Human Medullary Thyroid Carcinoma Research Articles

Abstract 4665: USP18 promotes anlotinib resistance in medullary thyroid carcinoma by stabilizing aurora B kinase

Abstract Targeted therapy resistance is the main cause of disease progression in advanced medullary thyroid carcinoma (MTC). However, the key molecules and mechanisms remain unclear. We previously performed a genome-wide in vitro screen of Anlotinib resistance-related genes in human MTC cell line TT using CRISPR-dCas9-SAM system and identified ubiquitin-specific protease 18 (USP18) as a key molecule mediating Anlotinib resistance in MTC. However, its downstream mechanism needs further clarification. Based on the high-throughput DNA sequencing results of the control group and the Anlotinib screening group, a group of key genes related to Anlotinib resistance in MTC was screened through the MAGeCK algorithm and the enrichment ratio of sgRNA in the Anlotinib screening group. Among them, USP18 ranked first in the enrichment results. Analysis of the correlation between the expression level of USP18 in MTC patient specimens and survival revealed that patients with high USP18 expression in tumor cells had poorer prognosis. Overexpression of USP18 in MTC tumor cells significantly promoted the growth of subcutaneous xenograft tumors in NOD/SCID mice treated with Anlotinib. In vitro cell experiments also showed that overexpression of USP18 promoted the proliferation of MTC tumor cells and significantly inhibited the proportion of cell apoptosis after Anlotinib treatment. Further research found that USP18, as a deubiquitinating enzyme that can specifically remove ISG15-like ubiquitin-like proteins from substrate proteins, can improve the kinase stability of Aurora B by removing ISG15-like ubiquitin modification and promote MTC tumor cell proliferation through the activation of PI3K-Akt signaling regulated by Aurora B kinase. Application of Aurora B kinase inhibitor in Anlotinib-resistant MTC cell lines significantly inhibited tumor cell proliferation, and Aurora B kinase inhibitor significantly increased the proportion of cell apoptosis in MTC tumor cells overexpressing USP18. In the subcutaneous xenograft tumor model of MTC in NOD/SCID mice, the growth of xenograft tumors in the Anlotinib combined with Aurora B kinase inhibitor treatment group was also significantly inhibited. In summary, overexpressed USP18 can activate PI3K-Akt signaling and promote tumor cell proliferation by increasing the stability of Aurora B kinase, ultimately leading to the formation of Anlotinib resistance in MTC patients. Combined use of Aurora B kinase inhibitor is a potentially effective combined treatment strategy for Anlotinib-resistant MTC patients. Citation Format: Xuan Qin, Xing Wan, Dapeng Li, Yimeng Liu, Xianhui Ruan, Fangkun Luan, Kejia Xu, Jia Li, Rong Xiang, Yongjun Piao, Yi Shi, Xiangqian Zheng. USP18 promotes anlotinib resistance in medullary thyroid carcinoma by stabilizing aurora B kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4665.

Preclinical Evaluation of Novel Tyrosine-Kinase Inhibitors in Medullary Thyroid Cancer.

