Hydrocortisone and purinergic signaling stimulate sodium/iodide symporter (NIS)-mediated iodide transport in breast cancer cells

The sodium/iodide symporter (NIS) mediates a remarkably effective targeted radioiodide therapy in thyroid cancer; this approach is an emerging candidate for treating other cancers that express NIS, whether endogenously or by exogenous gene transfer. Thus far, the only extrathyroidal malignancy known to express functional NIS endogenously is breast cancer. Therapeutic efficacy in thyroid cancer requires that radioiodide uptake be maximized in tumor cells by manipulating well-known regulatory factors of NIS expression in thyroid cells, such as TSH, which stimulates NIS expression via cAMP. Similarly, therapeutic efficacy in breast cancer will likely depend on manipulating NIS regulation in mammary cells, which differs from that in the thyroid. Human breast adenocarcinoma MCF-7 cells modestly express endogenous NIS when treated with all-trans-retinoic acid (tRa). We report here that hydrocortisone and ATP each markedly stimulates tRa-induced NIS protein expression and plasma membrane targeting in MCF-7 cells, leading to at least a 100% increase in iodide uptake. Surprisingly, the adenyl cyclase activator forskolin, which promotes NIS expression in thyroid cells, markedly decreases tRa-induced NIS protein expression in MCF-7 cells. Isobutylmethylxanthine increases tRa-induced NIS expression in MCF-7 cells, probably through a purinergic signaling system independent of isobutylmethylxanthine's action as a phosphodiesterase inhibitor. We also observed that neither iodide, which at high concentrations down-regulates NIS in the thyroid, nor cAMP has a significant effect on NIS expression in MCF-7 cells. Our findings may open new strategies for breast-selective pharmacological modulation of functional NIS expression, thus improving the feasibility of using radioiodide to effectively treat breast cancer.     

Dietary flavonoids and iodine metabolism

Flavonoids have inhibiting effects on the proliferation of cancer cells, including thyroidal ones. In the treatment of thyroid cancer the uptake of iodide is essential. Flavonoids are known to interfere with iodide organification in vitro, and to cause goiter. The influence of flavonoids on iodine metabolism was studied in a human thyroid cancer cell line (FTC-133) transfected with the human sodium/iodide transporter (NIS). All flavonoids inhibited growth, and iodide uptake was decreased in most cells. NIS mRNA expression was affected during the early hours after treatment, indicating that these flavonoids can act on NIS. Pendrin mRNA expression did not change after treatment. Only myricetin increased iodide uptake. Apeginin, luteolin, kaempferol and F21388 increased the efflux of iodide, leading to a decreased retention of iodide. Instead myricetin increased the retention of iodide; this could be of use in the radioiodide treatment of thyroid cancer.     

Regulation of sodium iodide symporter gene expression by Rac1/p38β mitogen-activated protein kinase signaling pathway in MCF-7 breast cancer cells

Activation of p38 MAPK is a key pathway for cell proliferation and differentiation in breast cancer and thyroid cells. The sodium/iodide symporter (NIS) concentrates iodide in the thyroid and lactating breast. All-trans-retinoic acid (tRA) markedly induces NIS activity in some breast cancer cell lines and promotes uptake of β-emitting radioiodide (131)I sufficient for targeted cytotoxicity. To identify a signal transduction pathway that selectively stimulates NIS expression, we investigated regulation by the Rac1-p38 signaling pathway in MCF-7 breast cancer cells and compared it with regulation in FRTL-5 rat thyroid cells. Loss of function experiments with pharmacologic inhibitors and small interfering RNA, as well as RT-PCR analysis of p38 isoforms, demonstrated the requirement of Rac1, MAPK kinase 3B, and p38β for the full expression of NIS in MCF-7 cells. In contrast, p38α was critical for NIS expression in FRTL-5 cells. Treatment with tRA or overexpression of Rac1 induced the phosphorylation of p38 isoforms, including p38β. A dominant negative mutant of Rac1 abolished tRA-induced phosphorylation in MCF-7 cells. Overexpression of p38β or Rac1 significantly enhanced (1.9- and 3.9-fold, respectively), the tRA-stimulated NIS expression in MCF-7 cells. This study demonstrates differential regulation of NIS by distinct p38 isoforms in breast cancer cells and thyroid cells. Targeting isoform-selective activation of p38 may enhance NIS induction, resulting in higher efficacy of (131)I concentration and treatment of breast cancer.     

Molecular Mechanism of 17-Allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL Receptor Tyrosine Kinase Degradation

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [³⁵S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.     

