4A2 was associated with abnormally increased levels of IMP1, a target of Ccr4-Not, the complex that interacts with eIF4A2 to mediate microRNA-dependent translational repression. By complementing the analyses with fibroblasts bearing EIF4A2 biallelic mutations, we established a correlation between IMP1 expression alterations and eIF4A2 functional dosage. Moreover, eIF4A2 and Ccr4-Not displayed significantly diminished colocalization in dystonia patient cells. Review of international databases identified EIF4A2 deletion variants (c.470_472del, c.1144_1145del) in another two dystonia-affected pedigrees. Our findings demonstrate that EIF4A2 haploinsufficiency underlies a previously unrecognized dominant dystonia-tremor syndrome. The data imply that translational deregulation is more broadly linked to both early neurodevelopmental phenotypes and later-onset dystonic conditions. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

PO-01-145 DYNAMIC UTR COMPOSITION IMPACTS RNA BINDING PROTEIN COMPLEXING TO CONTROL TRANSLATION AND LIMIT CARDIAC GAP JUNCTION FORMATION DURING STRESS

Translational control has emerged as a critical yet dynamic process, representing one of the most rapid means by which the cardiomyocyte can alter its proteome. Several key cardiac mRNAs, including connexin43 (Cx43, gene name GJA1) are understood to undergo alternative translation initiation with resulting products (ie, GJA1-20k) impacting cardiac function. We previously found alterations to GJA1 mRNA untranslated region (UTR) length occur in aged and stressed heart tissue, suppressing internal translation of GJA1-20k, and limiting Cx43 gap junction formation. Having therefore identified dynamic mRNA cis element-based regulation of Cx43/GJA1-20k translation, we next asked what trans-acting factors are at play to alter GJA1 mRNA translation during stress. The IMP family of RNA binding proteins (RBPs) regulate alternative and canonical translation initiation of several genes, which led us to investigate how they may impact GJA1 mRNA. Using GJA1 as a model RNA, we test the hypothesis that alternative transcription start site usage controls inclusion of cis-acting elements in the mRNA 5’UTR affecting RBP binding and regulation of translation. Pathological hypertrophy was induced by isoproterenol deliverey to adult mice via implanted minipumps. Mouse models are complemented with matured human IPSC-derived cardiomyocytes, primary mouse ventricular cardiomyocytes, exposed to chronic hypoxia or TGF-β in vitro. Single molecule fluorescence in situ hybridization (smFISH) coupled with super-resolution microscopy localize GJA1 mRNA and RBPs. Hypertrophic hearts show reduced GJA1-20k and IMP1 expression. Using smFISH to label GJA1 mRNA, we find IMP1 complexes with GJA1 mRNA predominantly in comparison to IMP2 and 3, and that this complexing is reduced during stress, concomitant with loss of GJA1-20k internal translation. Biochemical assays confirm GJA1 mRNA/IMP1 binding by RNA-IP and synthetic reduction of IMP1 expression by RNAi or CRISPR/Cas9 alters GJA1 mRNA localization, reduces internal translation of GJA1-20k, and limits gap junction formation. We find comparable results during hypoxia in primary mouse cardiomyocytes and human induced pluripotent stem cell derived cardiomyocytes. Dynamic GJA1-5’UTR composition therefore represents a powerful mechanism connecting alterations in transcription to gene-specific translational regulation by RBPs such as IMP1, disruption of intercellular communication during stress, and arrhythmogenesis.

Feedforward loop between IMP1 and YAP/TAZ promotes tumorigenesis and malignant progression in glioblastoma.

YAP/TAZ have been identified as master regulators in malignant phenotypes of glioblastoma (GBM); however, YAP/TAZ transcriptional disruptor in GBM treatment remains ineffective. Whether post-transcriptional dysregulation of YAP/TAZ improves GBM outcome is currently unknown. Here, we report that insulin-like growth factor 2 (IGF2) mRNA-binding protein 1 (IGF2BP1 or IMP1) is upregulated in mesenchymal GBM compared with proneural GBM and correlates with worse patient outcome. Overexpression of IMP1 in proneural glioma stem-like cells (GSCs) promotes while IMP1 knockdown in mesenchymal GSCs attenuates tumorigenesis and mesenchymal signatures. IMP1 binds to and stabilizes m6A-YAP mRNA, leading to activation of YAP/TAZ signaling, depending on its m6A recognition and binding domain. On the other hand, TAZ functions as enhancer for IMP1 expression. Collectively, our data reveal a feedforward loop between IMP1 and YAP/TAZ maintaining GBM/GSC tumorigenesis and malignant progression and a promising molecular target in GBM.

