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Selective Antitumor Activity of Datelliptium toward Medullary Thyroid Carcinoma by Downregulating RET Transcriptional Activity.

ABSTRACT: Medullary thyroid carcinoma (MTC) is a rare aggressive form of thyroid cancer with high rates of metastasis. Sporadic and hereditary MTC are strongly driven by somatic and germline mutations, respectively, in the transmembrane REarranged during Transfection (RET) proto-oncogene, which encodes a receptor tyrosine kinase. Our previous study identified datelliptium as a novel RET transcription inhibitor, which stabilizes the RET G-quadruplex structures and suppresses RET oncogene transcription. The present study aimed to elucidate the effect of datelliptium on the suppression of epithelial-to-mesenchymal transition (EMT) and metastasis-related behaviors of MTC cells, including cell migration and formation of cancer stem cells (CSCs). Our results demonstrated that datelliptium downregulated the expression of the mesenchymal markers, including N-cadherin, vimentin, slug, snail, and claudin-1. Compared to untreated cells, datelliptium significantly decreased the migration of TT cells in a dose-dependent manner in a wound healing assay. Additionally, datelliptium significantly reduced the size of preformed spheroids from TT cells over the time course. Finally, datelliptium inhibited approximately 75% of MTC xenograft growth with minimal systemic toxicity. In conclusion, datelliptium exerts its antitumor activity against MTC cells by reducing the EMT program, migratory ability, and self-renewal capacity of TT cells, thus preventing invasive and metastatic behavior of MTC.

SUBMITTER: Alqahtani T

PROVIDER: S-EPMC8267783 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDifferent pathogenic germline mutations in the RET oncogene are identified in MEN 2, a hereditary syndrome characterized by medullary thyroid carcinoma (MTC) and other endocrine tumors. Although genetic predisposition is recognized, not all RET mutation carriers will develop the disease during their lifetime or, likewise, RET mutation carriers belonging to the same family may present clinical heterogeneity. It has been suggested that a single germline mutation might not be sufficient for development of MEN 2-associated tumors and a somatic bi-allelic alteration might be required. Here we investigated the presence of somatic second hit mutation in the RET gene in MTC.MethodsWe integrated Multiplex Ligation-dependent Probe Amplification (MLPA) and whole exome sequencing (WES) to search for copy number alteration (CNA) in the RET gene in MTC samples and medullary thyroid cell lines (TT and MZ-CR-1). We next found reads spanning exon-exon boundaries on RET, an indicative of retrocopy. We subsequently searched for RET retrocopies in the human reference genome (GRCh37) and in the 1000 Genomes Project data, by looking for reads reporting joined exons in the RET locus or distinct genomic regions. To determine RET retrocopy specificity and recurrence, DNA isolated from sporadic and MEN 2-associated MTC (n = 37), peripheral blood (n = 3) and papillary thyroid carcinomas with RET fusion (n = 10) samples were tested using PCR-sequencing methodology.ResultsThrough MLPA we have found evidence of CNA in the RET gene in MTC samples and MTC cell lines. WES analysis reinforced the presence of the CNA and hinted for a retroposed copy of RET not found in the human reference genome and 1.000 Genomes Project. Extended analysis confirmed the presence of a somatic MTC-related retrocopy of RET in both sporadic and hereditary tumors. We further unveiled a recurrent (28%) novel point mutation (p.G548 V) found exclusively in the retrocopy of RET. The mutation was also found in cDNA of mutated samples, suggesting it might be functional.ConclusionWe here report a somatic specific RET retroposed copy in MTC samples and cell lines. Our results support the idea that generation of retrocopies in somatic cells is likely to contribute to MTC genesis and progression.