Simple SummaryMedullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular calcitonin-secreting C cells of the thyroid. Most of the patients affected by MTC, especially the familial form, harbor a mutation of the RET proto-oncogene. In patients with advanced disease, medical therapy is represented by two tyrosine-kinase inhibitors: cabozantinib and vandetanib. However, their usage is limited by several adverse events and drug-resistance onset. The aim of this preclinical study was to evaluate the antitumor activity of novel molecules for the therapy of MTC: SU5402, an inhibitor of the fibroblast growth factor receptor type 1 (FGFR-1) and vascular endothelial growth factor receptor (VEGFR)-2; sulfatinib, a multi-target kinase inhibitor selective for FGFR-1 and the VEGFR-1, -2, and -3; SPP86, a RET-specific inhibitor. Our results suggest a potential role in targeting the FGFR and VEGFR signaling pathways as an alternative strategy for resistant tumors and a significative antitumor activity of this new RET-specific inhibitor.Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor arising from parafollicular C cells of the thyroid gland. In this preclinical study, we tested three tyrosine-kinase inhibitors (TKIs): SU5402, a selective inhibitor of fibroblast growth factor receptor (FGFR)-1 and vascular endothelial growth factor receptor (VEGFR)-2; sulfatinib, an inhibitor of FGFR-1 and VEGFR-1, -2, -3; and SPP86, a RET-specific inhibitor. The effects of these compounds were evaluated in vitro in two human MTC cell lines (TT and MZ-CRC-1), and in vivo using xenografts of MTC cells in zebrafish embryos. SU5402, sulfatinib and SPP86 decreased cell viability. Sulfatinib and SPP86 significantly induced apoptosis in both cell lines. Sulfatinib and SPP86 inhibited the migration of TT and MZCRC-1 cells, while SU5402 was able to inhibit migration only in TT cells. In vivo we observed a significant reduction in TT cell-induced angiogenesis in zebrafish embryos after incubation with sulfatinib and SPP86. In conclusion, sulfatinib and SPP86 displayed a relevant antitumor activity both in vitro and in vivo. Moreover, this work suggests the potential utility of targeting FGFR and VEGFR signaling pathways as an alternative therapy for MTC.

Analogs of the Heat Shock Protein 70 Inhibitor MKT-077 Suppress Medullary Thyroid Carcinoma Cells.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor mainly caused by mutations in the RET proto-oncogene. We previously demonstrated that depletion of the mitochondrial molecular chaperone, mortalin, can effectively suppress human MTC cells in culture and in mouse xenografts, by disrupting mitochondrial bioenergetics and subsequently inducing apoptosis and RET downregulation. Similar effects were induced by MKT-077, a water-soluble rhodocyanine dye analog known to inhibit mortalin, but with notable toxicity in animals. These observations led us to evaluate recently developed MKT-077 analogs that exhibited higher selectivity to HSP70 proteins and improved bioavailability. We validated the MTC cell-suppressive effects of mortalin depletion in three-dimensional cultures of the human MTC lines, TT, and MZ-CRC-1, and then evaluated different MKT-077 analogs in two- and three-dimensional cell cultures, to show that the MKT-077 analogs, JG-98 and JG-194, effectively and consistently inhibited propagation of TT and MZ-CRC-1 cells in these cultures. Of note, these compounds also effectively suppressed the viability of TT and MZ-CRC-1 progenies resistant to vandetanib and cabozantinib. Moreover, JG-231, an analog with improved microsomal stability, consistently suppressed TT and MZ-CRC-1 xenografts in mice. These data suggest that mortalin inhibition may have therapeutic potential for MTC.

Characterization of Human Medullary Thyroid Carcinoma Glycosphingolipids Identifies Potential Cancer Markers.

Medullary thyroid carcinoma (MTC) accounts for only 1–2% of thyroid cancers; however, metastatic MTC is a mortal disease with no cure. In this study, glycosphingolipids were isolated from human MTCs and characterized by mass spectrometry and binding of carbohydrate recognizing ligands. The tissue distribution of selected compounds was investigated by immunohistochemistry. The amount of acid glycosphingolipids in the MTCs was higher than in the normal thyroid glands. The major acid glycosphingolipid was the GD3 ganglioside. Sulfatide and the gangliosides GM3 and GD1a were also present. The majority of the complex non-acid glycosphingolipids had type 2 (Galβ4GlcNAc) core chains, i.e., the neolactotetraosylceramide, the Lex, H type 2 and x2 pentaosylceramides, the Ley and A type 2 hexaosylceramides, and the A type 2 heptaosylceramide. There were also compounds with globo (GalαGalβ4Glc) core, i.e., globotriaosylceramide, globotetraosylceramide, the Forssman pentaosylceramide, and the Globo H hexaosylceramide. Immunohistochemistry demonstrated an extensive expression av Ley in the MTC cells and also a variable intensity and prevalence of Globo H and Lex. One individual with multiple endocrine neoplasia type 2B expressed the Forssman determinant, which is rarely found in humans. This study of human MTC glycosphingolipids identifies glycans that could serve as potential tumor-specific markers.