IMP1 Enhances Extracellular Vesicle Secretion in a Transformation‐Dependent Manner

Background RNA binding proteins regulate protein expression through coordinated binding to a series of related transcripts referred to as an RNA operon. Previous work demonstrates context and tissue specific roles for the RNA binding protein insulin-like growth factor 2 mRNA binding protein 1 (IMP1) in the context of cancer, especially colorectal cancer (CRC). Recent studies have linked IMP1 to extracellular vesicles (EV) and demonstrated that Imp1 is a potential regulator of autophagy during intestinal repair in response to damage. The endosome, EV, and autophagy pathways are interrelated; however, a direct role for IMP1 in coordinating these pathways in the GI tract has not been explored and is the objective of this study. Hypothesis We hypothesized that IMP1 is a mechanistic link between the endosome and autophagy pathways in CRC. Methods Ribosomal profiling and RNA sequencing compared transcript and translational efficiency profiles between IMP1 null and IMP1-overexpressing CRC cell lines. EVs were assessed by nanoparticle tracking analysis, western blot, and electron microscopy in CRC cells and non-transformed, mouse intestinal enteroids. Effects of IMP1 on endosome, EV, and autophagy pathways were assessed by electron microscopy, immunofluorescence, and western blot. Results: EV, endocytic, and exosome-related pathways were the most significant differentially regulated pathways by gene ontogeny analysis of RNA sequencing data in IMP1 null vs IMP1-overpressing CRC cells (p= 1.78x10-10, 2.43x10-10). Over 42% of the changed transcripts were associated with the vesicle pathway and differentially regulated by IMP1 expression. We found that IMP1 increased EV secretion in HT-29 (4483±403 vs 1934±414, p=0.006) and SW480 (3604±399 vs 2293±464, p=0.049) CRC cells vs null controls. IMP1 modulated translational efficiency or protein levels of early endosome proteins, including EEA1, but did not alter LC3II or p62 levels. When autophagic flux was inhibited with BafilomycinA1, the ability of IMP1 to enhance EV release in CRC cells was magnified. By contrast, Imp1 overexpression in non-transformed enteroids did not enhance EV secretion. Conclusions: Our novel findings suggest that the effects of IMP1 expression on EV biology differ between non-transformed and CRC cells. In CRC cells, IMP1 plays an important role in EV secretion and enhances early endosomal pathway protein translation and expression. Our findings have implications for the development of novel early detection and therapeutic approaches in CRC where IMP1 is overexpressed.

Posttranscriptional regulation of colonic epithelial repair by RNA binding protein IMP1/IGF2BP1.

RNA binding proteins, including IMP1/IGF2BP1, are essential regulators of intestinal development and cancer. Imp1 hypomorphic mice exhibit gastrointestinal growth defects, yet the specific role for IMP1 in colon epithelial repair is unclear. Our prior work revealed that intestinal epithelial cell-specific Imp1 deletion (Imp1ΔIEC ) was associated with better regeneration in mice after irradiation. Here, we report increased IMP1 expression in patients with Crohn's disease and ulcerative colitis. We demonstrate that Imp1ΔIEC mice exhibit enhanced recovery following dextran sodium sulfate (DSS)-mediated colonic injury. Imp1ΔIEC mice exhibit Paneth cell granule changes, increased autophagy flux, and upregulation of Atg5. In silico and biochemical analyses revealed direct binding of IMP1 to MAP1LC3B, ATG3, and ATG5 transcripts. Genetic deletion of essential autophagy gene Atg7 in Imp1ΔIEC mice revealed increased sensitivity of double-mutant mice to colonic injury compared to control or Atg7 single mutant mice, suggesting a compensatory relationship between Imp1 and the autophagy pathway. The present study defines a novel interplay between IMP1 and autophagy, where IMP1 may be transiently induced during damage to modulate colonic epithelial cell responses to damage.

IMP1 3' UTR shortening enhances metastatic burden in colorectal cancer.

