HER2-PI9 and HER2-I12: two novel and functionally active splice variants of the oncogene HER2 in breast cancer.
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ABSTRACT:
SUBMITTER: Hart V
PROVIDER: S-EPMC8397700 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background and purposeIn humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthermore uncovered and characterized constitutive activity of both isoforms.Experimental approachExpression levels of the hGPR17 isoforms were determined in several brain regions as well as heart and kidney using quantitative RT-PCR. A CREB reporter assay and [(35)S]-GTPgammaS binding were employed to assess the constitutive activity and the activation by UDP, UDP-glucose and -galactose and the cysteinyl leukotrienes LTC(4) and LTD(4). Leukotriene binding and induction of internalization were furthermore tested using homologous competition binding and antibody-feeding experiments respectively.Key resultsThe short isoform (hGPR17-S) was expressed more abundantly (eight- to 23-fold) in the brain than the long isoform (hGPR17-L), whereas the opposite was observed in heart and kidney. As previously reported, the uracil nucleotides activated hGPR17-S with micromolar potencies. However, much lower potencies were observed for hGPR17-L with a 50- to 170-fold increase in EC(50). Furthermore, contrary to previous reports, neither of the isoforms was activated or bound by the cysteinyl leukotrienes. Finally, both receptors were demonstrated to be constitutively active through Galpha(i).Conclusions and implicationsWe present the first isoform-specific characterization of GPR17 and show that differences exist between the isoforms, in both expression pattern and pharmacological profile. In turn, our results indicate that the two human isoforms might serve tissue-specific functions.
| S-EPMC2839267 | biostudies-literature
Project description:The human epidermal growth factor receptor 2 (HER2)/receptor tyrosine-protein kinasebB-2 (ERBB2) is overexpressed in 20-30% of breast tumors leading to faster growing and more aggressive tumors. Alternative splicing generates a functionally distinct HER2 variant called Herstatin, which is produced by the inclusion of intron 8. Herstatin acts as a tumor suppressor by effectively blocking HER2 activity and cell proliferation, while promoting apoptosis. In the present study we investigated HER2 pre-mRNA regulatory sequences and splicing factors which regulate the alternative splicing of Herstatin. A Herstatin minigene, comprising exon 8/intron 8/exon 9 of HER2 was generated and subsequent in vitro splicing assays revealed that RNA secondary structure and somatic mutations did not impact on inclusion of intron 8. However, using RNase-assisted RNA chromatography, followed by mass spectrometry, we identified six RNA-binding proteins (splicing factors) that bind to RNA sequences surrounding exon 8/intron 8 and intron 8/exon 9 boundaries; these included hnRNP I, H1, D, A2/B1 and hnRNPA1 plus the SR protein SRSF1. Specifically, overexpression of hnRNP A1 significantly increased retention of intron 8 resulting in higher levels of Herstatin in SKBR3 breast cancer cells whereas SRSF1 only had a marginal effect in decreasing Herstatin but increased exogenous HER2 levels under these experimental conditions. In conclusion, we have identified the first splicing factors and regulatory sequences that are involved in the production of Herstatin.
| S-EPMC5449083 | biostudies-other
Project description:BackgroundPeroxisome proliferator-activated receptor gamma coactivator 1alpha (PPARGC1A) is a coactivator with a vital and central role in fat and energy metabolism. It is considered to be a candidate gene for meat quality in pigs and is involved in the development of obesity and diabetes in humans. How its many functions are regulated, is however still largely unclear. Therefore a transcription profile of PPARGC1A in 32 tissues and 4 embryonic developmental stages in the pig was constructed by screening its cDNA for possible splice variants with exon-spanning primers.FindingsThis led to the discovery of 2 new splice variants in the pig, which were subsequently also detected in human tissues. In these variants, exon 8 was either completely or partly (the last 66 bp were conserved) spliced out, potentially coding for a much shorter protein of respectively 337 and 359 amino acids (aa), of which the first 291 aa would be the same compared to the complete protein (796 aa).ConclusionConsidering the functional domains of the PPARGC1A protein, it is very likely these splice variants considerably affect the function of the protein and alternative splicing could be one of the mechanisms by which the diverse functions of PPARGC1A are regulated.