Salinomycin and its derivatives as potent RET transcriptional inhibitors for the treatment of medullary thyroid carcinoma (MTC).

Rearranged during transfection kinase (RET) is a validated molecular target in medullary thyroid cancer (MTC), as activating mutations in RET are often associated with the development of MTC. The present study reports the first preclinical characterization of salinomycin and selected analogs as potent RET transcriptional inhibitors. Reverse transcription‐PCR and immunoblotting revealed that salinomycin profoundly decreased RET expression in the TT human MTC cell line by inhibiting RET transcription. Moreover, salinomycin resulted in remarkable anti‐proliferative activity against MTC that is driven by RET (gain of function mutation) by selectively inhibiting the intracellular PI3K/Akt/mTOR signaling pathway. Also, flow cytometry and fluorescence‐activated cell sorting showed that salinomycin induces G1 phase arrest and apoptosis by reducing the expression of retinoblastoma protein, E2F1, cyclin D and CDK4. The structure‐activity relationship of salinomycin was investigated in this study. Some of the salinomycin derivatives showed the ability to reduce RET expression where others fail to alter RET expression. These results suggest that the RET‐suppressing effect of salinomycin may be largely attributed to disruption of the Wnt pathway, presumably through interference with the ternary LRP6‐Frizzled‐Wnt complex. Furthermore, these findings support the further preclinical evaluation of salinomycin and its analogs as a promising new class of therapeutic agents for the improved treatment of MTCSupport or Funding InformationAmerican Cancer Society for their support of this study (grant no. RSGM‐12‐046‐01‐CDD).

Expression of the ret Proto-oncogene in Human Medullary Thyroid Carcinomas and Pheochromocytomas of MEN 2A

We studied the expression of the ret proto-oncogene (proto-ret) in human medullary thyroid carcinomas (MTCs) and pheochromocytomas of multiple endocrine neoplasia type 2A (MEN 2A) by Northern blot analysis. Expression of the normal-sized transcripts was detected in all 12 MTCs and in 6 of 8 pheochromocytomas. In situ localization of proto-ret mRNA revealed that the signal was confined to the cytoplasm of MTC cells. By Southern blot analysis neither amplification nor gross genetic changes of proto-ret were found in the tumors. Although no transcripts were detected in the normal portion of the thyroid from one MEN 2A patient, faint signals were detected in normal adrenal glands by Northern blot analysis, probably due to minor populations of C-cells and chromaffin cells in specimens from which MTC and pheochromocytoma might later develop. Proto-ret may play an important role in differentiation of a specific cell lineage from neuroectoderm, and it may be involved in development of MEN 2A tumors.

Cofilin is a mediator of RET-promoted medullary thyroid carcinoma cell migration, invasion and proliferation

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells and accounts for 5% of thyroid cancers. In inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell proliferation, survival and motility, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated.Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression.The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling.We found that cofilin transfection in human MTC cell line TT significantly increased migration (178 ± 44%, p < 0.001), invasion (165 ± 28%, p < 0.01) and proliferation (146 ± 18%, p < 0.001), accompanied by an increase of ERK1/2 phosphorylation (2.23-fold) and cyclin D1 levels (1.43-fold). Accordingly, all these responses were significantly reduced after genetic silencing of cofilin (−55 ± 10% migration, p < 0.001, −41 ± 8% invasion, p < 0.001, −17 ± 3% proliferation, p < 0.001). These results have been confirmed in primary cells cultures obtained from human MTCs.The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (−37 ± 8%, p < 0.001 and −31 ± 16%, p < 0.01, respectively). Furthermore, RPI-1 inhibitory effects on TT cell migration (−57 ± 13%, p < 0.01), but not on cell proliferation, were completely abolished in cells transfected with cofilin.In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.

Cofilin is a mediator of RET-promoted medullary thyroid carcinoma cell migration, invasion and proliferation

Abstract Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells and accounts for 5% of thyroid cancers. In inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell proliferation, survival and motility, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated. Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression. The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling. We found that cofilin transfection in human MTC cell line TT significantly increased migration (178 ± 44%, p The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (−37 ± 8%, p In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.