The RNA-binding protein insulin-like growth factor 2 mRNA binding protein 1 (IMP1) is overexpressed in colorectal cancer (CRC); however, evidence for a direct role for IMP1 in CRC metastasis is lacking. IMP1 is regulated by let-7 microRNA, which binds in the 3' untranslated region (UTR) of the transcript. The availability of binding sites is in part controlled by alternative polyadenylation, which determines 3' UTR length. Expression of the short 3' UTR transcript (lacking all microRNA sites) results in higher protein levels and is correlated with increased proliferation. We used in vitro and in vivo model systems to test the hypothesis that the short 3' UTR isoform of IMP1 promotes CRC metastasis. Herein we demonstrate that 3' UTR shortening increases IMP1 protein expression and that this in turn enhances the metastatic burden to the liver, whereas expression of the long isoform (full length 3' UTR) does not. Increased tumor burden results from elevated tumor surface area driven by cell proliferation and cell survival mechanisms. These processes are independent of classical apoptosis pathways. Moreover, we demonstrate the shifts toward the short isoform are associated with metastasis in patient populations where IMP1-long expression predominates. Overall, our work demonstrates that different IMP1 expression levels result in different functional outcomes in CRC metastasis and that targeting IMP1 may reduce tumor progression in some patients.

IMP1 regulates UCA1-mediated cell invasion through facilitating UCA1 decay and decreasing the sponge effect of UCA1 for miR-122-5p

BackgroundLong noncoding RNAs (LncRNAs) represent a class of widespread and diverse endogenous RNAs that can posttranscriptionally regulate gene expression through the interaction with RNA-binding proteins and micro RNAs (miRNAs). Here, we report that in breast carcinoma cells, the insulin-like growth factor 2 messenger RNA binding protein (IMP1) binds to lncRNA urethral carcinoma-associated 1 (UCA1) and suppresses the UCA1-induced invasive phenotype.MethodsRT-qPCR and RNA sequence assays were used to investigate the expression of UCA1 and miRNAs in breast cancer cells in response to IMP1 expression. The role of IMP1-UCA1 interaction in cell invasion was demonstrated by transwell analysis through loss-of-function and gain-of-function effects. RNA pull-down and RNA binding protein immunoprecipitation (RIP) were performed to confirm the molecular interactions of IMP1-UCA1 and UCA1-miR-122-5p involved in breast cancer cells.ResultsIn breast cancer cells, IMP1 interacts with UCA1 via the “ACACCC” motifs within UCA1 and destabilizes UCA1 through the recruitment of CCR4-NOT1 deadenylase complex. Meanwhile, binding of IMP1 prevents the association of miR-122-5p with UCA1, thereby shifting the availability of miR-122-5p from UCA1 to the target mRNAs and reducing the UCA1-mediated cell invasion. Accordingly, either IMP1 silencing or UCA1 overexpression resulted in reduced levels of free miR-122-5p within the cytoplasm, affecting miR-122-5p in regulating its target mRNAs.ConclusionsOur study provides initial evidence that interaction between IMP1 and UCA1 enhances UCA1 decay and competes for miR-122-5p binding, leading to the liberation of miR-122-5p activity and the reduction of cell invasiveness.

Abstract 1136: Cooperative functional roles of RNA binding proteins LIN28B and IMP1 in the pathogenesis of colorectal cancer

