Novel Therapeutic Target Identified in Aggressive Thyroid Cancers

In vivo models of anaplastic thyroid cancer (ATC) and poorly differentiated thyroid cancer (PDTC) show that downregulation of miR-30a-5p leads to overexpression of lysyl oxidase (LOX), a target which appears amenable to treatment with β-aminopropionitrile fumarate (BAPN). These findings suggest that a new therapeutic option could be on the horizon for patients with these aggressive forms of thyroid cancer.

"ATC, the most aggressive and lethal form of thyroid cancer, is resistant to current therapies. ATC commonly metastasizes to the lungs, bone, and brain, and patients with ATC typically have a median survival time of 1 year or less. Novel therapeutic approaches are needed to treat ATC and improve patient outcomes," said Myriem Boufraqech, PhD, of the Endocrine Oncology Branch of the National Cancer institute/National Institutes of Health in Bethesda, Maryland, at the 2014 ATA Annual Meeting.

This work sought to further elucidate the role of microRNAs (miRNA) and LOX expression in thyroid cancer progression. LOX initiates the process of collagen and elastin crosslinking, functions in remodeling and stiffening the fibrous deposits of proteins in the extracellular matrix of tumors, and, as such, is thought to play a key role in promoting tumor motility, migration, and rigidity, particularly in a hypoxic environment of a solid tumor.

To identify miRNA(s) and pathways involved in ATC, human thyroid cancer tissue samples were assessed using microRNA microarray and real-time polymerase chain reaction (RT-PCR). MiR-30a-5p was overexpressed in ATC cell lines, and luciferase assays were used to validate LOX as a direct target of miR-30a-5p. An in vivo metastatic ATC mouse model was then used to evaluate the role of miR-30a-5p and LOX in ATC progression.

Profiling of miRNA expression revealed that miR-30a-5p is significantly downregulated in both ATC and PDTC. Furthermore, 454 primary thyroid cancer samples from the Cancer Genome Atlas dataset showed that upregulation of LOX and downregulation of miR-30a were associated with more aggressive thyroid cancer and a higher mortality rate, suggesting that LOX and miR-30a may be useful as prognostic markers for aggressive thyroid cancer.

In the in vitro setting, ectopic expression of miR-30a-5p reduced the invasion and migration of ATC cells and downregulated the expression of epithelial mesenchymal transition (EMT) markers and LOX, which is a predicted target of miR-30a-5p.

Using an in vivo mouse model of ATC metastases, miR-30a-5p significantly reduced lung metastases with decreased vimentin, CD44, and LOX expression in lung metastases and tumor tissue. Additionally, both LOX and its downstream target TWIST-1 are highly expressed in ATC. In both ATC and PDTC samples, LOX overexpression was significantly inversely correlated with miR-30a-5p expression.

To demonstrate that miR-30a-5p directly targets LOX, the 3'-UTR region of LOX was cloned into a luciferase reporter plasmid and transfected along with miR-30a-5p into ATC cell lines. Compared with negative control miRNA, co-transfection of 3'UTR-LOX with miR-30a led to a decrease in luciferase activity, which demonstrates that LOX is, in fact, a direct target of miR-30a-5p.

Furthermore, direct knockdown of LOX not only inhibited cell proliferation, but also increased cellular apoptosis and decreased the invasion and migration of ATC cells. In the in vitro setting, treatment of four ATC cell lines with the irreversible LOX inhibitor BAPN did not affect cell proliferation but significantly inhibited cell invasion and migration and reduced EMT markers and CD44 expression. In vivo, mice with metastatic human ATC tumors that were treated with BAPN exhibited significantly fewer tumors, and lung metastases were smaller in size.

In general, miR-30a-5p suppresses migration, invasion, and in vivo metastases in ATC. Its role in thyroid cancer correlates with other work that suggests that LOX may play a role in other solid tumors, such as those in cervical and breast cancers, by promoting metastasis.

"Together, our data support an important role of the miR-30a-5p-LOX axis in thyroid cancer progression. The current development of agents targeting LOX could be useful for ATC and PDTC cancer therapy," Boufraqech concluded.

Boufraqech M, nilubol N, Zhang L, et al. Tumor suppressor miR-30a-5p inhibits LOX expression and cancer progression in thyroid cancer. Presented at: the Annual Meeting of the American Thyroid Association; October 29 — November 2, 2014; Coronado, CA. Abstract 121.

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