| S-EPMC2631024 | biostudies-literature
Project description:Cerebrotendinous Xanthomatosis (CTX), a rare lipid storage disorder, is caused by recessive loss-of-function mutations of the 27-sterol hydroxylase (CYP27A1), producing an alteration of the synthesis of bile acids, with an accumulation of cholestanol. Clinical characteristics include juvenile cataracts, diarrhea, tendon xanthomas, cognitive impairment and other neurological manifestations. Early diagnosis is critical, because treatment with chenodeoxycholic acid may prevent neurological damage. We studied the CYP27A1 gene in two Chilean CTX patients by sequencing its nine exons, exon-intron boundaries, and cDNA from peripheral blood mononuclear cells. Patient 1 is a compound heterozygote for the novel substitution c.256-1G > T that causes exon 2 skipping, leading to a premature stop codon in exon 3, and for the previously-known pathogenic mutation c.1183C > T (p.Arg395Cys). Patient 2 is homozygous for the novel mutation c.1185-1G > A that causes exon 7 skipping and the generation of a premature stop codon in exon 8, leading to the loss of the crucial adrenoxin binding domain of CYP27A1.
| S-EPMC4415556 | biostudies-literature
Project description:Hedgehog (HH) signaling is one of the key pathways with major significance for embryogenesis, tumorigenesis, and stem cell maintenance. Glioma-associated oncogene 1 (GLI1) is a transcription factor that acts as the terminal signaling effector but also represents a pathway target gene. Here we report the identification and functional properties of novel GLI1 splice variants generated by skipping exons 2 and 3 and encoding an N-terminal truncated GLI1 protein (GLI1DeltaN). Analysis of the GLI1DeltaN mRNAs in adult human tissues revealed comparable expression levels to the full-length GLI1 (GLI1FL), whereas in tumor cell lines a generally lower and more variable expression pattern was observed. Furthermore, GLI1DeltaN is up-regulated by HH signaling to the same extent as GLI1FL but has a weaker capacity to activate transcription. However, in specific cellular contexts GLI1DeltaN may be more potent than GLI1FL in activating endogenous gene expression. Moreover, the dual-specificity tyrosine phosphorylation-regulated kinase 1 (Dyrk1) potentiates the transcriptional activity of GLI1FL but not GLI1DeltaN. Interestingly, GLI1FL, in contrast to GLI1DeltaN, is localized solely at the nucleus, in line with its increased transcriptional capacity. The negative regulator of the pathway, Suppressor of Fused (SUFU), elicits a cytoplasmic retention of the GLI1 isoforms, which is more pronounced for GLI1FL, as this contains an N-terminal SUFU binding domain. Collectively, our findings reveal that the activation mechanism of the terminal transducer of the pathway, GLI1, is mediated not only by GLI1FL but also by the GLI1DeltaN variant.
| S-EPMC2386930 | biostudies-literature
Project description:The Nopp140 gene of Drosophila maps within 79A5 of chromosome 3. Alternative splicing yields two variants. DmNopp140 (654 residues) is the sequence homolog of vertebrate Nopp140. Its carboxy terminus is 64% identical to that of the prototypical rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains a glycine/arginine-rich domain that is often found in RNA-binding proteins such as vertebrate nucleolin. Both Drosophila variants localize to nucleoli in Drosophila Schneider II cells and Xenopus oocytes, specifically within the dense fibrillar components. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, whereas DmNopp140 partitions HeLa nucleoli into phase-light and phase-dark regions. The phase-light regions contain DmNopp140 and endogenous fibrillarin, whereas the phase-dark regions contain endogenous nucleolin. When coexpressed, both Drosophila variants colocalize to HeLa cell nucleoli. Both variants fail to localize to endogenous Cajal bodies in Xenopus oocyte nuclei and in HeLa cell nuclei. Endogenous HeLa coilin, however, accumulates around the periphery of phase-light regions in cells expressing DmNopp140. The carboxy truncation (DmNopp140DeltaRGG) also fails to localize to Cajal bodies, but it forms similar phase-light regions that peripherally accumulate endogenous coilin. Conversely, we see no unusual accumulation of coilin in cells expressing DmNopp140-RGG.
