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.
Papillary thyroid cancer (PTC) is the most common cancer in women under 25 years of age and is increasing in incidence. While PTC is often slow-growing and surgically resectable, recurrence years to decades after initial surgery occurs in ~20% of patients (now only in their 40s/50s), with mortality of 38-69%. The identification of both prognostic biomarkers and actionable therapeutic targets for high-risk PTC at diagnosis is critically important and an unmet need. RNASeq analysis of 45 PTC and matched normal patient tissues from our biobank identified collagen 26A1 (COL26A1) as significantly upregulated in patients with higher risk extrathyroidal extension, lymph node metastasis, and multifocal tumors, compared to those with localized disease. Kaplan Meier survival curves of The Cancer Genome Atlas (TCGA) data confirmed that increased COL26A1 expression significantly decreases survival probability by 25% (p=0.0086). Further analyses of TCGA data indicated that COL26A1 expression significantly correlated with clinical attributes including MACIS score (q=0.001), differentiation score (q=0.025), and tumor stage (q=0.025). Data from our in vitro culture PTC model system indicated that COL26A1 expression was increased 3X in PTC cell line K1, compared to immortalized “normal” thyroid epithelial cells, NThy-ori-3-1. Two CRISPR guides designed to target COL26A1 introduced into K1 resulted in repression of COL26A1 RNA and protein levels by 50% and 70%, respectively. COL26A1 repression decreased critical tumor phenotypes of aggressiveness and metastasis including proliferation (30%), clonogenicity (33%), anchorage-independent growth (37%), cell motility (43%), invasion (30%) and migration (33%). Cell-to-cell adhesion and cell-matrix adhesion also decreased (36% and 25%, respectively). Thus, COL26A1 may have use as a prognostic marker and actionable target for small molecule inhibitors in PTC. Citation Format: Michelle Carnazza, Danielle Quaranto, Nicole DeSouza, Sina Dadafarin, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Collagen COL26A1 promotes papillary thyroid cancer invasion and metastasis and correlates with poor survival and clinical outcomes [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 1397.
Papillary thyroid cancer (PTC) accounts for the vast majority of thyroid cancer cases and despite high survival rates, disease recurrence and metastasis remain prominent issues. Therefore, there is a need to identify potential molecular markers associated with PTC that aid in early diagnosis, prognosis, and that may serve as therapeutic targets. One area of focus for cancer study is trending towards the epigenome and its functional regulators. The largest subclass of RNA molecules in the human genome consists of long intergenic non-coding RNAs (lincRNAs) with many of their functions yet to be identified. Evidence supports their roles in the regulation of gene expression, as these molecules can act as scaffolds for RNA and protein localization as well as act as molecular “sponges” for binding to and inhibiting action of specific miRNAs. Recently, long non-coding RNA molecules have been demonstrated to play roles in both the development and progression of cancer, deeming them as both attractive and specific biomarkers and/or therapeutic targets for study. Data from our patient sample biobank identified LINC01614 as being significantly upregulated (~12 fold increase) in PTC, when compared to normal, matched thyroid tissue. There was also ~30 fold increase in LINC01614 expression in male PTC, compared to ~5.5 fold increase in female PTC, highlighting a potential sex correlation for study. Thus, we are studying LINC01614 as a potential PTC biomarker. Functional analysis of LINC01614 in vitro found it to be upregulated in various thyroid cancer cell lines, specifically two PTC cell lines: TPC1 (~5 fold; RET/PTC rearrangement; female) and K1 (~2 fold; BRAFV600E; male) when compared to the immortalized “normal” thyroid cell line (Nthy-ori-3-1). To establish a phenotype for LINC01614 expression, we utilized the CRISPRi technique to knockdown expression of this lincRNA in both TPC1 and K1. Knockdown resulted in decreased healing capacity (~20% for both), clonogenicity (~50% and ~34%, respectively), and proliferation (~55% and ~45%, respectively) in both the TPC1 and K1 PTC cell lines. Studies are underway to further assess migration, invasion, and morphology in both cell lines as well as studies to elucidate potential protein interactions and mechanisms of action of LINC01614 in PTC. Studying lincRNAs and their regulatory roles in cancer cell function can aid in the identification of various molecular markers for therapeutic intervention. Elucidation of how these molecules impact PTC development and progression is a promising avenue of exploration. Citation Format: Danielle Quaranto, Michelle Carnazza, Nicole DeSouza, Sina Dadafarin, Augustine Moscatello, Raj Tiwari, Jan Geliebter. Long non-coding RNA LINC01614 augments papillary thyroid cancer cell phenotype [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 1405.
Papillary thyroid cancer (PTC) accounts for 70-80% of all thyroid cancer cases and has been increasing in incidence over the last few decades. It is now the 8th most common cancer in women in the United States and the most common cancer in women under 25 years of age. PTC is often curable, as the tumors are most often slow-growing and surgically resectable and lymphatic spread does not worsen the generally favorable prognosis. However, there are aggressive forms of PTC that are not adequately treated by current therapeutic modalities and invasion into surrounding tissue next to the thyroid worsens the prognosis. Utilizing the NYMC biobank of PTC patient samples, meta-analysis of RNASeq data was performed, ranking differentially expressed genes between invasive vs noninvasive and lymph node positive vs lymph node negative samples. These genes were analyzed in conjunction with the GDC’s The Cancer Genome Atlas (TCGA) Thyroid Cancer (THCA) study, in which the Kaplan Meier survival curve identified genes with a significant impact on survival and those that were increased in metastatic tissue samples. Of the top differentially expressed genes that were increased in patients with extrathyroidal extension and lymph node metastasis, COL26A1 significantly inversely associated with survival (p = 0.0086). COL26A1 codes for collagen type XXVI α1 chain with known roles in degradation of the ECM and collagen chain trimerization. In TCGA datasets, COL26A1 expression was significantly correlated with clinical attributes including MACIS score (q = .001), differentiation score (q = 0.025), and tumor stage (q = 0.025). COL26A1 expression was increased 3-fold in papillary thyroid cancer cell line K1 when compared to normal thyroid cancer cells, NThy-ori 3-1. Two CRISPR guides designed to target COL26A1 were introduced into K1 resulting in repression of COL26A1 by 50% in both knockdowns. COL26A1 repression decreased the proliferation (33%), tumorigenicity (86%), invasion (33%), and migration capacity (73%). Furthermore, knockdown of COL26A1 leads to decreased cell-to-cell adhesion in culture. Thus, COL26A1 may have potential use as a prognostic marker and actionable therapeutic target for small molecule inhibitors. Long noncoding RNAs (lncRNAs) have vast implications in different cancer types with roles in gene expression via modulation of coding and noncoding RNAs. lncRNAs are tissue- and stage- specific and stable in serum alluding to their ability to be used in thyroid cancer prognosis and diagnosis. Therefore, we investigated the mRNA-miRNA-lncRNA axis involved in regulation of COL26A1 utilizing Qiagen Ingenuity Pathway Analysis (IPA). We have identified potential pathways linking COL26A1 with PTC, highlighting those involved in proliferation, migration, invasion, and adhesion. Investigation into these molecular interactions and pathways can elucidate the role of COL26A1 and allow for its optimal targeting for treatment of invasive papillary thyroid cancer. Citation Format: Michelle Carnazza, Danielle Quaranto, Nicole DeSouza, Sina Dadafarin, Augustine Moscatello, Raj K. Tiwari, Jan Geliebter. Role of COL26A1 in papillary thyroid cancer invasion [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 767.
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.
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