Mortalin (GRP75/HSPA9) Promotes Survival and Proliferation of Thyroid Carcinoma Cells.

We previously reported that upregulation of mortalin (HSPA9/GRP75), the mitochondrial HSP70 chaperone, facilitates tumor cell proliferation and survival in human medullary thyroid carcinoma (MTC), proposing mortalin as a novel therapeutic target for MTC. In this report, we show that mortalin is also upregulated in other thyroid tumor types, including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC), and that mortalin depletion can effectively induce growth arrest and cell death in human PTC (TPC-1), FTC (FTC133), and ATC (8505C and C643) cells in culture. Intriguingly, mortalin depletion induced varied effects on cell cycle arrest (G0/G1 phase arrest in TPC-1 and C643, G2/M phase arrest in 8505C, and mild G2/M phase arrest with increased sub-G0/G1 population in FTC133) and on the levels of TP53, E2F-1, p21CIP1, p27KIP1, and poly (ADP-ribose) polymerase cleavage in these cells, suggesting that thyroid tumor cells respond to mortalin depletion in a cell type-specific manner. In these cells, we also determined the efficacy of triphenyl-phosphonium-carboxy-proxyl (Mito-CP) because this mitochondria-targeted metabolism interfering agent exhibited similar tumor suppressive effects as mortalin depletion in MTC cells. Indeed, Mito-CP also induced robust caspase-dependent apoptosis in PTC and ATC cell lines in vitro, exhibiting IC50 lower than PLX4032 in 8505C cells and IC50 lower than vandetanib and cabozantinib in TPC-1 cells. Intriguingly, Mito-CP-induced cell death was partially rescued by mortalin overexpression, suggesting that Mito-CP may inactivate a mechanism that requires mortalin function. These findings support the significance of mortalin and mitochondrial activity in a broad spectrum of thyroid cancer.

SAT-566 Cofilin Is a Mediator of RET-Promoted Medullary Thyroid Carcinoma Cell Migration, Invasion and Proliferation

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells, accounting for 5% -10% of thyroid cancers. In all inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell motility, proliferation and survival, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated. Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression. The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling. We found that cofilin transfection in human MTC cell line TT significantly increased migration (178.5±44%, p<0.001), invasion (194±27%, p<0.05) and proliferation (145.9±18%, p<0.001), accompanied by an increase of ERK1/2 phosphorylation (1.8-fold) and cyclin D1 levels (1.43-fold). Accordingly, all these responses were significantly reduced after genetic silencing of cofilin (-55±10% migration, p<0.001, -40.7±8% invasion, p<0.001, -17±2.6% proliferation, p<0.001). The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RETΔTK) resulted in a reduction of cofilin expression (-30.8±11%, p<0.01 and -31±16%, p<0.01, respectively). Furthermore, RPI-1 inhibitory effects on TT cell migration (-70±18%, p<0.001) and proliferation (-29.4±9% , p<0.01) were completely abolished in cells transfected with cofilin. In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC.

Preclinical characterization of tyrosine kinase inhibitor-based targeted therapies for neuroendocrine thyroid cancer.

Medullary thyroid carcinoma (MTC) is a slow growing neuroendocrine (NE) tumor for which few treatment options are available. Its incidence is rising and mortality rates have remained unchanged for decades. Increasing the repertoire of available treatments is thus crucial to manage MTC progression. Scarcity of patient samples and of relevant animal models are two challenges that have limited the development of effective non-surgical treatments. Here we use a clinically accurate mouse model of MTC to assess the effects and mode of action of the tyrosine kinase inhibitor (TKI) Vandetanib, one of only two drugs currently available to treat MTC. Effects on tumor progression, histopathology, and tumorigenic signaling were evaluated. Vandetanib blocked MTC growth through an anti-angiogenic mechanism. Furthermore, Vandetanib had an apparent anti-angiogenic effect in a patient MTC sample. Vandetanib displayed minimal anti-proliferative effects in vivo and in human and mouse MTC tumor-derived cells. Based on these results, we evaluated the second-generation TKI, Nintedanib, alone and in combination with the histone deacetylase (HDAC) inhibitor, Romidepsin, as potential alternative treatments to Vandetanib. Nintedanib showed an anti-angiogenic effect while Romidepsin decreased proliferation. Mechanistically, TKIs attenuated RET-, VEGFR2- and PI3K/AKT/FOXO signaling cascades. Nintedanib alone or in combination with Romidepsin, but not Vandetanib, inhibited mTOR signaling suggesting Nintedanib may have broader anti-cancer applicability. These findings validate the MTC mouse model as a clinically relevant platform for preclinical drug testing and reveal the modes of action and limitations of TKI therapies.