Abstract Introduction: RNA binding proteins and miRNAs have emerged as crucial regulators of intestinal homeostasis by controlling the stability and translation of target mRNAs. LIN28B, an mRNA binding protein, plays a critical role in regulating growth and proliferation in the intestinal epithelium. Previous work in our lab revealed that LIN28B promotes growth and tumorigenesis of the intestinal epithelium via suppression of mature let-7 miRNAs.LIN28B suppression of let-7 promotes upregulation of let-7 targets, including IMP1 (Insulin-like growth factor II mRNA-binding protein 1). Indeed, previous studies from our lab have shown that transgenic mice expressing LIN28B from the mouse Vil1 promoter (Vil-Lin28b mice) have an increase in IMP1 protein levels that is increased further with conditional knockout of let-7. Mechanistically, Let-7 isoforms have been known to physically and functionally interact with IMP1; however, the specific role of IMP1 in Lin28b-let 7-mediated tumorigenesis remains unknown. The current study tested the hypothesis that IMP1 maybe required for LIN28B-mediated tumorigenesis and that LIN28B and IMP1 may cooperatively promote a tumor-initiating phenotype. Methods: We evaluated LIN28B and IMP1 expression and localization in colorectal cancer patient samples using tissue microarrays and clinical outcomes. We used intestinal epithelial cell lines with LIN28B overexpression and CRISPR-Cas9 mediated knockout of IMP1 to study the functional consequences on migration, invasion and proliferation. Results: LIN28B expression correlates with expression of IMP1 in colorectal cancer patient samples. Individually, LIN28B and IMP1 expression intensity each was associated with worse prognosis in stage II colon cancer. Knock-down of IMP1 in Lin28B overexpressing cells decreased migration of colorectal cancer cell lines, suggesting a relationship of IMP1 for the tumorigenic effects of LIN28B. Furthermore, our RNA target analyses show that Lin28b and IMP1 both bind to target mRNAs in the WNT and adherens junction pathways. Conclusions: These data implicate a novel role for the Lin28b-let7-IMP1 axis in colorectal cancer and support an emerging paradigm for a critical and cooperative role of RNA-binding proteins in intestinal homeostasis and cancer. We are using the cell lines generated for orthotopic xenograft studies to see the effects of IMP1 loss and Lin28b overexpression on tumor growth and dissemination. Furthermore, we are generating mice with intestinal epithelium specific Lin28b overexpression-IMP1 loss to study the effects in vivo. Citation Format: Priya Chatterji, Kathryn Hamilton, Sarah Andres, Rei Mizuno, Philip Hicks, Arjun Jeganathan, Monte M. Winslow, Antoni Castells, Miriam Cuatrecasas, Blair Madison, Anil Rustgi. Cooperative functional roles of RNA binding proteins LIN28B and IMP1 in the pathogenesis of colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1136.

IMP1 suppresses breast tumor growth and metastasis through the regulation of its target mRNAs.

We have previously reported the ability of IMP1 in inhibiting proliferation and invasiveness of breast carcinoma cells in vitro. In the current study, we utilized a mouse xenograft model to further investigate the function of IMP1 in breast tumor progression and its underlying mechanism. We demonstrated that IMP1 expression significantly suppressed the growth of MDA231 cell-derived xenograft tumors and subsequent lung metastasis. Microarray analyses and differential gene expression identified handful mRNAs, many of which were involved in breast tumor-growth and metastasis. Further studies revealed that these mRNAs were directly interacted with the KH34 domain of IMP1 and this interaction post-transcriptionally regulated their corresponding protein expression. Either deletion of the KH34 domain of IMP1 or alteration of the expression of IMP1-bound mRNAs affected cell proliferation and tumor growth, producing the same phenotypes as IMP1 knockdown. Correlation of increased IMP1 expression with the reduced levels of its bound mRNAs, such as PTGS2, GDF15 and IGF-2 transcripts, was also observed in human breast tumors. Our studies provide insights into a molecular mechanism that the positive function of IMP1 to inhibit breast tumor growth and metastasis could be through the regulation of its target mRNAs.

MRNA‐binding protein IMP1 is a novel regulator of autophagy following intestinal irradiation injury

BackgroundPost‐transcriptional regulation of intestinal homeostasis via mRNA‐binding proteins, including Igf2 mRNA‐binding protein 1 (IMP1), is an area of emerging interest. Imp1‐null mice exhibit severe intestinal growth defects, yet Imp1 deletion in intestinal epithelium (Imp1ΔIEC) reveals minimal phenotype. Imp1 expression is significantly increased in intestinal stem/progenitor cells post‐irradiation, and new data indicate that Imp1ΔIEC mice exhibit increased baseline autophagy. We hypothesized that Imp1 is essential for intestinal response to irradiation, possibly via modulation of autophagy.MethodsWe treated Imp1ΔIEC and control mice with 12Gy irradiation and evaluated small intestine 96 hours later. Epithelia were evaluated for changes in autophagy via western blot and FACS. Enteroids plated from crypts of Imp1ΔIEC and control mice evaluated stem cell growth.ResultsAutophagy is up‐regulated in control mice following irradiation; however, Imp1ΔIEC mice exhibit repressed autophagy. In control mice, irradiation increases the proportion of spheres (non‐differentiated) to budding enteroids (differentiated), reflecting stem cell expansion. Intriguingly, irradiated Imp1ΔIEC mice show a further increase in sphere‐like enteroids, indicating that Imp1 may function to modulate stemness/differentiation following injury.ConclusionsOur data suggest that Imp1 may regulate autophagy in response to irradiation in the intestine, representing a novel paradigm by which autophagy may influence stemness in intestinal homeostasis.

