Remodeling Anaplastic Thyroid Cancer\u27s Aggressive Profile and Metabolic Signature by Natural Alkaloid Berberine

Anaplastic thyroid cancer is a rare, fatal cancer with a five-year survival of 4%. Universally diagnosed at stage IV, anaplastic thyroid cancer is characterized by its lack of differentiation, rapid proliferative rate, highly inflammatory tumor microenvironment, and metabolic dysregulation. Refractory to all established therapies, anaplastic thyroid cancer requires a novel therapeutic approach that targets all of these drivers of anaplastic thyroid cancer carcinogenesis. We propose natural alkaloid berberine as a therapeutic with multitarget efficacy to alter mitochondrial metabolism and reprogram anaplastic thyroid cancer’s aggressive phenotype. Our in vitro model uses monocyte cell line U937, anaplastic thyroid cancer cell lines T238 and SW1736, and immortalized normal thyroid cell line Nthy-ori-3-1. Validation of in vitro findings via RNA Sequencing was conducted by Genewiz from Azenta and Qiagen’s Ingenuity Pathway Analysis was used for in silico modeling. In targeting the aggressiveness of anaplastic thyroid disease, berberine selectively slowed proliferation by 80% in anaplastic thyroid cancer cells from 48 to 72 hours while sparing normal cells. Berberine reduced migratory capacity by 33% in T238 cells and 51% in SW1736 cells after 24 hours. Berberine reduced both migration and invasion by 30% in T238. These observations were substantiated by Western blot analysis – berberine selectively decreased phosphorylation of MEK, ERK, and ribosomal protein S6, crucial downstream regulators of the pro-proliferative and pro-survival pathways in anaplastic thyroid cancer cells. Further, berberine specifically modulated cancer-associated metabolism as observed through an increase in AMPKα phosphorylation, a major rate-limiting protein in cancer-induced dysregulation with an anti-tumor effect. Modeling the anaplastic thyroid cancer tumor microenvironment, U937 cells were activated and polarized into a proinflammatory macrophage phenotype. Following berberine treatment at the activation/polarization stages, 19 soluble inflammatory mediators were significantly downregulated in the conditioned media compared to controls. U937 cells polarized using anaplastic thyroid cancer-conditioned media pre-treated with berberine also showed decreased IFN-γ and TNF-α secretion. Validation of in vitro findings via RNA Sequencing revealed more than 400 significantly differentially expressed genes involved in mitochondrial metabolism, glycometabolism, sirtuin signaling, apoptosis, and proliferation. Following a comprehensive analysis, we identified significant downregulation of 22 of 37 total mitochondrially encoded genes and 13 of 13 mitochondrially encoded protein-coding genes comprising the oxidative phosphorylation complexes, illuminating a clear link between berberine treatment and altered mitochondrial metabolism in anaplastic thyroid cancer. Additionally, protein expression of significantly downregulated mitochondrial genes identified via RNA Sequencing was validated via Western blot, demonstrating decreased mitochondrially-encoded protein expression related to oxidative phosphorylation. This work reveals a novel role for berberine as an inhibitor of mitochondrial metabolism that can be used to reprogram the aggressive nature of anaplastic thyroid cancer and open the door for promising combination therapy in treating fatal anaplastic thyroid cancer

Remodeling Anaplastic Thyroid Cancer’s Aggressive Profile and Metabolic Signature by Natural Alkaloid Berberine

