Target In Anaplastic Thyroid Cancer Research Articles

Abstract 4633: Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is always diagnosed as stage IV with a 5 year survival rate of less than 3% due to its aggressive metastatic phenotype. ATC lacks differentiation and loses its thyroid-like functions making it a difficult cancer to treat due to its lack of targetable genetic lesions and unresponsiveness to conventional treatments. Therefore, we investigated genes that had aberrant expression in ATC in relation to differentiation. Using NCBI Gene expression omnibus (GSE33630, GSE85457), which provides publicly available transcriptomic datasets, and bioinformatics software, GEO2R, we found that Homeobox D4 (HOXD4) has 12.5FC higher expression in ATC vs. normal tissue (p=0.000552) and 4.36FC higher in ATC vs. papillary thyroid cancer (PTC) in human patients (p=6.69e-08). HOXD4 is a transcription factor that plays a crucial role in cell differentiation and has been associated with a worse prognosis in cancers such as ovarian serous carcinoma. As loss of differentiation is essential to ATC’s refractory nature and therapeutic resistance, we are exploring modulation of differentiation by all-trans retinoic acid (ATRA). ATRA was used to induce differentiation in ATC cell line T238 therefore abrogating the aggressive phenotype of this cancer. T238 treated with ATRA had a 26% decrease in proliferation at 24 hours, 46% at 48 hours, and 87% at 72 hours measured using a trypan blue exclusion assay. T238 treated with ATRA also had a 44% decrease in migratory capacity at 24 hours and 24% at 48 hours measured by a cell scratch wound assay. Immortalized normal thyroid cell line NTHY-ori-3-1 did not have a significant decrease in either proliferation or migratory capacity when treated with ATRA. T238 cells treated with ATRA for 24 hours had a 30% decrease in HOXD4 protein levels. Lastly, immunohistochemistry staining of HOXD4 in human patient tissue samples confirmed the over-expression of HOXD4 in ATC (19.5% staining) vs. normal tissue (2.5%) and PTC (7.5%). Overall, we hypothesize that the aberrant expression of HOXD4 in ATC plays a crucial role in the aggressive metastatic phenotype and therefore differentiation agents such as ATRA can target this biomarker to modulate the ATC phenotype. Therefore, HOXD4 can be used as a potential prognostic indicator for early detection of ATC. Future studies will involve CRISPRi based knockdown of HOXD4 in vitro and validation of these results in animal in vivo models. Citation Format: Kaci Kopec, Tara Jarboe, Nicole R. DeSouza, Humayun K. Islam, Jan Geliebter, Raj K. Tiwari. Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer [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 4633.