OA166] A1M is a potential kidney protector in 177Lu-octreotate treatment of neuroendocrine tumours

Treatment of patients with neuroendocrine tumours (NET) using 177Lu-octreotate has resulted in prolonged survival and improved quality of life, but low cure rate. The treatment is limited by side effects on normal tissues, where kidneys and bone marrow are the major risk organs. Alpha-1-microglobulin (A1M) is an antioxidant with the ability to protect normal tissues from oxidative stress. A1M has recently shown beneficial effects on renal function after 177Lu-octreotate treatment in preclinical studies. When introducing a radiation protector in radiation therapy it is crucial to maintain high therapeutic effect on tumour tissue. Purpose To investigate if A1M affects the uptake of 177Lu in tumour tissue, and/or affects the treatment effect of 177Lu-octreotate in NET-bearing mice. Methods Study 1) The biodistribution of 177Lu-octreotate was studied in BALB/c mice bearing GOT2 tumours (human medullary thyroid carcinoma). The mice were i.v. injected with 5 MBq 177Lu-octreotate, and half of the mice also received 5 mg/kg A1M. The mice were killed 1 h to 7 days after treatment. The activity concentration in tumour and normal tissues was measured and compared between the two groups. Study 2) The effect of A1M on tumour volume was studied on BALB/c mice bearing GOT1 tumours (human small intestine neuroendocrine tumour). The mice were divided into three groups and treated with: 30 MBq 177Lu-octreotate or 5 mg/kg A1M or both combined (30 MBq 177Lu-octreotate and 5 mg/kg A1M). The tumour volume was followed over time and compared between the groups. Results Study 1) Overall, the biodistribution of 177Lu-octreotate was similar between the two groups. Study 2) No decrease in mean tumour volume in GOT1 bearing mice treated with A1M alone was observed. For both groups treated with 177Lu-octreotate, a strong tumour response was observed during the first 14 days after treatment, with a mean volume reduction of about 50%. Thereafter, the tumours re-grew with a similar growth rate for both groups. Conclusions A1M showed no negative effects on the biodistribution of 177Lu-octreotate or on the therapeutic response of 177Lu-octreotate, indicating that A1M is a good candidate for kidney protection during treatment with 177Lu-octreotate, and should be further investigated.

Abstract 4783: Selective antitumor activity of NSC311152 toward medullary thyroid cancer by downregulating the transcription of RET oncogene

Abstract The RET (REarranged during Transfection) proto-oncogene encodes a tyrosine-kinase receptor specifically expressed in tissues originated from neuroectodermal tissues. Dominant-activating mutations in this RET proto-oncogene is often associated with Multiple Endocrine Neoplasia type 2A and 2B and Familial Medullary Thyroid Carcinoma syndromes, characterized by the occurrence of medullary thyroid carcinomas. Human medullary thyroid carcinoma cell line TT cell line harbours a MEN2A-type mutation, so this cell line could be a useful cell system to investigate the antitumor activity of new agents interfering with the function of RET oncogene in medually thyroid cancer. Recently, ellipticine and its derivatives were identified as excellent RET G-quadruplex stabilizing agents. Furthermore, these compounds also interfered with the transcriptional mechanism of the RET gene in TT cells, subsequently decreasing the endogenous RET protein expression. In present study, we demonstrated the specificity of NSC311152 by using papillary thyroid carcinoma (PTC) cells, the K1, which carry BRAF and PIK3CA mutations but lacks the dominant-activating mutations in this RET proto-oncogene. NSC311152 selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. The systemic administration of NSC311152 in a mouse MTC xenograft model significantly inhibited the tumor growth through RET downregulation, whereas NSC31152 only showed a moderate antitumor activity against K1 xenograft tumors. Citation Format: Daekyu Sun, Vishnu Muthuraj Kumarasamy, Tariq Alqahtani. Selective antitumor activity of NSC311152 toward medullary thyroid cancer by downregulating the transcription of RET oncogene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4783.