Project description:BackgroundThe RET S836S variant has been associated with early onset and increased risk for metastatic disease in medullary thyroid carcinoma (MTC). However, the mechanism by which this variant modulates MTC pathogenesis is still open to discuss. Of interest, strong linkage disequilibrium (LD) between RET S836S and 3'UTR variants has been reported in Hirschsprung's disease patients.ObjectiveTo evaluate the frequency of the RET 3'UTR variants (rs76759170 and rs3026785) in MTC patients and to determine whether these variants are in LD with S836S polymorphism.MethodsOur sample comprised 152 patients with sporadic MTC. The RET S836S and 3'UTR (rs76759170 and rs3026785) variants were genotyped using Custom TaqMan Genotyping Assays. Haplotypes were inferred using the phase 2.1 program. RET mRNA structure was assessed by Vienna Package.ResultsThe mean age of MTC diagnosis was 48.5±15.5 years and 57.9% were women. The minor allele frequencies of RET polymorphisms were as follows: S836S, 5.6%; rs76759170, 5.6%; rs3026785, 6.2%. We observed a strong LD among S836S and 3'UTR variants (|D'| = -1, r2 = 1 and |D'| = -1, r2 = 0,967). Patients harboring the S836S/3'UTR variants presented a higher percentage of lymph node and distant metastasis (P = 0.013 and P<0.001, respectively). Accordingly, RNA folding analyses demonstrated different RNA secondary structure predictions for WT(TCCGT), S836S(TTCGT) or 3'UTR(GTCAC) haplotypes. The S836S/3'UTR haplotype presented a greater number of double helices sections and lower levels of minimal free energy when compared to the wild-type haplotype, suggesting that these variants provides the most thermodynamically stable mRNA structure, which may have functional consequences on the rate of mRNA degradation.ConclusionThe RET S836S polymorphism is in LD with 3'UTR variants. In silico analysis indicate that the 3'UTR variants may affect the secondary structure of RET mRNA, suggesting that these variants might play a role in posttranscriptional control of the RET transcripts.

Project description:Abstract The RET receptor tyrosine kinase proto-oncogene is activated by somatic or germline mutations in a majority of medullary thyroid cancers (MTC). However, treatment of MTC has been challenging due to the lack of effective and tolerable RET-specific therapy, thus testing tumors for the presence of somatic RET mutation has not been warranted. In a first-in-human, phase 1/2 clinical trial (LIBRETTO-001, NCT03157128), selpercatinib (LOXO-292), an investigational, highly selective, potent small molecule RET kinase inhibitor, demonstrated significant and durable anti-tumor activity in patients with advanced RET-mutant MTC or with diverse RET fusion-positive cancers (1). Among the primary analysis set of patients with RET-mutant MTC previously treated with cabozantinib and/or vandetanib (N=55), the investigator-assessed objective response rate (ORR) per RECIST 1.1 was 56% (95% CI 42.3-69.7, n=31/55). Duration of response was not reached with a 10.6-months median follow-up (data cutoff date 17-Jun-2019). Here, we evaluated investigator-assessed ORR per RECIST 1.1 and clinical benefit rate (CBR) in this previously treated patient population by RET alteration and by germline or somatic testing used for enrollment. The ORR remained consistent across subgroups with RET M918T (49%, 95% CI 30.8-66.5, n=16/33), V804M/L gatekeeper mutations (60%, 95% CI 14.7-94.7, n=3/5), extracellular cysteine mutations (43%, 95% CI 9.9-81.6, n=3/7), other mutations (90%, 95% CI 55.5-99.7, n=9/10), and germline (50%, 95% CI 6.8-93.2, n=2/4) or somatic (57%, 95% CI 42.2-70.7, n=29/51) testing. The CBR, defined as the proportion of patients with best overall response of confirmed complete response, confirmed or unconfirmed partial response, or stable disease lasting 16 weeks or more, in this patient set was 87% (95% CI 75.5-94.7, n=48/55). The CBR remained consistent across subgroups with RET M918T (88%, 95% CI 71.8-96.6, n=29/33), V804M/L gatekeeper mutations (80%, 95% CI 28.4-99.5, n=4/5), extracellular cysteine mutations (71%, 95% CI 29.0-96.3, n=5/7), other mutations (100%, 95% CI 69.2-100.0, n=10/10), and germline (75%, 95% CI 19.4-99.4, n=3/4) or somatic (88%, 95% CI 76.1-95.6, n=45/51) testing. The primary technologies used to identify RET alterations were tumor next-generation sequencing (n=43) and polymerase chain reaction (n=9). As previously reported, selpercatinib was well tolerated with an acceptable safety profile (1). These results indicate broad anti-tumor activity for selpercatinib in patients with RET-mutant MTC irrespective of the specific RET mutation, and support implementation of RET mutation testing for patients with advanced MTC, including somatic testing, to identify patients who may benefit from selpercatinib. Reference: (1) Wirth et al., Ann Oncol. 2019 Oct; 30(supplement 5): v933.

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Selective Antitumor Activity of Datelliptium toward Medullary Thyroid Carcinoma by Downregulating RET Transcriptional Activity.

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