The RCAN1.4 Metastasis Suppressor Is Hypermethylated at Intron 1 in Thyroid Cancer.

Background: Regulator of calcineurin 1.4 (RCAN1.4) is a functionally downregulated metastasis progression suppressor (MPS) in thyroid cancer; however, the mechanisms for RCAN1.4 loss in thyroid cancer have not yet been reported. The RCAN1.4 promoter and gene contain several cytosine-guanine (CG)-rich regions, some of which are reported to be hypermethylated in nonthyroid tissues. We, therefore, hypothesized that RCAN1.4 downregulation in thyroid cancer was in part due to hypermethylation. Methods: Studies were performed in 5 thyroid cancer cell lines (TPC1, FTC133, BCPAP, C643, and 8505C) with different genetic drivers, and in 18 paired normal and thyroid cancer human thyroid cancer tissues. Basal RCAN1.4 messenger RNA (mRNA) and protein levels were assessed in all of the cell lines. Cell lines with lowest RCAN1.4 expression levels were treated with the DNA methyl transferase inhibitor, decitabine. Normal/tumor tissue pairs were analyzed for methylation of three CG-rich regions both by capture of methylated DNA by MBD2 protein and by methylation-specific polymerase chain reaction (MSPCR). Results: In all assessed cell lines, RCAN1.4 mRNA and protein levels increased after decitabine treatment. In silico analysis of the RCAN1.4 gene identified 3 CG-rich regions as possible methylation targets: 1 in the proximal promoter and 2 in intron 1. Hypermethylation of the intron 1 CG-rich regions was identified by both the capture method and MSPCR. In contrast, hypermethylation of the CG-rich region of the proximal promoter was not identified. Gene expression confirmed that hypermethylation in thyroid cancer samples in intron 1 of RCAN1.4 was associated with lower levels of RCAN1.4 mRNA. Finally, the cancer samples demonstrated increased NFE2L3 expression, a downstream marker of functional RCAN1.4 loss. Conclusions: The MPS gene, RCAN1.4, is downregulated in thyroid cancer cells and human thyroid cancer in part by hypermethylation of CG-rich regions in intron 1.

Adrenal Near-Infrared Autofluorescence.

Context: Parathyroid tissue is one of the few tissues to have strong near-infrared (NIR) autofluorescence, which has been exploited to improve intraoperative parathyroid identification. The US Food and Drug Administration has approved 2 devices for this purpose. Adrenal glands can be difficult to distinguish from surrounding fat, an issue during total adrenalectomy. Objective: We hypothesized adrenal tissue may also possess considerable NIR autofluorescence. Methods: Resected patient adrenal specimens were examined after robotic adrenalectomy with an NIR camera intraoperatively. Patients did not receive fluorescent dye. Images were taken of both gross and sectioned specimens. Post hoc image analysis was performed with ImageJ software. Confocal microscopy was performed on selected tissues using immunofluorescence and hematoxylin-eosin staining. Results: Resected tissue was examined from 22 patients undergoing surgery for pheochromocytomas (6), primary aldosteronism (3), adrenocorticotropin-independent hypercortisolism (10), and a growing or suspicious mass (3). Normal adrenal tissue demonstrated strong NIR autofluorescence. The intensity ratio compared to background (set as 1) for gross images was 2.03 ±â€…0.51 (P < .0001) compared to adjacent adipose of 1.24 ±â€…0.18. Autofluorescence from adrenal tumors was also detected at variable levels of intensity. Cortisol-producing tumors had the highest fluorescence ratio of 3.01 ±â€…0.41. Confocal imaging localized autofluorescence to the cytosol, with the highest intensity in the zona reticularis followed by the zona fasciculata. Conclusion: Normal and abnormal adrenal tissues possess natural NIR autofluorescence. Highest autofluorescence levels were associated with cortisol-producing tumors. Confocal imaging demonstrated the highest intensity in the zona reticularis. NIR cameras may have the potential to improve identification of adrenal tissue during surgery.

Progression and dormancy in metastatic thyroid cancer: concepts and clinical implications.

Distant metastasis classically has been defined as a late-stage event in cancer progression. However, it has become clear that metastases also may occur early in the "lifetime" of a cancer and that they may remain stable at distant sites. This stability of metastatic cancer deposits has been termed "metastatic dormancy" or, as we term it, "metastatic progression dormancy" as the progression either may reflect growth of already existing metastases or new cancer spread. Biologically, dormancy is the presence of nongrowing, static metastatic cells that survive over time. Clinically, dormancy is defined by stability in tumor markers, imaging, and clinical course. Metastatic well-differentiated thyroid cancer offers an excellent tumor type to understand these processes for several reasons: (1) primary therapy often includes removal of the entire gland with ablation of residual normal tissue thereby removing one source for new metastases; (2) the presence of a sensitive biochemical and radiographic monitoring tests enabling monitoring of metastasis throughout the progression process; and (3) its tendency toward prolonged clinical dormancy that can last for years or decades be followed by progression. This latter factor provides opportunities to define therapeutic targets and/or markers of progression. In this review, we will discuss concepts of metastatic progression dormancy and the factors that drive both long-term stability and loss of dormancy with a focus on thyroid cancer.

MAPK- and AKT-activated thyroid cancers are sensitive to group I PAK inhibition.

The number of individuals who succumb to thyroid cancer has been increasing and those who are refractory to standard care have limited therapeutic options, highlighting the importance of developing new treatments for patients with aggressive forms of the disease. Mutational activation of MAPK signaling, through BRAF and RAS mutations and/or gene rearrangements, and activation of PI3K signaling, through mutational activation of PIK3CA or loss of PTEN, are well described in aggressive thyroid cancer. We previously reported overactivation and overexpression of p21-activated kinases (PAKs) in aggressive human thyroid cancer invasive fronts and determined that PAK1 functionally regulated thyroid cancer cell migration. We reported mechanistic crosstalk between the MAPK and PAK pathways that are BRAF-dependent but MEK independent, suggesting that PAK and MEK inhibition might be synergistic. In the present study, we tested this hypothesis. Pharmacologic inhibition of group I PAKs using two PAK kinase inhibitors, G-5555 or FRAX1036, reduced thyroid cancer cell viability, cell cycle progression and migration and invasion, with greater potency for G-5555. Combination of G-5555 with vemurafenib was synergistic in BRAFV600E-mutated thyroid cancer cell lines. Finally, G-5555 restrained thyroid size of BRAFV600E-driven murine papillary thyroid cancer by >50% (P < 0.0001) and reduced carcinoma formation (P = 0.0167), despite maintenance of MAPK activity. Taken together, these findings suggest both that group I PAKs may be a new therapeutic target for thyroid cancer and that PAK activation is functionally important for BRAFV600E-mediated thyroid cancer development.