Abstract 501: A RET-related microRNA, miR-153-3p, acts as a tumor suppressor in medullary thyroid carcinoma (MTC) via S6K signaling

Abstract Background Medullary thyroid carcinoma (MTC), which comprises 3-5% of all thyroid cancers, originates from a small population of neuroendocrine calcitonin C-cells of thyroid gland. Gain-of-function mutations of the human Rearranged during Transfection (RET) proto-oncogene have been recognized as the key driver of MTC tumorigenesis. RET has been targeted by tyrosine kinase inhibitors (TKIs), however with modest efficacy. Many of the microRNAs (miRNAs) identified in human genome have been associated with cancer and have prompted interest in the establishment of miRNA-based therapeutics. However miRNA studies in MTC has been hampered due to the lack of normal C-cell tissue as a differential expression comparator. Aims We aimed to characterize differentially expressed miRNAs, which play fundamental roles in MTC tumorigenesis and in modulation of TKI responses. Methods RET of human MTC cells were silenced using TKI, Cabozantinib, or siRNA. Small RNA sequencing was performed (AGRF) to identify differentially expressed miRNAs before and after RET inhibition. MiRNA effects on cell proliferation (MTS assay), cell cycle and apoptosis (flow cytometry, Western blot) were investigated with gain-of-function studies. For in vivo study, MTC xenografts were established and miRNAs were intravenously delivered using nanoparticle delivery vehicles invented by biotechnology company - EnGeneIC Ltd. Results miR-153-3p was identified to be under-expressed in MTC following RET inhibition. Restoration of miR-153-3p significantly reduced cell proliferation, induced G2 cell arrest and increased cell apoptosis. Furthermore, combined treatment of miR-153-3p and Cabozantinib caused greater cell proliferation inhibition compared to individual treatment. miR-153-3p targets ribosomal protein S6 kinase B1 (RPS6KB1) and reduced phosphorylation of Bcl-2 associated death promoter (BAD) protein. In vivo delivery of miR-153-3p significantly impeded tumor growth compared to scrambled sequence. Combination of miR-153-3p delivery and oral administration of Cabozantinib resulted in further tumor stabilization effect. Conclusion miR-153-3p reveals a significant tumor suppressive role in MTC via S6K signaling, demonstrating its important therapeutic potential for MTC patients. Citation Format: Lauren Jin Suk Joo, Jocelyn Weiss, Anthony J. Gill, Roderick Clifton-Bligh, Himanshu Brahmbhatt, Jennifer A. MacDiarmid, Matti L. Gild, Bruce G. Robinson, Stan B. Sidhu, Jing Ting Zhao. A RET-related microRNA, miR-153-3p, acts as a tumor suppressor in medullary thyroid carcinoma (MTC) via S6K signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 501.

A whole-animal platform to advance a clinical kinase inhibitor into new disease space.

Synthetic tailoring of approved drugs for new indications is often difficult, as the most appropriate targets may not be readily apparent and therefore few roadmaps exist to guide chemistry. Here, we report a multidisciplinary approach for accessing novel target and chemical space starting from an FDA-approved kinase inhibitor. Combining chemical and genetic modifier screening with computational modeling, we identify distinct kinases that strongly enhance (‘pro-targets’) or limit (‘anti-targets’) whole animal activity of the clinical kinase inhibitor sorafenib in a Drosophila medullary thyroid carcinoma (MTC) model. We demonstrate that RAF—the original intended sorafenib target—and MKNK kinases function as pharmacological liabilities due to inhibitor-induced transactivation and negative feedback, respectively. Through progressive synthetic refinement, we report a novel class of ‘Tumor Calibrated Inhibitors’ with unique polypharmacology and strongly improved therapeutic index in fly and human MTC xenograft models. This platform provides a rational approach for creating new high efficacy/low toxicity drugs.