CSPG4 Is a Potential Therapeutic Target in Anaplastic Thyroid Cancer

CSPG4 Is a Potential Therapeutic Target in Anaplastic Thyroid Cancer

CSPG4 Is a Potential Therapeutic Target in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is a rare cancer with poor prognosis and few treatment options. The objective of this study was to investigate new immune-associated therapeutic targets by identifying ATC-derived, human leukocyte antigen (HLA) class II-presenting peptides. One protein that generated multiple peptides in ATC was chondroitin sulfate-proteoglycan-4 (CSPG4), a transmembrane proteoglycan with increased expression in multiple aggressive cancers, but not yet investigated in ATC. Methods: We applied autologous peripheral blood T cells to ATC patient-derived xenografted mice to examine whether ATC induces a tumor-specific T cell response. We then identified peptide antigens eluted from the HLA-DQ complex in ATC patient-derived cells using mass spectrometry, detecting abundant CSPG4-derived peptides specific to the ATC sample. Next, we analyzed the surface expression level of CSPG4 in thyroid cancer cell lines and primary cell culture using flow cytometry. In addition, we used immunohistochemistry to compare the expression level and localization of the CSPG4 protein in ATC, papillary thyroid cancer, and normal thyroid tissue. We then investigated the correlation between CSPG4 expression and clinicopathological features of patients with thyroid cancer. Results: We found that ATC tissue had a high level of HLA-DQ expression and that the patient's CD4+ T cells showed activation when exposed to ATC. By eluting the HLA-DQ complex of ATC tissue, we found that CSPG4 generated one of the most abundant and specific peptides. CSPG4 expression at the cell surface of thyroid cancer was also significantly high when determined by flow cytometry, with the majority of ATC cell lines exhibiting ∼10-fold higher mean fluorescence intensity. Furthermore, most ATC patient cases expressed CSPG4 in the cytoplasm or membrane of the tumor cells. CSPG4 expression was correlated with tumor size, extrathyroidal extension, and intercellular adhesion molecule-1 (ICAM-1) circumferential expression. CSPG4 mRNA overexpression was associated with worse overall survival in patients with ATC and poorly differentiated thyroid cancer. Conclusions: CSPG4 expression is significantly elevated in aggressive thyroid cancers, with a strong correlation with a poor prognosis. The vast number of HLA-DQ eluted CSPG4 peptides was identified in ATC, demonstrating the potential of CSPG4 as a novel immunotherapeutic target for ATC.

Dsg2-mediated c-Met activation in anaplastic thyroid cancer motility and invasion.

Anaplastic thyroid cancer (ATC) is a rapidly growing, highly metastatic cancer with limited therapeutic alternatives, thus targeted therapies need to be developed. This study aimed to examine desmoglein 2 (Dsg2) expression in ATC and its biological role and potential as a therapeutic target in ATC. Consequently, Dsg2 was downregulated or aberrantly expressed in ATC tissues. ATC patients with low Dsg2 expression levels also presented with distant metastasis. Dsg2 depletion significantly increased cell migration and invasion, with a relatively limited effect on ATC cell proliferation in vitro and increased distant metastasis in vivo. Dsg2 knockdown induced cell motility through the hepatocyte growth factor receptor (HGFR, c-Met)/Src/Rac1 signaling axis, with no alterations in the expression of EMT-related molecules. Further, specific targeting of c-Met significantly inhibited the motility of shDsg2-depleted ATC cells. Decreased membrane Dsg2 expression increased the metastatic potential of ATC cells. These results indicate that Dsg2 plays an important role in ATC cell migration and invasiveness. Therapies targeting c-Met might be effective among ATC patients with low membrane Dsg2 expression levels, indicating that the analysis of Dsg2 expression potentially provides novel insights into treatment strategies for ATC.

Novel role of ASH1L histone methyltransferase in anaplastic thyroid carcinoma

Anaplastic thyroid cancer (ATC) is one of the most aggressive human malignancies, with an average life expectancy of ∼6 months from the time of diagnosis. The genetic and epigenetic changes that underlie this malignancy are incompletely understood. We found that ASH1-like histone lysine methyltransferase (ASH1L) is overexpressed in ATC relative to the much less aggressive and more common differentiated thyroid cancer. This increased expression was due at least in part to reduced levels of microRNA-200b-3p (miR-200b-3p), which represses ASH1L expression, in ATC. Genetic knockout of ASH1L protein expression in ATC cell lines decreased cell growth both in culture and in mouse xenografts. RNA-Seq analysis of ASH1L knockout versus WT ATC cell lines revealed that ASH1L is involved in the regulation of numerous cancer-related genes and gene sets. The pro-oncogenic long noncoding RNA colon cancer-associated transcript 1 (CCAT1) was one of the most highly (approximately 68-fold) down-regulated transcripts in ASH1L knockout cells. Therefore, we investigated CCAT1 as a potential mediator of the growth-inducing activity of ASH1L. Supporting this hypothesis, CCAT1 knockdown in ATC cells decreased their growth rate, and ChIP-Seq data indicated that CCAT1 is likely a direct target of ASH1L's histone methyltransferase activity. These results indicate that ASH1L contributes to the aggressiveness of ATC and suggest that ASH1L, along with its upstream regulator miR-200b-3p and its downstream mediator CCAT1, represents a potential therapeutic target in ATC.