RNA Protein Granules Modulate tau Isoform Expression and Induce Neuronal Sprouting

The neuronal microtubule-associated protein Tau is expressed in different variants, and changes in Tau isoform composition occur during development and disease. Here, we investigate a potential role of the multivalent tau mRNA-binding proteins G3BP1 and IMP1 in regulating neuronal tau expression. We demonstrate that G3BP1 and IMP1 expression induces the formation of structures, which qualify as neuronal ribonucleoprotein (RNP) granules and concentrate multivalent proteins and mRNA. We show that RNP granule formation leads to a >30-fold increase in the ratio of high molecular weight to low molecular weight tau mRNA and an ∼12-fold increase in high molecular weight to low molecular weight Tau protein. We report that RNP granule formation is associated with increased neurite formation and enhanced process growth. G3BP1 deletion constructs that do not induce granule formation are also deficient in inducing neuronal sprouting or changing the expression pattern of tau. The data indicate that granule formation driven by multivalent proteins modulates tau isoform expression and suggest a morphoregulatory function of RNP granules during health and disease.

High-Throughput Fluorescence Anisotropy Screen for Inhibitors of the Oncogenic mRNA Binding Protein, IMP-1

Cancer cell proliferation is regulated by oncogenes, such as c-Myc. An alternative approach to directly targeting individual oncogenes is to target IMP-1, an oncofetal protein that binds to and stabilizes messenger RNAs (mRNAs), leading to elevated expression of c-Myc and other oncogenes. Expression of IMP-1 is tightly correlated with a poor prognosis and reduced survival in ovarian, lung, and colon cancer. Small-molecule inhibitors of IMP-1 have not been reported. We established a fluorescence anisotropy/polarization microplate assay (FAMA) for analyzing binding of IMP-1 to a fluorescein-labeled 93 nucleotide c-Myc mRNA target (flMyc), developed the assay as a highly robust (Z′ factor = 0.60) FAMA-based high-throughput screen for inhibitors of binding of IMP-1 to flMyc, and carried out a successful pilot screen of 17,600 small molecules. Our studies support rapidly filtering out toxic nonspecific inhibitors using an early cell-based assay in control cells lacking the target protein. The physiologic importance of verified hits from the in vitro high-throughput screen was demonstrated by identification of the first small-molecule IMP-1 inhibitor, a lead compound that selectively inhibits proliferation of IMP-1–positive cancer cells with very little or no effect on proliferation of IMP-1–negative cells.

A network of heterochronic genes including Imp1 regulates temporal changes in stem cell properties

Stem cell properties change over time to match the changing growth and regeneration demands of tissues. We showed previously that adult forebrain stem cell function declines during aging because of increased expression of let-7 microRNAs, evolutionarily conserved heterochronic genes that reduce HMGA2 expression. Here we asked whether let-7 targets also regulate changes between fetal and adult stem cells. We found a second let-7 target, the RNA binding protein IMP1, that is expressed by fetal, but not adult, neural stem cells. IMP1 expression was promoted by Wnt signaling and Lin28a expression and opposed by let-7 microRNAs. Imp1-deficient neural stem cells were prematurely depleted in the dorsal telencephalon due to accelerated differentiation, impairing pallial expansion. IMP1 post-transcriptionally inhibited the expression of differentiation-associated genes while promoting the expression of self-renewal genes, including Hmga2. A network of heterochronic gene products including Lin28a, let-7, IMP1, and HMGA2 thus regulates temporal changes in stem cell properties. DOI: http://dx.doi.org/10.7554/eLife.00924.001.