Anaplastic thyroid cancer is a rare, fatal cancer with a five-year survival of 4%. Universally diagnosed at stage IV, anaplastic thyroid cancer is characterized by its lack of differentiation, rapid proliferative rate, highly inflammatory tumor microenvironment, and metabolic dysregulation. Refractory to all established therapies, anaplastic thyroid cancer requires a novel therapeutic approach that targets all of these drivers of anaplastic thyroid cancer carcinogenesis. We propose natural alkaloid berberine as a therapeutic with multitarget efficacy to alter mitochondrial metabolism and reprogram anaplastic thyroid cancer’s aggressive phenotype. Our in vitro model uses monocyte cell line U937, anaplastic thyroid cancer cell lines T238 and SW1736, and immortalized normal thyroid cell line Nthy-ori-3-1. Validation of in vitro findings via RNA Sequencing was conducted by Genewiz from Azenta and Qiagen’s Ingenuity Pathway Analysis was used for in silico modeling. In targeting the aggressiveness of anaplastic thyroid disease, berberine selectively slowed proliferation by 80% in anaplastic thyroid cancer cells from 48 to 72 hours while sparing normal cells. Berberine reduced migratory capacity by 33% in T238 cells and 51% in SW1736 cells after 24 hours. Berberine reduced both migration and invasion by 30% in T238. These observations were substantiated by Western blot analysis – berberine selectively decreased phosphorylation of MEK, ERK, and ribosomal protein S6, crucial downstream regulators of the pro-proliferative and pro-survival pathways in anaplastic thyroid cancer cells. Further, berberine specifically modulated cancer-associated metabolism as observed through an increase in AMPKα phosphorylation, a major rate-limiting protein in cancer-induced dysregulation with an anti-tumor effect. Modeling the anaplastic thyroid cancer tumor microenvironment, U937 cells were activated and polarized into a proinflammatory macrophage phenotype. Following berberine treatment at the activation/polarization stages, 19 soluble inflammatory mediators were significantly downregulated in the conditioned media compared to controls. U937 cells polarized using anaplastic thyroid cancer-conditioned media pre-treated with berberine also showed decreased IFN-γ and TNF-α secretion. Validation of in vitro findings via RNA Sequencing revealed more than 400 significantly differentially expressed genes involved in mitochondrial metabolism, glycometabolism, sirtuin signaling, apoptosis, and proliferation. Following a comprehensive analysis, we identified significant downregulation of 22 of 37 total mitochondrially encoded genes and 13 of 13 mitochondrially encoded protein-coding genes comprising the oxidative phosphorylation complexes, illuminating a clear link between berberine treatment and altered mitochondrial metabolism in anaplastic thyroid cancer. Additionally, protein expression of significantly downregulated mitochondrial genes identified via RNA Sequencing was validated via Western blot, demonstrating decreased mitochondrially-encoded protein expression related to oxidative phosphorylation. This work reveals a novel role for berberine as an inhibitor of mitochondrial metabolism that can be used to reprogram the aggressive nature of anaplastic thyroid cancer and open the door for promising combination therapy in treating fatal anaplastic thyroid cancer

Profiling of Traditional Chinese Medicine Compound Berberine as Anticancer Agent in Melanoma

Over the last three decades, melanoma has been diagnosed with an increased incidence. Melanoma is a cancer that arises from a wealth of mutations, including a BRAFV600E genetic lesion present in 50-60% of melanoma cases. The accumulation of mutations in melanoma makes it difficult to treat by conventional therapies and allows it to easily confer resistance to therapeutics. Despite the rise of novel immunotherapies that target melanoma, including small molecule inhibitors and checkpoint inhibitors, high side effects, low response rates, and acquired resistance have been observed. This bolsters the necessity of identifying alternative, supportive therapeutic strategies for potential combination therapies. Traditional Chinese Medicine (TCM) and Ayurveda, ancient systems of holistic medicine, have used formulations of compounds derived from plants and natural sources as treatment agents for thousands of years. Amongst these compounds are those that possess anti-inflammatory and anti-cancer properties. One such compound, Berberine (BBR), is a plant alkaloid isolated from Hydrastis canadensis (goldenseal), Coptis chinensis (goldenthread), Berberis aquifolium (Oregon grape), Berberis vulgaris (barberry), and Berberis aristate. Traditionally, it has been widely used for its antimicrobial and antiprotozoal properties. Current pre-clinical and clinical research on the impact of BBR in heart disease, diabetes, neurological disorders, aging, allergies, and many types of cancer is underway. We studied the effect of BBR on the proliferative abilities of three melanoma cells lines with different BRAF status: SKMEL28 (BRAFV600E+), SKMEL37 (BRAFV600E+/-), and SKMEL103 (BRAF wildtype). IC50 for cells was determined by XTT assay, which showed SKMEL103 has the lowest IC50 of 2.5 μL, making it the most susceptible to low concentration BBR treatment. Following this, cells were treated with their individual IC50 concentration (20-25 μL for BRAFV600E+ and 2.5 μL for BRAF wildtype) and effects on key cellular signaling pathways, including MAPK and PI3K/AKT/mTOR were studied by western blots. BBR treatment differentially downregulated phospho-MEK and phospho-ERK 1/2 dependent on presence of the BRAF genetic lesion. BBR treatment also downregulated expression of mTOR, which could consequentially result in regaining control of apoptosis and survival in cancer cells. BBR also arrested cell cycle progression in the G1 phase of the cell cycle after 72 hours of treatment in SKMEL37, making it a promising supportive therapeutic modality that may be used to supplement immunotherapies and small molecule inhibitors

