Gene Expression Profiling In Thyroid Cancer

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A limited number of studies have reported on DNA microarray studies in thyroid cancer: two on follicular tumors, one on papillary carcinoma (PTC) and only one that examined a range of benign and malignant thyroid disease. The numbers of samples analyzed in each study was small to moderate in size. Most of the studies quoted are, however, robust and pass muster for the stringent rules stipulated for reporting of gene expression studies (4).

Papillary thyroid carcinoma

Huang et al. (12) used oligonucleotide DNA chips containing more than 12,000 genes to profile 8 papillary carcinomas and matching normal thyroid tissue. They found the expression of 8 genes to be suppressed in 7/8 samples and that of 19 genes in 6/8 samples. The genes whose expression was suppressed fell in a number of categories: tumor suppressor genes (e.g. bcl-2, gas-1 and fos-B), thyroid metabolism (e.g. dio-1, dio-2, tpo), cell adhesion (dpt and fgl-2), fatty acid binding (apo-B and fabp-4) and signal transduction (stc-1 and itpr-1).

24 genes were overexpressed in all 8 papillary thyroid cancer samples and an additional 22 genes in 7/8 specimens. Among the genes found to be overexpressed were several previously reported and include fibronectin-1, the met oncogene, dipeptidylpep-tidase IV, a -- - a ss titryps i ss, keratin-19 and galectin 3. Other overexpressed genes fell in the categories of cellular adhesion/extracellular matrix, cytoskeleton, growth factors and their receptors as well as those involved in signal transduction.

Several genes found to be over-expressed in papillary carcinoma were not previously reported in any neoplasia or the thyroid and include: ADORA1 (adenosine A1 receptor), SCEL (sciellin), ODZ1 (Odz 1, Drosophila), PROS1 (vitamin K-dependent plasma protein S), K1AA0937, CST6 (cystatin E/M), SDC4 ( ryudocan core protein), P4HA2 (propyl-4-hydroxylase alpha (II) subunit), DUSP6 ( dual specificity phos-phatase 6), TSSC3 (tumor suppressing subtransferable candidate 3). Changes in gene expression of selected genes were confirmed by multiplex semiquantitative RT PCR and consistent and highly correlated patterns of gene expression pattern in tumor samples relative to normal was also verified by hierarchial cluster analysis. The latter aspect of the results was unexpected given the previously noted heterogeneity of individual gene expression in PTC.

Specificity for PTC of two gene products (Cbp/p300-interacting transactivator [C1TED1] and surfactant, pulmonary-associated protein B [SFTPB]) previously associated with other neoplasia was explored by immunoflorescence in a large number archival of PTC tissue and other thyroid malignant tumors (12).

Follicular tumors

Using the same array system as did Huang et al. (12), Barden et al. (13) compared RNA from 10 follicular adenomas with those from 9 follicular carcinomas (FTC), two minimally invasive, one poorly differentiated and one Hurthle cell carcinomas. The authors identified 105 genes whose expression significantly differed between adenomas and carcinomas (overexpressed in one or the other). They found that many previously unidentified genes contributed to the distinction between adenomas and carcinomas. Interestingly, very few of the genes suppressed or overexpressed in follicular tumors were identified as important in the Huang et al. study (12).

The authors (13) chose 5 genes with >3 fold overexpression for further verification of expression by semiquantitative RT-PCR and in the case of one gene, product extracellular matrix metalloproteinase inducer (EMMPRIN), by Western blotting of the extracted protein. The gene products overexpressed in follicular carcinomas compared to adenomas were adrenomedullin, autotoxin, EMMPRIN, transforming growth factor II receptor and the met oncogene. Met was previously reported to be relevant to thyroid carcinogenesis (12,14, also see below). Adrenomedullin is important in growth and survival of several human cancers whereas autoxin promotes tumor cell growth and angiogenesis. EMMPRIN is a surface glycoprotein that is associated with metastatic behavior.

Aldred et al. (15,16) selected genes found to be differentially regulated in 19 FTCs. They chose genes mapping to regions of loss of heterozygozity (LOH), previously reported in FTC (15). They also monitored the downregulation of peroxisome proliferator-activated receptor gamma (16). Because of the questions asked, the authors focussed on down-regulated genes. In contrast to the initial study from this group (12), there was not enough material to test the samples in duplicate nor was there enough paired normal thyroid tissues for samples. The authors had, therefore, to respectively normalize their results to mean intensity and to carry out pairwise comparisons between all normal and tumorous thyroid tissue (15).