Dynamics of survival of motor neuron (SMN) protein interaction with the mRNA‐binding protein IMP1 facilitates its trafficking into motor neuron axons

Spinal muscular atrophy (SMA) is a lethal neurodegenerative disease specifically affecting spinal motor neurons. SMA is caused by the homozygous deletion or mutation of the survival of motor neuron 1 (SMN1) gene. The SMN protein plays an essential role in the assembly of spliceosomal ribonucleoproteins. However, it is still unclear how low levels of the ubiquitously expressed SMN protein lead to the selective degeneration of motor neurons. An additional role for SMN in the regulation of the axonal transport of mRNA-binding proteins (mRBPs) and their target mRNAs has been proposed. Indeed, several mRBPs have been shown to interact with SMN, and the axonal levels of few mRNAs, such as the β-actin mRNA, are reduced in SMA motor neurons. In this study we have identified the β-actin mRNA-binding protein IMP1/ZBP1 as a novel SMN-interacting protein. Using a combination of biochemical assays and quantitative imaging techniques in primary motor neurons, we show that IMP1 associates with SMN in individual granules that are actively transported in motor neuron axons. Furthermore, we demonstrate that IMP1 axonal localization depends on SMN levels, and that SMN deficiency in SMA motor neurons leads to a dramatic reduction of IMP1 protein levels. In contrast, no difference in IMP1 protein levels was detected in whole brain lysates from SMA mice, further suggesting neuron specific roles of SMN in IMP1 expression and localization. Taken together, our data support a role for SMN in the regulation of mRNA localization and axonal transport through its interaction with mRBPs such as IMP1.

IMP1 promotes tumor growth, dissemination and a tumor-initiating cell phenotype in colorectal cancer cell xenografts

Igf2 mRNA binding protein 1 (IMP1, CRD-BP, ZBP-1) is a messenger RNA binding protein that we have shown previously to regulate colorectal cancer (CRC) cell growth in vitro. Furthermore, increased IMP1 expression correlates with enhanced metastasis and poor prognosis in CRC patients. In the current study, we sought to elucidate IMP1-mediated functions in CRC pathogenesis in vivo. Using CRC cell xenografts, we demonstrate that IMP1 overexpression promotes xenograft tumor growth and dissemination into the blood. Furthermore, intestine-specific knockdown of Imp1 dramatically reduces tumor number in the Apc (Min/+) mouse model of intestinal tumorigenesis. In addition, IMP1 knockdown xenografts exhibit a reduced number of tumor cells entering the circulation, suggesting that IMP1 may directly modulate this early metastatic event. We further demonstrate that IMP1 overexpression decreases E-cadherin expression, promotes survival of single tumor cell-derived colonospheres and promotes enrichment and maintenance of a population of CD24+CD44+ cells, signifying that IMP1 overexpressing cells display evidence of loss of epithelial identity and enhancement of a tumor-initiating cell phenotype. Taken together, these findings implicate IMP1 as a modulator of tumor growth and provide evidence for a novel role of IMP1 in early events in CRC metastasis.

Barley (Hordeum vulgare L.) inositol monophosphatase: gene structure and enzyme characteristics

The cellular myo-inositol (Ins) pool is important to many metabolic and signaling pathways in plants. Ins monophosphatase (IMPase; EC 3.1.3.25) activity is essential for the de novo synthesis of myo-Inositol (Ins), and for recycling of Ins in Ins(1,4,5)P3. However, proteins encoded by at least one family of IMP genes also have L-galactose-1-P phosphatase activity important to ascorbic acid synthesis, indicating a bifunctionality that links these two branches of carbon metabolism. As part of research into the regulation of Ins synthesis and supply during seed development, the barley IMP-1 gene and gene products were studied. The 1.4 kb barley IMP-1 promoter contains one low temperature response element (RE), two heat shock REs, one gibberellin and two auxin REs, and five sugar REs. Barley IMP-1 is expressed in all tissues assayed, and expression levels were not greatly altered by abiotic stress treatments. Reduced use of Ins for Ins P6 synthesis in developing seed of barley low phytic acid (lpa) mutants results in Ins accumulation, and IMP-1 expression is reduced in proportion to the increase in Ins level. The barley recombinant enzyme had a lower Km, indicating higher affinity, for D/L-Ins(3)P1 (Km = 9.7 microM) as compared with reported Km (Ins P1) values for other eukaryotic IMPases (43-330 microM) or with a reported Km (L-Gal-1P) of 150 microM for a kiwifruit (Actinidia deliciosa) enzyme. These and other data indicate that the barley IMP-1 gene is regulated at least in part in response to Ins metabolic needs, and that the enzyme it encodes displays catalytic properties well suited for a role in Ins synthesis, in addition to other roles as an L-gal-1-P phosphatase important to ascorbate synthesis, or as an IMPase important to Ins(1,4,5)P3 signal recycling.