Steroid receptor coactivator-3 as a target for anaplastic thyroid cancer.

Anaplastic thyroid carcinoma (ATC) is an aggressive malignancy without effective therapeutic options to improve survival. Steroid receptor coactivator-3 (SRC-3) is a transcriptional coactivator whose amplification and/or overexpression has been identified in many cancers. In this study, we explored the expression of SRC-3 in ATCs and the effects of a new class of SRC-3 inhibitor-2 (SI-2) in human ATC cells (THJ-11T and THJ-16T cells) and mouse xenograft models to assess therapeutic potential of SI-2 for the treatment of ATC. SRC-3 protein abundance was significantly higher in human ATC tissue samples and ATC cells than in differentiated thyroid carcinomas or normal controls. SI-2 treatment effectively reduced the SRC-3 expression in both ATC cells and ATC xenograft tumors induced by these cells. Cancer cell survival in ATC cells and tumor growth in xenograft tumors were significantly reduced by SI-2 treatment through induction of cancer cell apoptosis and cell cycle arrest. SI-2 also reduced cancer stem-like cells as shown by an inhibition of tumorsphere formation, ALDH activity, and expression of stem cell markers in ATC. These findings indicate that SRC-3 is a potential therapeutic target for treatment of ATC patients and that SI-2 is a potent and promising candidate for a new therapeutic agent.

Abstract 2137: JAK2 as a novel therapeutic target in anaplastic thyroid cancer

Abstract Introduction: Thyroid carcinoma is the most common endocrine malignancy. Anaplastic thyroid cancer (ATC) is rare (1.3%) and represents arguably the most lethal human malignancy with 1-year survival rates of only 10%. There are currently no effective treatments for the majority of patients, highlighting an urgent need for novel therapeutics to manage this disease. Objective: To validate the functional importance of JAK2 as a therapeutic target in ATC. Methods: In this study, we have used siRNA knockdown to interrogate the JAK2 signaling pathway as a therapeutic target in ATC. We investigated the mechanism of action and cell death, and assayed for migration and invasion, in vitro. The chick chorioallantoic membrane (CAM) model was also utilized for drug testing, whereby 1x106 Cal62 ATC cells were on-planted to the chick embryo membrane. Two days post on-plant, CAM models were treated with the vehicle (DMSO) or lestaurtinib to measure outcomes including tumor volume and vascularity. Results: We identified the JAK2 inhibitor lestaurtinib as an inhibitory agent controlling cell line proliferation at submicromolar mean inhibitory concentrations. Immunoblotting revealed the inhibition of phosphorylation of the downstream signaling molecule STAT5 in a dose-dependent manner. Treatment of Cal62 cells resulted in a decrease in cell migration using the scratch-wound assay. The anti-proliferative effective of lestaurtinib did not cause apoptosis, autophagy or cell senescence. CAM models treated with a 4 uM dose of lestaurtinib showed a significant decrease in both tumor volume and vascularity. Conclusions: Lestaurtinib was found to provide potent control of ATC cell proliferation and migration, and was also found to decrease tumor growth and vascularity in a CAM model. Knockout studies are underway to confirm that the anticancer effect of this drug is indeed mediated through JAK2 signaling. If validated, JAK2 represents a novel therapeutic target for the treatment of aggressive thyroid cancers. Citation Format: Nicole C. Pinto, Kara Ruicci, Stephenie Prokopec, Karlee Searle, Matthew Lowerison, John Yoo, Kevin Fung, Danielle MacNeil, Hon S. Leong, Alessandro Datti, Paul C. Boutros, John W. Barrett, Anthony C. Nichols. JAK2 as a novel therapeutic target in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2137. doi:10.1158/1538-7445.AM2017-2137

Phase II trial of ribociclib and everolimus in p16 low anaplastic thyroid cancer (ATC).

