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RNA biology has gained extensive recognition in the last two decades due to the identification of novel transcriptomic elements and molecular functions. Cancer arises, in part, due to the accumulation of mutations that greatly contribute to genomic instability. However, the identification of differential gene expression patterns of wild-type loci has exceeded the boundaries of mutational study and has significantly contributed to the identification of molecular mechanisms that drive carcinogenic transformation. Non-coding RNA molecules have provided a novel avenue of exploration, providing additional routes for evaluating genomic and epigenomic regulation. Of particular focus, long non-coding RNA molecule expression has been demonstrated to govern and direct cellular activity, thus evidencing a correlation between aberrant long non-coding RNA expression and the pathological transformation of cells. lncRNA classification, structure, function, and therapeutic utilization have expanded cancer studies and molecular targeting, and understanding the lncRNA interactome aids in defining the unique transcriptomic signatures of cancer cell phenotypes.

Anaplastic thyroid cancer's (ATC) undifferentiated, inflammatory nature makes it one of the most aggressive cancers, with a five-year survival rate of only 4% when metastatic. ATC is a rare cancer that is refractory to conventional therapeutic modalities. Without high expression of targetable genetic lesions, small molecule inhibitors alone have been insufficient in trials. Treatment of ATC could benefit from a holistic approach that reprograms the inflammatory tumor microenvironment (TME). Berberine (BBR), a natural plant-derived alkaloid used extensively in Traditional Chinese Medicine, is a compound shown to exhibit anti-microbial, anti-inflammatory, and anti-cancer properties. Our work aims to exploit the anti-inflammatory activity of BBR in ATC. As the inflammatory status of ATC defines its intractable nature, remodeling its secretome, including its cytokine and chemokine profile and its exosomal cargo, in the TME fundamentally targets ATC's progressive determinant - inflammation. Exosomes, membrane-bound extracellular vesicles, are secreted by cells in the TME, including activated tumor-associated macrophages and ATC cells. Exosomal cargo primarily consists of miRNAs. We observed distinct miRNA expression from ATC-secreted exosomes when compared to papillary thyroid cancer (PTC)-secreted exosomes. Comparative analysis revealed ten miRNAs specifically downregulated in exosomes secreted from anaplastic-like 8505C compared to papillary BCPAP, including: hsa-miR-26b-5p, hsa-miR-125b-5p, hsamiR-138-5p, hsa-miR-148a-5p, hsamiR-152-5p, hsa-miR-191-5p, hsa-miR-9-5p, hsa-miR-21-5p, hsa-miR-134-5p, and hsa-miR-379-5p. The first six miRNAs listed are tumor suppressors, and as such, their downregulation may be contributory to the metastatic propensity and aggressiveness of ATC and its refractory nature towards conventional treatments. In activated macrophage-derived exosomes, miR-21-5p and miR-138-5p were also upregulated. ATC-secreted exosomes activate macrophages, subsequently priming the TME to be pro-tumorigenic and pro-inflammatory. This reciprocal interaction between inflammatory macrophages and ATC cells mediated by exosomal miRNAs defines its metastatic and inflammatory phenotype. We also observed that BBR significantly downregulates phosphorylation of MEK, ERK, and ribosomal protein S6 in proliferating ATC cells with as low as 10 μM BBR treatment. These are important downstream regulators of the pro-proliferative, pro-survival, and metabolic MAPK and PI3K-PTEN-AKT signaling pathways. Overall, the ability for BBR to alleviate the pro-inflammatory phenotype of ATC and remodel its immune environment, while simultaneously depressing overactive signaling in these cell survival pathways, may mark it as an important agent to make ATC amenable to combination therapy with small molecule inhibitors (MEKi) or other immunotherapeutics. Citation Format: Tara Jarboe, Nicole DeSouza, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari, Xiu-Min Li. Berberine-mediated reprogramming of the inflammatory environment in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 317.

Thyroid cancer incidence is increasing at an alarming rate almost tripling every decade. Although the majority of thyroid tumors are treatable, about 2-3% of thyroid cancers, mainly anaplastic thyroid cancer (ATC) is refractory to mainstream therapies. Driver mutations like BRAFV600E, PI3KCAE545K prompted development of targeted therapies with kinase inhibitors for ATC, but several reports suggest development of resistance. Given the refractory nature of ATC, it is imperative to explore alternative targets using accessory cell surface molecules that modulate the tumor immune microenvironment (TIME). Immune checkpoint molecules have revolutionized the field of cancer therapeutics, but are underexplored in ATC as an alternative therapeutic target. To this end, 4 ATC cell lines 8505C, T238, SW1736 and HTh74; 3 papillary thyroid cancer (PTC) cell lines TPC-1, BCPAP and K1 and 1 follicular thyroid cancer (FTC) cell line CGTH-W-1 were screened for expression of 29 immune-checkpoint molecules by qRT PCR, immunocytochemistry, western blot and flow cytometry. We noted a differential expression of HVEM, BTLA, CD160, TIM3, LGALS9 and PD-L1 across the cell lines. The expression level of these genes was much higher in ATC cell lines compared to the rest. Expression level of HVEM was more than 30 fold higher compared to the other cell lines on average. HVEM is a member of TNFRSF which interacts with BTLA, CD160 and LIGHT in a cis or trans manner. Given the highly inflammatory nature of ATC, expression of HVEM on tumor cells was suggestive of a possible immune regulation in the TME by inflammatory cytokines. To evaluate this regulation, cells were treated with IL8 and TNFα for 24 hrs and HVEM expression was monitored on cell surface and in conditioned media. Interestingly, we observed a reduced surface expression and increased solubilization of HVEM in ATC in presence of these inflammatory cytokines. To explore possible tumor intrinsic signaling transduced by HVEM, we treated the cells with soluble form of its cognate ligand, LIGHT and observed a significant increase in pIκβ, pJNK and p-c-Jun suggestive of increased proliferative potential of these cells triggered by this interaction with LIGHT. In order to assess possible tumor intrinsic role of this molecule, we knocked down HVEM in 8505C by CRISPRi and performed clonogenic assay. HVEM knocked down cells had significantly reduced potential for clonal expansion compared to WT HVEM+ 8505C. Currently we are conducting co-culture experiments to validate its role in immunomodulation and trying to understand its regulation in case of acquired resistance against small molecule inhibitors, like vemurafenib. We believe our study has identified HVEM as an immunotherapeutic target, soluble form of which could be a potential biomarker in ATC patients. Citation Format: Sanjukta Chakraborty, Tara Jarboe, Sina Dadafarin, Jan Geliebter, Augustine Moscatello, Raj K. Tiwari. Implications of HVEM/BTLA/LIGHT signaling in anaplastic thyroid cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2235.