Only 2% of thyroid cancer (TC) cases are anaplastic thyroid cancer (ATC), but this rare, undifferentiated cancer with a highly inflamed tumor microenvironment (TME) presents universally as stage IV with a 5-year survival rate of 3%. Due to its loss of differentiation, ATC is resistant to radioiodine therapy, refractory to external beam radiation, and insufficiently treated by chemotherapy or immunotherapy. Berberine (BBR), a natural plant alkaloid, has vast pharmacological activities, including anti-inflammatory and anti-cancer roles. BBR blocks proliferation, induces cell cycle arrest, promotes apoptosis, and hinders invasion and metastasis. BBR may also inhibit cell proliferation through miRNA interactions and regulate the TME through its anti-inflammatory and antioxidant properties. Research in ATC using BBR is limited, specifically investigation on how BBR reprograms the inflammatory TME via altering miRNA cargo of secretory exosomes and polarization of tumor associated macrophages (TAMs). ATC tissues have extensive infiltration of a mixed TAM population. In ATC, infiltration with classically anti-tumorigenic, inflammatory M1 macrophages has a pro-tumorigenic role. We assessed the secretory profile of ATC cells and found a high expression of pro-inflammatory cytokines, such as TNFα, IL-6, IL-1, MCP-1/2, and MIP-1α/β in ATC conditioned media (CM), supporting the existence of an inflammatory TME. As this chronic inflammation is partially mediated by the presence of M1 TAMs in the TME, we activated monocyte cell line U937 with TPA and polarized with IFNγ and LPS into an M1 phenotype. In the presence of BBR at the activation and polarization stages, inflammatory mediators IL-1β, IL-6, IL-6R, CXCL9, MIP-1α/β, and TNFR1/2 were downregulated in the CM compared to M1 macrophages activated and polarized without BBR. BBR also appeared to prevent some level of activation and polarization of these cells into the M1 phenotype altogether. BBR may have an essential role in lessening the burden of inflammation in ATC through remodeling its tumor immune infiltrates, potentially priming it for greater success in combination therapy. Intervention prior to metastasis warrants secretome analysis to define ATC disease progression. Exosomal cargo, particularly miRNA, secreted by ATC cells was evaluated as exosomes have potential as biomarkers to detect ATC earlier improving prognosis, guide treatment, and avoid unneeded surgeries. Distinct miRNA expression from ATC-secreted exosomes compared to papillary thyroid cancer (PTC)-secreted exosomes revealed six miRNAs specifically downregulated in ATC-secreted exosomes that are known tumor suppressors. We are currently evaluating how BBR alters the miRNA profile of ATC- and PTC-secreted exosomes. The ability of BBR to alleviate inflammation in ATC and remodel its TME releases its chronic hold on ATC progression and may prime the tumor for increased responsiveness to therapy. Citation Format: Tara Jarboe, Kaci Kopec, Nicole R. DeSouza, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Xiu-Min Li, Raj K. Tiwari. Berberine eases inflammation in the anaplastic thyroid cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 243.

Anaplastic thyroid cancer (ATC) is an undifferentiated thyroid cancer. A hallmark of ATC is increased inflammatory cytokine expression and inflammatory cell infiltration in the tumor microenvironment that is responsible for its aggressive nature. In contrast, papillary thyroid cancer (PTC) is well-differentiated and subsequently significantly less aggressive and more responsive to available therapies. The metabolic differences between ATC and PTC that correlate with differentiation status, and hence metastatic propensity, are poorly understood. Owing to its lack of differentiation, ATC is radioiodine therapy resistant, whereas differentiated thyroid cancers are susceptible. AMPK in the thyroid gland is responsible for glucose and iodine uptake. The AMPK pathway is a potential target for modulation due to the higher glucose uptake and metabolism associated with treatment resistance seen in ATC. Berberine, a naturally occurring plant alkaloid, regulates glycometabolism and lipid metabolism, improves energy expenditure, and reduces body weight. Exploration of a possible connection between the metabolic regulation displayed by BBR and AMPKα expression was warranted. Increased AMPKα signaling, observed through increased AMPKα phosphorylation, has an anti-tumor effect. When comparing PTC (TPC1) and ATC (T238) cell lines there was a 4.75- and 2.34-fold change, respectively, in phosphorylation of AMPKα upon BBR treatment. This demonstrates that BBR increases metabolic regulation in thyroid cancer cell lines, importantly, even when poorly differentiated. Further analysis of the correlation between differentiation status and metabolic characteristics between ATC and PTC was investigated using RNASeq. Differential gene expression was conducted to compare ATC and PTC using human thyroid cancer cell lines T238 and K1, respectively. These thyroid cancer cell lines were treated with 100 μM BBR or the same concentration of DMSO as a vehicle control for 24 hours. RNA was isolated from T238 and K1 cells and RNASeq was conducted by GeneWiz. This differential gene expression will help identify specific genes that are regulating metabolic patterns and is currently ongoing. Berberine (BBR) is a natural compound used widely in Traditional Chinese Medicine for its many pharmacological properties including anti-inflammatory, metabolic, antioxidant, and anti-cancer. BBR may be able to mitigate the metabolic pattern of ATC to potentially affect differentiation status as an intervening product. Intervention of the differentiation status of ATC will impact its progression, specifically metastasis. Therefore, identifying gene patterns that correlate with differentiation status will identify novel intervention targets for BBR. Citation Format: Kaci Kopec, Tara Jarboe, Mordechai Sternman, Nicole R. DeSouza, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari, Xiu-Min Li. Metabolic modulation of anaplastic thyroid cancer by Berberine: A targeted intervention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1410.