Effect of parathyroid hormone on apoptosis of human medullary thyroid carcinoma cells*

Abstract Objective The aim of the study was to investigate the effect of parathyroid hormone (PTH) on the apoptosis of human medullary thyroid carcinoma (MTC) cells. Methods In vitro cultured medullary thyroid carcinoma cell lines were treated with parathyroid hormone and parathyroid hormone receptor-monoclonal antibody, and the apoptosis of cells was detected by flow cytometry. Results The cell morphology changed significantly after treatment based on the observation using the inverted phase-contrast microscope. Various concentrations of parathyroid hormone and parathyroid hormone receptor-monoclonal antibody effectively induced apoptosis in a time- and concentration-dependent manner. When the concentration of parathyroid hormone was 2.0 µmol/L and that of parathyroid hormone receptor-monoclonal antibody was 1.0 µmol/L, the apoptotic rate was 13.24% and 20.78%, respectively, representing a statistically significant difference from that of the control cells (P &lt; 0.05). Conclusion PTH plays a role in inducing apoptosis of human MTC cells.

Upregulated miR-9-3p Promotes Cell Growth and Inhibits Apoptosis in Medullary Thyroid Carcinoma by Targeting BLCAP.

Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer derived from parafollicular C cells in the thyroid gland. It has great interest as a research focus because of its unusual genetic, clinical, and prognostic characteristics. However, the pathogenesis in MTC is not completely clear. We investigated the role of miR-9-3p and bladder cancer-associated protein (BLCAP) in MTC TT cells. First, miR-9-3p expression was upregulated in human MTC tissues and TT cells and compared to the control by RT-PCR. Flow cytometric analysis indicated that the cell cycle progression in TT cells was significantly inhibited by the miR-9-3p inhibitor but was increased by the miR-9-3p mimic. On the contrary, the apoptosis of TT cells was significantly increased by the miR-9-3p inhibitor and suppressed by the miR-9-3p mimic. A similar change pattern was observed in the expression of apoptosis-regulated protein caspase 3 induced by the miR-9-3p mimic or inhibitor in TT cells. We then identified that BLCAP is a target of miR-9-3p by bioinformatic prediction and luciferase reporter assay. The expression of BLCAP was also significantly downregulated by the miR-9-3p mimic while being upregulated by the miR-9-3p inhibitor in TT cells. Furthermore, we confirmed that the inhibited apoptosis of TT cells induced by the miR-9-3p mimic was enhanced by BLCAP overexpression. The levels of apoptosis were strongly decreased by BLCAP silencing in TT cells, which were not further influenced by the miR-9-3p inhibitor. In summary, upregulated miR-9-3p has a positive role in human MTC progression by regulating the growth and apoptosis of cancer cells via targeting BLCAP. This might represent a possible diagnosis or therapeutic target for MTC.

Vandetanib and cabozantinib potentiate mitochondria-targeted agents to suppress medullary thyroid carcinoma cells

ABSTRACTAlthough the FDA-approved receptor tyrosine kinases inhibitors, vandetanib and cabozantinib, are used to treat surgically inoperable progressive medullary thyroid carcinoma (MTC), not all patients are responsive while the disease sometimes progresses after an initial response. To better understand MTC drug resistance at molecular and biochemical levels, we have generated drug-resistant subpopulations of the human MTC cell lines, TT and MZ-CRC-1, via prolonged exposure to vandetanib and cabozantinib. These drug-resistant progenies exhibited substantial cross-resistance to vandetanib and cabozantinib, suggesting that these inhibitors may invoke an overlapping resistance mechanism(s) in MTC cells. Of note, vandetanib and cabozantinib increased mitochondrial membrane potential (Δψm) in drug-naïve as well as drug-resistant cells but only drug-naïve cells exhibited substantially altered oxygen consumption and extracellular acidification rates. Therefore, these inhibitors appear to cause a bioenergetics stress to which drug-resistant MTC cells are more tolerant. Given the ability of vandetanib and cabozantinib to increase Δψm, we hypothesized that these inhibitors can augment growth inhibitory effects of mitochondria-targeted carboxy-proxyl and ubiquinone by increasing their Δψm–dependent uptake/retention in MTC cells. Indeed, our in vitro and mouse xenograft data strongly support this possibility.