RET/PTC Rearrangements Are Associated with Elevated Postoperative TSH Levels and Multifocal Lesions in Papillary Thyroid Cancer without Concomitant Thyroid Benign Disease

RET/PTC rearrangements, resulting in aberrant activity of the RET protein tyrosine kinase receptor, occur exclusively in papillary thyroid cancer (PTC). In this study, we examined the association between RET/PTC rearrangements and thyroid hormone homeostasis, and explored whether concomitant diseases such as nodular goiter and Hashimoto''s thyroiditis influenced this association. A total of 114 patients diagnosed with PTC were enrolled in this study. Thyroid hormone levels, clinicopathological parameters and lifestyle were obtained through medical records and surgical pathology reports. RET/PTC rearrangements were detected using TaqMan RT-PCR and validated by direct sequencing. No RET/PTC rearrangements were detected in benign thyroid tissues. RET/PTC rearrangements were detected in 23.68% (27/114) of PTC tissues. No association between thyroid function, clinicopathological parameters and lifestyle was observed either in total thyroid cancer patients or the subgroup of patients with concomitant disease. In the subgroup of PTC patients without concomitant disease, RET/PTC rearrangement was associated with multifocal cancer (P = 0.018). RET/PTC rearrangement was also correlated with higher TSH levels at one month post-surgery (P = 0.037). Based on likelihood-ratio regression analysis, the RET/PTC-positive PTC cases showed an increased risk of multifocal cancers in the thyroid gland (OR = 5.57, 95% CI, 1.39–22.33). Our findings suggest that concomitant diseases such as nodular goiter and Hashimoto''s thyroiditis in PTC may be a confounding factor when examining the effects of RET/PTC rearrangements. Excluding the potential effect of this confounding factor showed that RET/PTC may confer an increased risk for the development of multifocal cancers in the thyroid gland. Aberrantly increased post-operative levels of TSH were also associated with RET/PTC rearrangement. Together, our data provides useful information for the treatment of papillary thyroid cancer.     

RET/PTC (rearranged in transformation/papillary thyroid carcinomas) tyrosine kinase phosphorylates and activates phosphoinositide-dependent kinase 1 (PDK1): an alternative phosphatidylinositol 3-kinase-independent pathway to activate PDK1

Thyroid cancers are a leading cause of death due to endocrine malignancies. RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although the oncogenic potential of RET/PTC is related to intrinsic tyrosine kinase activity, the substrates for this enzyme are yet to be identified. In this report, we show that phosphoinositide-dependent kinase 1 (PDK1), a pivotal serine/threonine kinase in growth factor-signaling pathways, is a target of RET/PTC. RET/PTC and PDK1 colocalize in the cytoplasm. RET/PTC phosphorylates a specific tyrosine (Y9) residue located in the N-terminal region of PDK1. Y9 phosphorylation of PDK1 by RET/PTC requires an intact catalytic kinase domain. The short (iso 9) and long forms (iso 51) of the RET/PTC kinases (RET/PTC1 and RET/PTC3) induce Y9 phosphorylation of PDK1. Moreover, Y9 phosphorylation of PDK1 by RET/PTC does not require phosphatidylinositol 3-kinase or Src activity. RET/PTC-induced phosphorylation of the Y9 residue results in increased PDK1 activity, decrease of cellular p53 levels, and repression of p53-dependent transactivation. In conclusion, RET/PTC-induced tyrosine phosphorylation of PDK1 may be one of the mechanisms by which it acts as an oncogenic tyrosine kinase in thyroid carcinogenesis.     

Cooperative enhancement of radiosensitivity after combined treatment of 17-(allylamino)-17-demethoxygeldanamycin and celecoxib in human lung and colon cancer cell lines

We investigated whether the combined treatment of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat-shock protein 90 (hsp90), and celecoxib, an inhibitor of cyclooxygenase-2, can cooperatively enhance the radiosensitivity of various human cancer cells. Combined treatment with 17-AAG and celecoxib, at clinically relevant concentrations, cooperatively induced radiosensitization in all tested cancer cells, but not in normal cells. Cooperative radiosensitization by the drug combination was also shown in a human tumor xenograft system. We found that ataxia-telangiectasia and rad3-related (ATR) and ataxia-telangiectasia mutated (ATM) are novel client proteins of hsp90. Combined treatment with 17-AAG and celecoxib cooperatively induced downregulation of ATR and ATM. In conclusion, combined treatment with 17-AAG and celecoxib at clinically relevant concentrations may significantly enhance the therapeutic efficacy of ionizing radiation.     