TPS6098 Background: ATC is a rare thyroid cancer with a highly aggressive clinical course. Median OS is 3-4 months and 90% of patients die within 1 year of the diagnosis. There are no effective treatment options in metastatic disease. Targeted therapies to ALK and BRAF are occasionally associated with dramatic responses. Retinoblastoma (Rb) inhibits cell cycle progression and is inactivated by CDK 4/6, which is the target for ribociclib. Nearly all differentiated thyroid cancers are Rb negative, conversely nearly 100% of ATC expresses intact Rb which may be crucial for rapid cell cycle progression. Ribociclib is a CDK 4/6 inhibitors that slows cell cycle progression and DNA replication in tumors with functional Rb. The p16 protein similarly inhibits CDK4/6; if p16 levels are high Rb is phosphorylated and thus inactive. p16 is low in ATC, and from our comprehensive genomic analysis 30% of ATC lacks the p16 gene CDKN2a. Most ATC demonstrates PI3K/Akt/mTOR abnormalities, this pathway represents an attractive target in ATC. The mTOR inhibitor everolimus has shown promising efficacy in ATC cell lines, especially in those with TSC2 mutation whose wild type negatively regulates mTOR. Methods: The combination of everolimus and ribociclib targets mutations/abnormalities that are frequently seen in ATC, and is tolerable based on Phase I/II trial results in the latest arm of the biomarker/oncogene driven ATC Master Protocol . This open-label trial treats metastatic Rb+ ATC patients with p16-/ CDKN2a-. Treatment is ribociclib 400 mg + everolimus 5 mg QD. The primary endpoint is the overall response rate; secondary endpoints are PFS, OS, safety and toxicity. Exploratory objectives include if tissue biomarkers or mutations noted on Next Generation Sequencing correlate for enhanced/impaired response to combination therapy. Simon's two-stage design will be used with the null hypothesis that the true response rate is 5%, this will be tested against a one-sided alternative. In the first stage, 9 patients will be accrued. If no response in these 9 patients, the study will be stopped. Otherwise, 21 additional patients will be accrued for a total of 30. Clinical trial information: NCT02289144.

Integrin-linked kinase is a potential therapeutic target for anaplastic thyroid cancer

We investigated integrin-linked kinase (ILK), a focal adhesion serine-threonine protein kinase, as a new molecular target for treating anaplastic thyroid cancer. ILK mediates cell growth and survival signals and is overexpressed in a number of cancers. Therefore, we hypothesized that inhibition of ILK leads to growth arrest and apoptosis of thyroid cancer cells. According to Western blotting, the level of ILK protein was highly expressed in one papillary (NPA187) and four of five (Hth74, DRO, ARO, KAT4, and K4) anaplastic thyroid cancer cell lines. Immunohistochemical analysis of a human tissue microarray revealed that ILK was highly expressed in anaplastic thyroid cancer but not in normal human thyroid tissue. Treating thyroid cancer cell lines with a new ILK inhibitor, QLT0267, inhibited epidermal growth factor-induced phosphorylation of AKT, inhibited cell growth, and induced apoptosis in the NPA187, DRO, and K4 cell lines. QLT0267 also inhibited the kinase activity of immunoprecipitated ILK in four of five cell lines. Tumor volumes in mice treated with QLT0267 were significantly reduced compared with those in untreated mice. In immunohistochemical studies, QLT0267 suppressed phosphorylated p-AKT and angiogenesis (i.e., reduced mean vascular density) and induced apoptosis in both tumor cells and tumor-associated endothelial cells of the thyroid DRO xenografts. In summary, we found that ILK expression and activity were elevated in human anaplastic thyroid cancer and ILK inhibition led to growth arrest and apoptosis in vitro and in vivo. Our results provide preliminary evidence that ILK is a potential therapeutic target for treating anaplastic thyroid cancer.