Anaplastic Thyroid Cancer (ATC) is one of the most aggressive forms of thyroid cancer, with only a 4% survival rate with metastatic disease. Further, ATC is highly refractory and resistant to traditional therapeutics, resulting in poor patient outcomes. Therefore, the elucidation of molecular mechanisms that contribute to ATC’s metastatic propensity, and the identification of molecular biomarkers for the early diagnosis, prognosis, and therapeutic intervention are critical unmet needs. This is reinforced by the many challenges that accompany the use of small molecular inhibitors and targeted therapy to treat ATC. Recently, long non-coding RNAs (lncRNA) have been identified as having major regulatory roles in the fine-tuning of gene expression and cellular behavior of cancer cells, highlighting the importance of identifying non-coding profiles that potentially impact ATC behavior. Using the publicly available datasets (Gene Expression Omnibus; GSE33630, GSE85457), we have investigated gene expression profiles of ATC vs. normal thyroid tissue. Bioinformatic analyses with the GEO2R program identified the lncRNA, Double Homeobox A Pseudogene 10 (DUXAP10), to be highly upregulated in ATC patient-derived tissue samples when compared to normal thyroid tissue. DUXAP10 is structurally a pseudogene-derived lncRNA that has been identified to be upregulated in hepatocellular, bladder, esophageal, and gastric cancer, as well as papillary thyroid cancer. We found that the expression of DUXAP10 is also upregulated in the ATC cell line, T238, in comparison to Nthy-ori-3-1, an immortalized normal thyroid cell line. T238 is homozygous for the BRAF V600E mutation, which serves as a driver mutation. However, breakthrough alterations can limit the effectiveness of anti-BRAF therapeutics and highlight the utility of combination therapies. To understand the consequences of DUXAP10 overexpression in ATC, we used CRISPRi technology to determine the phenotypic and genotypic effects of DUXAP10 downregulation. CRISPRi knockdown of DUXAP10 significantly reduces the migratory capabilities of T238 by 87%. This provides evidence that elevated DUXAP10 expression may increase the probability and negative consequences of metastases. Thus, targeting DUXAP10 expression may have beneficial clinical consequences. The role of DUXAP10 on thyroid cell proliferation and invasion are currently in progress. Thus, we have identified DUXAP10 as a potential biomarker for ATC diagnosis and prognosis, as well as a potential therapeutic target. Further analysis remains to be determined if DUXAP10 can be targeted for standalone or combination therapy, as well as the elucidation of the full range of interactions that DUXAP10 has in ATC. Citation Format: Nicole DeSouza, Michelle Carnazza, Danielle Quaranto, Tara Jarboe, Kaci Kopec, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Functional analysis of lncRNA DUXAP10 in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 67.