Journey of the iodide transporter NIS: from its molecular identification to its clinical role in cancer

The Na+/I- symporter (NIS) is an intrinsic plasma membrane protein that mediates the active transport of I- in the thyroid, lactating mammary gland, stomach and salivary glands. The presence of NIS in the thyroid is exploited in diagnostic scintigraphic imaging and radioiodide therapy in thyroid cancer. The continued rapid progress in NIS research (aimed at the elucidation of the Na+-dependent I- transport mechanism, the analysis of NIS structure-function relations and the study of the tissue-specific regulation of NIS at all levels), holds potentially far-reaching medical applications beyond thyroid disease, in breast cancer and malignancies in other tissues.     

Influence of RET/PTC1 and RET/PTC3 oncoproteins in radiation-induced papillary thyroid carcinomas on amounts of cytoskeletal protein species

Radiation-induced human papillary thyroid carcinomas (PTCs) show a high prevalence of fusions of the RET proto-oncogene to heterologous genes H4 (RET/PTC1) and ELE1 (RET/PTC3), respectively. In contrast to the normal membrane-bound RET protein, aberrant RET fusion proteins are constitutively active oncogenic cytosolic proteins that can lead to malignant transformation of thyroid epithelia. To detect specific tumor-associated protein changes that reflect the effect of RET/PTC fusion proteins, we analyzed normal thyroid tissues, thyroid tumors of the RET/PTC1 and RET/PTC3 type and their respective lymph node metastases by a combination of high-resolution two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry. PTCs without RET rearrangements served as controls. Several cytoskeletal protein species showed quantitative changes in tumors and lymph node metastases harboring RET/PTC1 or RET/PTC3. We observed prominent C-terminal actin fragments assumedly generated by protease cleavages induced due to enhanced amounts of the active actin-binding protein cofilin-1. In addition, three truncated vimentin species, one of which was proven to be headless, were shown to be highly abundant in tumors and metastases of both RET/PTC types. The observed protein changes are closely connected with the constitutive activation of RET-rearranged oncoproteins and reflect the importance to elucidate disease-related typical signatures on the protein species level.     

XB130 Mediates Cancer Cell Proliferation and Survival through Multiple Signaling Events Downstream of Akt

XB130, a novel adaptor protein, mediates RET/PTC chromosome rearrangement-related thyroid cancer cell proliferation and survival through phosphatidyl-inositol-3-kinase (PI3K)/Akt pathway. Recently, XB130 was found in different cancer cells in the absence of RET/PTC. To determine whether RET/PTC is required of XB130-related cancer cell proliferation and survival, WRO thyroid cancer cells (with RET/PTC mutation) and A549 lung cancer cells (without RET/PTC) were treated with XB130 siRNA, and multiple Akt down-stream signals were examined. Knocking-down of XB130 inhibited G(1)-S phase progression, and induced spontaneous apoptosis and enhanced intrinsic and extrinsic apoptotic stimulus-induced cell death. Knocking-down of XB130 reduced phosphorylation of p21Cip1/WAF1, p27Kip1, FOXO3a and GSK3β, increased p21Cip1/WAF1protein levels and cleavages of caspase-8 and-9. However, the phosphorylation of FOXO1 and the protein levels of p53 were not affected by XB130 siRNA. We also found XB130 can be phosphorylated by multiple protein tyrosine kinases. These results indicate that XB130 is a substrate of multiple protein tyrosine kinases, and it can regulate cell proliferation and survival through modulating selected down-stream signals of PI3K/Akt pathway. XB130 could be involved in growth and survival of different cancer cells.     

RET tyrosine kinase signaling in development and cancer

The variety of diseases caused by mutations in RET receptor tyrosine kinase provides a classic example of phenotypic heterogeneity. Gain-of-function mutations of RET are associated with human cancer. Gene rearrangements juxtaposing the tyrosine kinase domain to heterologous gene partners have been found in sporadic papillary carcinomas of the thyroid (PTC). These rearrangements generate chimeric RET/PTC oncogenes. In the germline, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Both MEN 2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, activate the RET downstream targets. Loss-of-function mutations of RET cause Hirschsprung's disease (HSCR) or colonic aganglionosis. A deeper understanding of the molecular signaling of normal versus abnormal RET activity in cancer will enable the development of potential new treatments for patients with sporadic and inherited thyroid cancer or MEN 2 syndrome. We now review the role and mechanisms of RET signaling in development and carcinogenesis.     