Interactome of Long Non-Coding RNAs: Transcriptomic Expression Patterns and Shaping Cancer Cell Phenotypes

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

Berberine-Mediated Reprogramming of the Inflammatory Environment in Anaplastic Thyroid Cancer

Anaplastic thyroid cancer\u27s (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\u27s 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

Long Non-Coding RNAs as Determinants of Thyroid Cancer Phenotypes: Investigating Differential Gene Expression Patterns and Novel Biomarker Discovery

Thyroid Cancer (TC) is the most common endocrine malignancy, with increasing incidence globally. Papillary thyroid cancer (PTC), a differentiated form of TC, accounts for approximately 90% of TC and occurs predominantly in women of childbearing age. Although responsive to current treatments, recurrence of PTC by middle age is common and is much more refractive to treatment. Undifferentiated TC, particularly anaplastic thyroid cancer (ATC), is the most aggressive TC subtype, characterized by it being resistant and unresponsive to all therapeutic and surgical interventions. Further, ATC is one of the most aggressive and lethal malignancies across all cancer types. Despite the differences in therapeutic needs in differentiated vs. undifferentiated TC subtypes, there is a critical unmet need for the identification of molecular biomarkers that can aid in early diagnosis, prognosis, and actionable therapeutic targets for intervention. Advances in the field of cancer genomics have enabled for the elucidation of differential gene expression patterns between tumors and healthy tissue. A novel category of molecules, known as non-coding RNAs, can themselves be differentially expressed, and extensively contribute to the up- and downregulation of protein coding genes, serving as master orchestrators of regulated and dysregulated gene expression patterns. These non-coding RNAs have been identified for their roles in driving carcinogenic patterns at various stages of tumor development and have become attractive targets for study. The identification of specific genes that are differentially expressed can give insight into mechanisms that drive carcinogenic patterns, filling the gaps of deciphering molecular and cellular processes that modulate TC subtypes, outside of well-known driver mutations

Novel Immune Targets in Melanoma by Profiling of Co-Activators and Co-Receptors

Melanoma, the most aggressive form of skin cancer, is still a challenge despite the advent of recent immunotherapies using checkpoint inhibitors. The presence of checkpoint molecules on immune cells serve to regulate optimal immune responses, however, when present on tumor cells, they serve as immune evasion molecules. As such, this presents challenges to the specific inhibitors being used in relation to dose, side effects, and specificity. We hypothesize that tumor cell profiling of co-activators and co-inhibitors will serve as an important step in the identification and use of specific checkpoint inhibitors in personalized medicine. To this end, we used five patient-derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM, and GLM-2 as a model and screened them for the expression of a comprehensive list of twenty-nine co-stimulatory and co-inhibitory molecules. We compared the differentially expressed molecules on tumor cells with the expression of the co-stimulatory and co-inhibitors on normal non-transformed adult melanocytes. We see a differential mRNA expression of many of these immune-regulatory molecules, including BTLA, HVEM, CD160, CD226 and TIM1. Western blots and immunofluorescence confirmed the presence of these molecules at the protein level. A flow cytometry analysis demonstrated that BTLA, HVEM, CD160, TIM1 and CD226 are present on the membrane of these patient derived melanoma cells; implying that they are capable of engaging their respective ligands and exerting a functional role in immunomodulation. The expression of these molecules by immunohistochemistry in patient melanoma issues provided additional validation. Analysis of protein expression in various cancers from the Human protein atlas was conducted so as to evaluate the expression of these immune regulators across cancers. HVEM expression in melanoma was high. Interestingly, treatment of MEL-2, MEL-V, KFM and GLM-2, our BRAFV600E containing patient-derived cells, with BRAFV600E inhibitor PLX4032 led to the upregulation of these molecules. Since BRAFV600E is the most common mutation, and some response to small molecule inhibitors has been clinically verified, we undertook an analysis of the expression of immune regulators with overall survival using the cancer genome atlas. We also evaluated the interferon regulatory factor binding site so as to molecularly validate that expression of these molecules is dependent on secreted cytokine in the tumor microenvironment. We conclude that the BTLA-HVEM axis is a significant novel target in melanoma that can be used in combinatorial therapy with small molecule inhibitors of cell survival. Our results advocate the need for profiling of immune modulators on tumor cells prior to immunotherapy