Anaplastic Thyroid Cancer (ATC) is one of the most aggressive forms of cancer, with only a 4% survival rate with metastatic disease. ATC is highly refractory and resistant to both traditional therapeutic modalities and recently discovered small molecule inhibitors. The elucidation of molecular mechanisms that contribute to ATC’s metastatic propensity, and the identification of molecular biomarkers for early diagnosis, prognosis, and therapeutic intervention are critical unmet clinical needs-highlighting the importance of novel avenues of intervention. Recently, long non-coding RNAs (lncRNA) have been described as regulators of gene expression and cancer cell phenotypes, exhibiting the importance of identifying non-coding profiles that potentially impact ATC behavior. Publicly available datasets provided by Gene Expression Omnibus (GSE33630, GSE85457) enabled investigation of gene expression profiles of ATC vs. normal thyroid patient tissue. Bioinformatic analyses with the GEO2R program identified the lncRNA, Double Homeobox A Pseudogene 10 (DUXAP10), to be highly upregulated in ATC patient-derived tissue samples when compared to normal thyroid tissue by about 5logFC. Analysis using Gene Expression Profiling Interactive Analysis shows that high DUXAP10 expression is correlated with decreased survival in thyroid cancer patients. To recapitulate this patient data in an in vitro model, we found that the expression of DUXAP10 is also upregulated in the ATC cell line, T238, in comparison to Nthy-ori-3-1, an immortalized “normal” thyroid cell line. To understand the consequences of DUXAP10 overexpression in ATC, we used CRISPRi technology to determine the phenotypic and genotypic effects of DUXAP10 downregulation. CRISPRi knockdown of DUXAP10 significantly reduces the migratory capabilities of T238 by 50%, invasiveness by 37%, clonogenicity by 70%, cell size by 34%, and proliferation by 28%, 45%, and 36% at 24, 48, and 72 hours, respectively. This provides evidence that elevated DUXAP10 expression may increase the probability and negative consequences of metastases. Thus, targeting DUXAP10 expression may have beneficial clinical outcomes. Identification of the molecular innervations of DUXAP10 that orchestrate these pro-metastatic phenotypic changes are currently in progress. Immediate next steps are aimed toward determining if DUXAP10 can be utilized as a potential biomarker for ATC diagnosis and prognosis, as well as elucidating the full range of interactions that DUXAP10 has in shaping the ATC phenotype. Citation Format: Nicole R. DeSouza, Michelle Carnazza, Danielle Quaranto, Tara Jarboe, Kaci Kopec, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Long non-coding RNA DUXAP10 is a putative determinant of anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1406.

Anaplastic thyroid cancer (ATC) is universally diagnosed as Stage IV with a five-year-survival of 4%, illuminating its dreadful prognosis. ATC is undifferentiated, has an inflammatory tumor microenvironment (TME), and metabolic dysregulation. As it is refractory to all established therapies, we propose a novel therapeutic - Berberine (BBR), a natural plant alkaloid, with numerous cellular targets that can potentially reprogram ATC’s aggressive phenotype. This research comprehensively examines the multitarget efficacy of BBR. Following initial dose response studies, all assays used 100µM concentration of BBR or DMSO vehicle control for 24-hour exposure. To model the ATC tumor microenvironment, monocyte cell line U937 cells were activated and polarized into a proinflammatory macrophage phenotype. In the presence of berberine at the activation and polarization stages, 33 soluble inflammatory mediators were downregulated in the conditioned media compared to controls. Targeting the aggressiveness of anaplastic disease, berberine slowed proliferation by 50% selectively in ATC cells from 48 to 72 hours, while sparing the immortalized normal thyroid cells. BBR also delayed wound healing by 30% in ATC cells after 24, 48, and 72 hours. These observations were substantiated by Western blot analysis of ATC and immortalized normal cells where BBR decreased phosphorylation of MEK, ERK, and ribosomal protein S6, crucial downstream regulators of the pro-proliferative and pro-survival pathways and increased phosphorylation of AMPKα, activating its anti-tumor and regulatory effects. Validation of in vitro findings via RNA-Seq was conducted by Genewiz from Azenta Life Sciences and Qiagen’s Ingenuity Pathway Analysis was used for in silico modeling. Greater than 400 significantly differentially expressed genes were identified suggesting that BBR regulates mitochondrial metabolism, cholesterol biosynthesis, glycometabolism, apoptosis, inflammation, and proliferation. BBR’s ability to alleviate inflammatory mediators in the TME, depress overactive cell signaling, and regulate metabolism to make it a more targetable cancer reveals it as an interesting candidate to prime ATC for combination therapy. Citation Format: Tara Jarboe, Kaci Kopec, Nicole R. DeSouza, Jan Geliebter, Raj K. Tiwari, Xiu-Min Li. Remodeling of anaplastic thyroid cancer cell signaling and immune landscape by natural alkaloid berberine. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3824.