| S-EPMC65094 | biostudies-literature
Project description:Owing to the marked sexual dimorphism of hepatocellular carcinoma (HCC), sex hormone receptor signaling has been implicated in numerous aspects of liver cancer pathogenesis. We sought to reconcile the clear contribution of androgen receptor (AR) activity that has been established in preclinical models of HCC with the clinical failure of AR antagonists in patients with advanced HCC by evaluating potential resistance mechanisms to AR-targeted therapy. The AR locus was interrogated for resistance-causing genomic modifications using publicly available primary HCC datasets (1,019 samples). Analysis of HCC tumor and cell line RNA-seq data revealed enriched expression of constitutively active, treatment-refractory AR splice variants (AR-SV). HCC cell lines expressed C-terminal-truncated AR-SV; 28 primary HCC samples abundantly expressed AR-SV. Low molecular weight AR species were nuclear localized and constitutively active. Furthermore, AR/AR-SV signaling promoted AR-mediated HCC cell progression and conferred resistance to AR antagonists. Ligand-dependent and -independent AR signaling mediated HCC epithelial-to-mesenchymal transition by regulating the transcription factor SLUG. These data suggest that AR-SV expression in HCC drives HCC progression and resistance to traditional AR antagonists. Novel therapeutic approaches that successfully target AR-SVs may be therapeutically beneficial for HCC. SIGNIFICANCE: Treatment-refractory, constitutively active androgen receptor splice variants promote hepatocellular carcinoma progression by regulating the epithelial-to-mesenchymal transition pathway.
| S-EPMC7002251 | biostudies-literature
Project description:Oncogene amplification confers a growth advantage to tumor cells for clonal expansion. There are several, recurrently amplified oncogenes throughout the human genome. However, it remains unclear whether this recurrent amplification is solely a manifestation of increased fitness resulting from random amplification mechanisms, or if a genomic locus-specific amplification mechanism plays a role. Here we show that the ERBB2 oncogene at 17q12 is susceptible to palindromic gene amplification, a mechanism characterized by the inverted (palindromic) duplication of genomic segments, in HER2-positive breast tumors. We applied two genomic approaches to investigate amplification mechanisms: sequencing of DNA libraries enriched with tumor-derived palindromic DNA (Genome-wide Analysis of Palindrome Formation) and whole genome sequencing (WGS). We observed significant enrichment of palindromic DNA within amplified ERBB2 genomic segments. Palindromic DNA was particularly enriched at amplification peaks and at boundaries between amplified and normal copy-number regions. Thus, palindromic gene amplification shaped the amplified ERBB2 locus. The enrichment of palindromic DNA throughout the amplified segments leads us to propose that the ERBB2 locus is amplified through the mechanism that repeatedly generates palindromic DNA, such as Breakage-Fusion-Bridge cycles. The genomic architecture surrounding ERBB2 in the normal genome, such as segmental duplications, could promote the locus-specific mechanism.
| S-EPMC5314454 | biostudies-literature
Project description:Daxx is involved in transcriptional control and apoptosis. It comprises several domains, including a regulatory C terminus that is responsible for the interaction with numerous proteins such as p53, promyelocytic leukemia protein (PML), and Hsp27. Here, we describe the identification and characterization of two novel variants of Daxx termed Daxx-? and Daxx-?, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a consequence, Daxx-? and Daxx-? show a markedly decreased affinity to PML, which in turn is associated with a different subnuclear localization of these proteins compared with Daxx. Although Daxx is localized mainly in PML-oncogenic domains (PODs) Daxx-? and Daxx-? display a distinct distribution pattern. Furthermore, Daxx-? and Daxx-? show a decreased affinity to p53 also due to the truncated C terminus. We provide evidence that the p53 recruitment into PODs is Daxx isoform-dependent. The decreased affinity of Daxx-?/-? to p53 and PML results in a diffuse localization of p53 throughout the nucleus. In contrast to Daxx, Daxx-? and Daxx-? are unable to repress p53-mediated transcription. Therefore, alternative splicing of Daxx might indicate an additional level in the cellular apoptosis network.
| S-EPMC3103337 | biostudies-literature
Project description:Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.
| S-EPMC4556188 | biostudies-literature