Three genes coordinately downregulated, caveolin-1, caveolin-2 and GDF10/ BMP3b were further studied on the basis of their localization to two chromosomal regions, 7q31.1 and l0q11.1, that commonly show LOH in FTC. The authors also selected for further analysis two additional genes (glypican-3: Xq26.1 and a novel chordin-like:Xq22) involved in bone morphogenesis signalling and possible interaction with GDF10. Each of the 5 genes was downregulated in at least 15/19 of samples by RT-PCR. The authors followed in greater detail the relevance of caveolin-1, thought to be involved in the regulation of the dual-specificity phosphatase PTEN, suppressed in FTC. They found that it is the (3 isoform of calveolin-1 that is specifically down-regulated and that the reduced expression is specific to FTC, including insular and Hurthle-cell varieties (15). On the other hand, the expression of the 3 genes involved in bone morphogenesis signalling were downregulated also in benign adenomas and multinodular goitre and glypican-3 in PTC suggesting that they are early events in pathologic thyroid cell growth.

In apparently the same set of tumors, Aldred et al (16) found that whereas only 2/19 FTC exhibited the re-arrangement (see Chapter 4), the majority

(13/17) of the remainder showed greatly reduced expression of PPARy by microarray and semiquantitative RT-PCR. Reduction of immunoreactivity was found not only in FTCs but also in Hurthe cell carcinomas and PTCs. Down regulation of is probably related to repression by one or more upstream regulatory protein, as the results could not be explained on the basis of gene deletion, mutation or hypermethylation of the regulatory sequences.

A spectrum of thyroid tumors

We have used human cDNA microarray constructed in-house by spotting previously PCR-amplified and purified gene-specific samples to study RNA from a range of thyroid tissues. We examined samples from multinodular goitre (MNG), Graves' disease, Hashimoto's thyroiditis, papillary carcinoma, follicular carcinoma and follicular adenoma and compared these to normal perinodular thyroid tissue. Hierarchical cluster analysis (Figure 1) showed clear separation of various clinical pathological entities according to variation in the expression of 1322 genes, many of which were unidentified at the time.

We selected 26 genes whose expression showed variation in thyroid tumors for further examination using semiquantitative RT-PCR. Given that we used a completely

MNG Hashimoto Papillary carcinoma Microfollicular Follicular

Figure 1. Hierarchical clustering of 1322 genes separates the thyroid diseases studied. Specific clusters were arrived at according to thyroid disease. The closest relatives were mircofollicular adenomas and follicular carcinoma, while papillary carcinoma exhibited some relationship to this subfamily. Multinodular goiter and Hashimoto's thyroiditis were distinct from each other and from other thyroid disease types.

Hierarchical cluster analysis was done using the Omniviz Gene Expression software package (Omniviz Inc.)

different panel of genes than did Huang et al. (12), we found remarkable agreement in genes differentially regulated in PTC. The expression of some of these genes was, however, also regulated in other thyroid tissues studied, thus (and as expected) met expression was increased in FTC, as was that of galacetin3 whereas that of cartilage glycoproteins was overexpressed in adenomas, MNG and FTC. That of Cpb/p300-interacting protein was overexpressed in FTC but 5 fold less in PTC.

Type I iodothyronine deiodinase which is down-regulated in PTC was enhanced in FTC and benign nodular tissue, CRAB1 cellular retinoic add binding protein expression was also reduced in adenoma, FTC and MNG but less than in PTC whereasfatty add binding protein 4, suppressed in PTC, appears to up-regulated in MNG.

We found the C8FW phosphoprotein, regulated in the thyroid gland by TSH and EGF (17), to be specifically overexpressed in FTC and so was proprotein convertase subtilisin/kexin 2. The latter is a furin involved in the processing of prohormones (18) but which is also involved in the digestion of pre-metalloproteases and cadherins and might be involved in tumor invasion and metastatic behavior. S100 calcium binding protein (mts 1 or metastasin), the subject ofintense recent interest in carcinogensis (19), was specifically increased in PTC. By contrast the small GTPase Rapl (20), which is involved in the regulation of cell-adhesion and Glycerol kinase 2 (testis specific), a key enzyme in adipose tissue metabolism are increased in FTC.

The 15 Kda selenoprotein (21) whose abundance is modified in transformed cells was increased in PTC but not in FTC and less so in MNG.

We found secretedfrizzled related protein (22,23), that is involved in the regulation of wnt signalling, to be specifically reduced in PTC. In contrast, the expression of tight junction protein 1, important in maintaining cellular polarity (24) was curiously suppressed specifically in FTC. Eosinophil-derived neurotoxin, which plays a role in allergic reactions and has an anti retroviral activity is reduced in all tumorous tissues studied (25,26). We speculate that it may be involved in evasion by tumors of immune cells. Keratin, Type II cytoskeletal 7 important in maintaining cellular structure is reduced in all except follicular adenomas (27). In an independent study (28), we found that the cannabinoid receptor 2 (CNR2) as well as met to relevant to the metastatic behavior of anaplastic thyroid carcinoma.

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