Disruption of Cell-Cell Communication in Anaplastic Thyroid Cancer as an Immunotherapeutic Opportunity

Thyroid cancer incidence is increasing at an alarming rate, almost tripling every decade. About 44,280 new cases of thyroid cancer (12,150 in men and 32,130 in women) are estimated to be diagnosed in 2021, with an estimated death toll of around 2200. Although most thyroid tumors are treatable and associated with a favorable outcome, anaplastic thyroid cancer (ATC) is extremely aggressive with a grim prognosis of 6-9 months post-diagnosis. A large contributing factor to this aggressive nature is that ATC is completely refractory to mainstream therapies. Analysis of the tumor microenvironment (TME) associated with ATC can relay insight to the pathological realm that encompasses tumors and aids in cancer progression and proliferation. The TME is defined as a complex niche that surrounds a tumor and involves a plethora of cellular components whose secretions can modulate the environment in order to favor tumor progression. The cellular heterogeneity of the TME contributes to its dynamic function due to the presence of both immune and nonimmune resident, infiltrating, and interacting cell types. Associated immune cells discussed in this chapter include macrophages, dendritic cells (DCs), natural killer (NK) cells, and tumor-infiltrating lymphocytes (TILs). Nonimmune cells also play a role in the establishment and proliferation of the TME, including neuroendocrine (NE) cells, adipocytes, endothelial cells (ECs), mesenchymal stem cells (MSCs), and fibroblasts. The dynamic nature of the TME contributes greatly to cancer progression.Recent work has found ATC tissues to be defined by a T cell-inflamed hot tumor immune microenvironment (TIME) as evidenced by presence of CD3+ and CD8+ T cells. These tumor types are amenable to immune checkpoint blockade (ICB) therapy. This therapeutic avenue, as of 2021, has remained unexplored in ATC. New studies should seek to explore the therapeutic feasibility of a combination therapy, through the use of a small molecule inhibitor with ICB in ATC. Screening of in vitro model systems representative of papillary, anaplastic, and follicular thyroid cancer explored the expression of 29 immune checkpoint molecules. There are higher expressions of HVEM, BTLA, and CD160 in ATC cell lines when compared to the other TC subtypes. The expression level of HVEM was more than 30-fold higher in ATC compared to the others, on average. HVEM is a member of tumor necrosis factor (TNF) receptor superfamily, which acts as a bidirectional switch through interaction with BTLA, CD160, and LIGHT, in a cis or trans manner. Given the T cell-inflamed hot TIME in ATC, expression of HVEM on tumor cells was suggestive of a possibility for complex crosstalk of HVEM with inflammatory cytokines. Altogether, there is emerging evidence of a T cell-inflamed TIME in ATC along with the expression of immune checkpoint proteins HVEM, BTLA, and CD160 in ATC. This can open doors for combination therapies using small molecule inhibitors targeting downstream effectors of MAPK pathway and antagonistic antibodies targeting the HVEM/BTLA axis as a potentially viable therapeutic avenue for ATC patients. With this being stated, the development of adaptive resistance to targeted therapies is inevitable; therefore, using a combination therapy that targets the TIME can serve as a preemptive tactic against the characteristic therapeutic resistance that is seen in ATC. The dynamic nature of the TME, including the immune cells, nonimmune cells, and acellular components, can serve as viable targets for combination therapy in ATC. Understanding the complex interactions of these associated cells and the paradigm in which their secretions and components can serve as immunomodulators are critical points of understanding when trying to develop therapeutics specifically tailored for the anaplastic thyroid carcinoma microenvironment