Differential interaction of Enigma protein with the two RET isoforms

The receptor tyrosine kinase RET, with a known role in embryonic development and in human pathologies, is alternatively spliced to yield at least two functional isoforms, which differ only in their carboxyl terminal. Enigma protein is a member of the PDZ-LIM family and is known to interact with the short isoform of RET/PTC2, a chimeric oncoprotein isolated from papillary thyroid carcinoma. Here, we show that Enigma also interacts in intact cells with the short isoform of RET-wt and of its pathologic mutants associated to MEN2 syndromes, RET-C634R and RET-M918T. In contrast, Enigma binds all the corresponding RET long isoforms very poorly and colocalizes with short but not long RET/PTC2 isoforms. The RET docking tyrosine for Enigma is the last but one before the divergence between the two isoforms and we demonstrated that short-isoform-specific amino acid residues +2 to +4 to this tyrosine are required for the interaction of RET/PTC2 with Enigma.     

Genetic factors predisposing to the development of papillary thyroid cancer

According to classic theory of neogenesis, cancer arises from well-differentiated cell that in response to variety of factors de-differentiates, becomes able to proliferate without control and/or loses its ability to undergo apoptosis. According to another theory, cancers (at least cancers of some organs) originate from stem cells, which "by definition" are poorly differentiated and able to proliferate indefinitely. Therefore a lower number of abnormal events is necessary for these cells to escape proliferation-controlling mechanisms. With regard to papillary thyroid cancers it is still thought that it arises from well-differentiated thyreocyte. One of the characteristic features of cancer cell is chromosomal instability. Lowest number of such abnormalities is observed in well-differentiated thyroid cancers (including papillary cancer), intermediate - in poorly-differentiated cancers, while highest - in anaplastic cancers. Microarray analysis shows that despite of clinical heterogeneity, gene expression profiles of papillary cancers are very similar. Genetic anomalies predisposing to the development of papillary cancer most commonly regard proteins that possess kinase activity. Kinases phosphorylate other proteins, and play an extremely important role in signal transduction from outside the cell as well as inside the cell. Constitutive activation of some kinases may lead to the excessive and/or permanent activation of some transduction pathways specific for mitogens or growth factors. This results in excessive proliferation. The best known protein of such type which function is altered in papillary thyroid cancers is RET - a membrane-located growth factor-receptor with kinase activity. RET gene undergoes different rearrangements in this type of cancer. There are approximately 10 RET rearrangements known, with RET/PTC3 and RET/PTC1 being most common. In this anomaly kinase domain-encoding 3' end of RET gene is aberrantly bound to 5' end of another gene. Fusion protein synthesized on such hybrid template is not present in the cell membrane but in the cytoplasm, where it permanently activates transduction pathway specific for RET. NTRK1 gene encoding a member of family of neuronal growth factor receptors containing thyrosine kinase domain is also rearranged in papillary cancers. However, genes fused to its kinase domain-encoding sequence are different from the ones fused to RET. MET, a gene encoding another membrane protein with thyrosine kinase activity, which acts as a growth factor-receptor, is overexpressed in 70%-90% of papillary thyroid cancers. BRAF gene encoding another yet kinase transducing signals from RAS and RAF to the cell is mutated at position 1796 (T/A, amino acid substitution V599E) in 38-69% of papillary cancers. The presence of this activatory mutation is associated with higher degree of clinical advancement of the disease. In addition, in majority of papillary cancers tested, mutations of the genes encoding nuclear triiodothyronine receptors were found. Transgenic mice with both TRB allele replaced with dominant-negative TRB mutants develop aggressive thyroid cancers. Progression from papillary to anaplastic cancer is most possibly caused by the occurrence of additional anomalies within P53, RAS, NM23,b-catenin gene and other genes.     

Mammary radioiodine accumulation due to functional sodium iodide symporter expression in a benign fibroadenoma

The sodium iodide symporter (NIS) has been characterized to mediate the active transport of iodide not only in the thyroid gland but also in various non-thyroidal tissues, including lactating mammary gland and the majority of breast cancers, thereby offering the possibility of diagnostic and therapeutic radioiodine application in breast cancer. In this report, we present a 57-year-old patient with multifocal papillary thyroid carcinoma, who showed focal radioiodine accumulation in a lesion in the right breast on a posttherapy (131)I scan following radioiodine therapy. CT and MR-mammography showed a focal solid lesion in the right breast suggestive of a fibroadenoma, which was confirmed by histological examination. Immunostaining of paraffin-embedded tumor tissue sections using a human NIS antibody demonstrated NIS-specific immunoreactivity confined to epithelial cells of mammary ducts. In conclusion, in a thyroid cancer patient we identified a benign fibroadenoma of the breast expressing high levels of functionally active NIS protein as underlying cause of focal mammary radioiodine accumulation on a posttherapy (131)I scan. These data show for the first time that functional NIS expression is not restricted to lactating mammary gland and malignant breast tissue, but can also be detected in benign breast lesions, such as fibroadenomata of the breast.