Identification of Let-7–Regulated Oncofetal Genes

MicroRNAs (miRNA) are small RNA molecules of approximately 20 to 22 nucleotides that reduce expression of proteins through mRNA degradation and/or translational silencing. Each known miRNA has a large number of predicted targets. Members of the let-7/miR-98 family of miRNAs are up-regulated at the end of embryonic development. Let-7 is often down-regulated early during cancer development, suggesting that let-7-regulated oncofetal genes (LOG) may become reexpressed in cancer cells. Using comparative bioinformatics, we have identified 12 conserved LOGs that include HMGA2 and IMP-1/CRD-BP. IMP-1 has growth-promoting activities through stabilization of c-myc mRNA. We experimentally confirmed that IMP-1 is a direct let-7 target that promotes cell growth and motility of tumor cells, and we confirmed by proteomics analysis that IMP-1 and HMGA2 are major miRNA targets. Our data suggest that a substantial part of the growth inhibitory activities of let-7 comes from suppressing the expression of IMP-1. LOGs could be novel therapeutic targets and potential biomarkers for cancer treatment.

Expression of the RNA-binding protein IMP1 correlates with poor prognosis in ovarian carcinoma

The IMP (IGFII mRNA-binding protein) family comprises a group of three RNA-binding proteins involved in the regulation of cytoplasmic mRNA-fate. Recent studies identified IMP proteins as oncofetal factors in various neoplasias, but knowledge of a potential role in ovarian carcinomas is still lacking. The immunohistochemical analysis of 107 ovarian carcinomas, 30 serous borderline tumors of the ovary and five normal ovaries revealed de novo synthesis of IMP1 in 69% of ovarian carcinomas. Elevated IMP1 expression was observed preferentially in high-grade and high-stage cases and was a significant prognostic indicator for reduced recurrence-free and overall survival. Phenotypic studies in ovarian carcinoma-derived ES-2 cells demonstrated that IMP1 knockdown affects proliferation and cell survival. Reduced proliferation was associated with decreased c-myc mRNA half-life, suggesting IMP1 as an oncogenic factor that is involved in promoting elevated proliferation by stabilizing the c-myc mRNA in ovarian carcinoma cells.

Increased Expression of Insulin-like Growth Factor-II Messenger RNA–Binding Protein 1 Is Associated with Tumor Progression in Patients with Lung Cancer

To identify novel biomarkers and therapeutic targets for lung cancers, we screened for genes that were highly transactivated in a large proportion of non-small cell lung cancers (NSCLC) using a cDNA microarray representing 27,648 genes. A gene encoding insulin-like growth factor-II mRNA-binding protein 1 (IMP-1) was selected as a candidate (> or =3-fold expression than in normal lung tissue in about 70% of NSCLCs). Tumor tissue microarray was applied to examine expression of IMP-1 protein in archival lung cancer samples from 267 patients and investigated its clinicopathologic significance. A role of IMP-1 in cancer cell growth and/or survival was examined by small interfering RNA experiments. Cellular invasive activity of IMP-1 on mammalian cells was examined using Matrigel assays. mRNAs associated with IMP-1 in cancer cells were also isolated by RNA immunoprecipitation followed by cDNA microarray analysis. Positive immunostaining of IMP-1 was correlated with male (P = 0.0001), tumor size (P = 0.0003), non-adenocarcinoma histology (P < 0.0001), smoking history (P = 0.0005), non-well-differentiated tumor grade (P = 0.0001), and poor prognosis (P = 0.0053). Suppression of IMP-1 expression with small interfering RNA effectively suppressed growth of NSCLC cells. In addition, we identified that exogenous expression of IMP-1 increased the migratory activity of mammalian cells. IMP-1 was able to bind to mRNAs encoding a variety of proteins involved in signal transduction, cell cycle progression, cell adhesion and cytoskeleton, and various types of enzymatic activities. These results suggest that IMP-1 expression is likely to play important roles in lung cancer development and progression, and that IMP-1 is a prognostic biomarker and a promising therapeutic target for lung cancer.