Identification of microRNAs associated with medullary thyroid carcinoma by bioinformatics analyses

The present study aimed to investigate the microRNA (miRNA) profile in human medullary thyroid carcinoma (MTC) tissue. The GSE40807 data profile was downloaded from the Gene Expression Omnibus database. Following preprocessing, differentially expressed microRNAs (DEMs) between MTC and healthy tissues were identified. Based on the obtained DEMs, transcription factor (TF)‑miRNA and miRNA‑target gene regulatory association pairs were predicted. Finally, functional enrichment analysis was performed on target genes of DEMs. Fifteen upregulated and 17 downregulated DEMs were identified. In the constructed TF‑miRNA regulatory network, hsa‑miR‑9‑5p was regulated by 9 TFs and hsa‑miR‑1 was regulated by 8 TFs. TFs of nuclear factor of κ light polypeptide gene enhancer in B‑cells 1 (NF‑κB1) and v‑myc avian myelocytomatosis viral oncogene homolog (MYC) regulated 4 and 3 DEMs, respectively. In the miRNA‑target gene regulatory network, hsa‑miR‑1, hsa‑miR‑9‑5p, hsa‑miR‑96‑5p and hsa‑miR‑590‑5p were most upregulated. The target genes of these 4 miRNAs were primarily enriched in the mitogen activated protein kinase (MAPK) signaling pathway. Therefore, MAPK signaling pathway may serve important roles in MTC progression. In conclusion, the DEMs hsa‑miR‑1 and hsa‑miR‑9‑5p, and TFs of NF‑κB1 and MYC may be used as biomarkers for the diagnosis and treatment of MTC.

Multilayer OMIC Data in Medullary Thyroid Carcinoma Identifies the STAT3 Pathway as a Potential Therapeutic Target in RETM918T Tumors.

Purpose: Medullary thyroid carcinoma (MTC) is a rare disease with few genetic drivers, and the etiology specific to each known susceptibility mutation remains unknown. Exploiting multilayer genomic data, we focused our interest on the role of aberrant DNA methylation in MTC development.Experimental Design: We performed genome-wide DNA methylation profiling assessing more than 27,000 CpGs in the largest MTC series reported to date, comprising 48 molecularly characterized tumors. mRNA and miRNA expression data were available for 33 and 31 tumors, respectively. Two human MTC cell lines and 101 paraffin-embedded MTCs were used for validation.Results: The most distinctive methylome was observed for RETM918T-related tumors. Integration of methylation data with mRNA and miRNA expression data identified genes negatively regulated by promoter methylation. These in silico findings were confirmed in vitro for PLCB2, DKK4, MMP20, and miR-10a, -30a, and -200c. The mutation-specific aberrant methylation of PLCB2, DKK4, and MMP20 was validated in 25 independent MTCs by bisulfite pyrosequencing. The methylome and transcriptome data underscored JAK/Stat pathway involvement in RETM918T MTCs. Immunostaining [immunohistochemistry (IHC)] for the active form of signaling effector STAT3 was performed in a series of 101 MTCs. As expected, positive IHC was associated with RETM918T-bearing tumors (P < 0.02). Pharmacologic inhibition of STAT3 activity increased the sensitivity to vandetanib of the RETM918T-positive MTC cell line, MZ-CRC-1.Conclusions: Multilayer OMIC data analysis uncovered methylation hallmarks in genetically defined MTCs and revealed JAK/Stat signaling effector STAT3 as a potential therapeutic target for the treatment of RETM918T MTCs. Clin Cancer Res; 23(5); 1334-45. ©2016 AACR.