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The FGFR1 gene transcript is alternatively processed to produce functionally different receptor forms. Previously, we identified a 69-nucleotide exonic splicing enhancer (ESE) required for α-exon inclusion in JEG3 cells. In the present study, we found that this sequence is composed of three independent elements, two smaller ESE sequences flanking an exonic splicing silencer sequence. Ultraviolet cross-linking and immunoprecipitation identified ESE-specific binding of the splicing regulator SRp55. A RNA interference-mediated decrease in SRp55 confirmed the significance of this interaction. There was a 6- to 14-fold decrease in exon inclusion on ablation of SRp55. In SNB19 glioblastoma cells, which normally skip this exon, SRp55 was also demonstrated to play a role in exon inclusion after the removal of intronic splicing silencer sequences. These observations indicate that SRp55 plays a major role in maintaining normal FGFR1 α-exon inclusion, which is subject to dominant intronic splicing silencer-mediated and exonic splicing silencer-mediated inhibition in SNB19 cells.

Medullary thyroid carcinoma (MTC) is derived from the calcitonin-producing C-cells of the thyroid gland. Oncogenic mutations of the RET proto-oncogene are found in all hereditary MTC and ∼45% of the sporadic cases. However, it is clear that additional genetic lesions contribute to the development of both RET-dependent and RET independent MTC. We analyzed 30 primary medullary thyroid cancer (MTC) tumors by comparative genomic hybridization (CGH) arrays (Agilent 244K) and a subset of those tumors by ultra-high-resolution SNP arrays (Affymetrix SNP6.0). A consistent finding with both arrays was a loss localized to 22q13.1 in 40% of tumors. This region contains the ATF4 gene and loss of this gene was validated by real time PCR. ATF4 is a central mediator of integrated stress response pathway and activates expression of a cohort of downstream target genes known to be involved in cell survival, apoptosis, autophagy and senescence. We hypothesize that the loss of ATF4 expression contributes to MTC progression through mechanisms involving aberrant proliferation and escape from apoptosis. We present several findings to support this. First, ATF4 protein levels were found downregulated in 50% MTC tumor tissues (n = 43) and low ATF4 expression was associated with poor overall survival in MTC patients (HR = 5.015, 95% CI: 1.38-10.64), P = 0.013). A negative correlation was observed between RET and ATF4 protein expression in MTC tumors (r = -0.89, R square = 0.799, p Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Elizabeth G. Grubbs, Michelle D. Williams, Steven I. Sherman, Robert F. Gagel. ATF4 is a putative tumor suppressor gene in medullary thyroid 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 3660.

Exclusion of the alpha-exon by alternative RNA splicing of the fibroblast growth factor receptor 1 (FGFR1) primary transcript leads to the production of FGFR1beta. Glial cell transformation is associated with a progressive increase in FGFR1beta expression that coincides with a dramatic increase in the expression of the splicing factor polypyrimidine tract-binding protein (PTB). Cell-specific overexpression of PTB increased alpha-exon skipping, and a reduction in PTB increased alpha-exon inclusion. Targeted disruption of PTB interaction with FGFR1 precursor RNA in vivo by an antisense oligonucleotide also increased alpha-exon inclusion. These results suggest that PTB plays a direct role in alpha-exon splicing.

RET proto-oncogene, a receptor tyrosine kinase (RTK), activates a complex network of signal transduction pathways that when unregulated contributes to cell transformation. Germ line missense mutations, which give rise to constitutively active oncogenic RET, were found to cause multiple endocrine neoplasia type 2 (MEN2), a dominant inherited cancer syndrome that affects multiple neuroendocrine organs. Recent findings also suggest that impairment of RTK down-regulation or deactivation may be involved in tumorigenesis. The molecular mechanism that functions to attenuate RET signaling remain elusive. ATF4 is a central mediator of integrated stress response pathway. Activation of ATF4 results in tumor cell death under stress conditions thereby suggesting that ATF4 agonists constitute a potential therapeutic strategy for inhibiting tumor growth. Here, we report that ATF4 overexpression in medullary thyroid cancer cells harboring RET C634W mutation decreases RET protein expression but not the mRNA levels. In addition, ATF4 accelerates RET turnover and promotes its ubiquitination. Moreover, a RET kinase dead mutant is more stable than wild type or M918T RET suggesting that kinase activity is required for ATF4-mediated degradation. Finally, ATF4 depletion upregulates RET protein levels and RET autophosphorylation. Our findings unravel ATF4 as a feedback negative attenuator of RET signaling that could be used as a potential therapeutic target for medullary thyroid cancer or other malignancy with RET abnormalities. Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Robert F. Gagel. Activating transcription factor 4 regulates RET autophosphorylation and ubiquitin-dependent degradation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5262. doi:10.1158/1538-7445.AM2014-5262

Neuroendocrine tumors are rare forms of cancers originating from hormone-producing cells that are scattered throughout the body. Medullary thyroid carcinoma (MTC) develops from calcitonin-secreting, C cells, in the thyroid gland. Surgical removal of the thyroid is the best treatment currently available for early stage cancer patients but advanced forms of MTC are often incurable and fatal. Thus, better therapies are needed. A lack of relevant animal models for this disease has slowed the development of new treatments. We have recently reported a novel conditional mouse model for MTC. In these mice, overexpression of the cyclin-dependent kinase 5 (Cdk 5), activator, p25 in C cells induces MTC tumors. Furthermore, interruption of p25 overexpression results in growth arrest of these tumors. Here we take advantage of our conditional mouse model to investigate the molecular pathways underlying MTC tumorigenesis. We used Illumina Mouse WG-6 v2.0 Expression BeadChip to compare the gene expression profile of proliferating versus arrested mouse MTC tumors. We found that a total of 920 genes encoding transcription factors, cell cycle proteins, motor proteins and angiogenesis factors were differentially regulated in growing versus arrested tumors. Particularly the genes encoding cell cycle proteins that are involved in the regulation of Rb pathway were significantly up-regulated in the proliferating tumors. Selected genes with higher degrees of change were evaluated by quantitative PCR and immunoblotting analyses. Taken together these findings suggests that the Rb signaling pathways is involved in MTC tumorigenesis and that Cdk5 may act as a critical regulator of the Rb signaling pathway in this cancer. Thus, targeting Cdk5 and/or the Rb pathway may be a valid strategy to stop the progression of neuroendocrine thyroid cancer. Citation Format: Karine Pozo, Antje Hillmann, Alexander Augustyn, Tanvir Singh, Florian Plattner, John Minna, Herbert Chen, Gilbert J. Cote, James A. Bibb, Fiemu E. Nwariaku. Molecular characterization of an animal model for neuroendocrine thyroid cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3923. doi:10.1158/1538-7445.AM2014-3923