Thyroid Hormone, Cancer, and Apoptosis

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1 Thyroid Hormone, Cancer, and Apoptosis Hung-Yun Lin, *1,2 Yu-Tan Chin, 1 Yu-ChenS.H.Yang, 3 Hsuan-Yu Lai, 1 Jacqueline Whang-Peng, 4 Leroy F. Liu, 1 Heng-Yuan Tang, 5 and Paul J. Davis, 5,6* ABSTRACT Thyroid hormones play important roles in regulating normal metabolism, development, and growth. They also stimulate cancer cell proliferation. Their metabolic and developmental effects and growth effects in normal tissues are mediated primarily by nuclear hormone receptors. A cell surface receptor for the hormone on integrin αvβ3 is the initiation site for effects on tumor cells. Clinical hypothyroidism may retard cancer growth, and hyperthyroidism was recently linked to the prevalence of certain cancers. Local levels of thyroid hormones are controlled through activation and deactivation of iodothyronine deiodinases in different organs. The relative activities of different deiodinases that exist in tissues or organs also affect the progression and development of specific types of cancers. In this review, the effects of thyroid hormone on signaling pathways in breast, brain, liver, thyroid, and colon cancers are discussed. The importance of nuclear thyroid hormone receptor isoforms and of the hormone receptor on the extracellular domain of integrin αvβ3 as potential cancer risk factors and therapeutic targets are addressed. We analyze the intracellular signaling pathways activated by thyroid hormones in cancer progression in hyperthyroidism or at physiological concentrations in the euthyroid state. Determining how to utilize the deaminated thyroid hormone analog (tetrac), and its nanoparticulate derivative to reduce risks of cancer progression, enhance therapeutic outcomes, and prevent cancer recurrence is also deliberated American Physiological Society. Compr Physiol 6: , Introduction Thyroid hormones (THs) are iodinated endogenous compounds known to regulate a wide range of cellular activities through thyroid hormone receptors (TRs). Thyroid hormones, such as L-thyroxine (T 4 ), are major secretory products of a normal thyroid gland. Although T 4 has certain physiological roles for example, in the actin cytoskeleton (52) and angiogenesis (14, 112) its principal function is thought to be that of a prohormone, undergoing conversion to 3,5,3 - triiodo-l-thyronine (T 3 ) by deiodination to effect genomic hormonal actions that require nuclear receptors for thyroid hormone receptors (17). Nuclear receptors are sequencespecific ligand-dependent transcription factors that trigger many of the downstream effects of thyroid hormones, such as T 3, by activating or repressing target genes. THs are critical to organism development, tissue differentiation and growth, and maintenance of a cell s metabolic balance (169). These actions are thyroid hormone receptor dependent. Severe disruption of THs actions during fetal and early neonatal development leads to permanent functional deficits (53), particularly of the nervous system. Integrins are heterodimeric structural components of plasma membranes that are able to bind to a large number of extracellular matrix (ECM) proteins as ligands. Certain ones of these ligands contain Arg-Gly-Asp (RGD) sequences that allow recognition of ECM proteins by a group of specific integrins and permit integrins to generate intracellular (outside-in) signals specific to ECM molecules (38). In addition to protein ligands, integrin αvβ3 was shown to have small-molecule receptor sites for thyroid hormones and hormone analogs, dihydrotestosterone, and resveratrol, a polyphenol with certain estrogen-like features. These binding sites are close to the RGD recognition site on αvβ3. The TH receptor contains discrete binding domains, one of which binds both T 4 and T 3, and the other is specific for T 3 ; the two sites have discrete functions with regard to regulating signal transduction pathways and cell functions (32, 101) (see later). In dividing endothelial cells and tumor cells, the plasma membrane expresses large amounts of integrin αvβ3, the extracellular domain of which bears the thyroid hormone * Correspondence to linhy@tmu.edu.tw or pdavis.ordwayst@gmail.com 1 Taipei Cancer Center, Taipei Medical University, Taipei, Taiwan 2 PhD program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 3 Joint Biobank, Office of Human Research, Taipei Medical University, Taipei, Taiwan 4 Center of Excellence Cancer Research, Taipei Medical University, Taipei, Taiwan 5 Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, New York, USA 6 Albany Medical College, Albany, New York, USA Published online, July 2016 (comprehensivephysiology.com) DOI: /cphy.c Copyright American Physiological Society. Volume 6, July

2 receptor (5, 32). The receptor site is not structurally related to the thyroid hormone receptor-binding site for iodothyronines, and functions of this receptor on the integrin are distinct from those of nuclear receptors. Like other plasma membrane proteins, integrin αvβ3 is internalized and recycled. In the presence of T 4 at the cell surface, however, integrin αvβ3 in tumor cells is internalized in a very specific manner; that is, the interaction between the thyroid hormone and integrin αvβ3 induces (i) import of integrin αvβ3 into the cytoplasm and resolution into its component monomers that do not bind T 4 and (ii) activation of extracellular signal-regulated kinase (ERK)1/2 (100) which may support trafficking to the nuclear compartment and phosphorylation of cytoplasmic TRβ1 (39). In addition, the process is associated with the formation of intranuclear complexes of coactivator proteins that are relevant to genomic hormonal actions involving thyroid hormone receptor-regulated genes (37). The αv monomer in T 4 -treated cells is taken up by nuclei and may participate in coactivator or corepressor complexes. The integrin thyroid hormone-binding site contains two thyroid hormone-binding domains, designated S1 and S2, which differentially translate the T 4 and T 3 signals (32, 56, 101). S1 exclusively binds to T 3 at physiological concentrations leading to activation (phosphorylation) of phosphatidylinositol 3-kinase (PI3K). S2 binds to T 4, and to a lesser extent to T 3, and ultimately activates the ERK1/2 signal transduction pathway (101). Activation of the oncogenic ERK1/2 pathway by thyroid hormones, primarily T 4, via the S2 site of αvβ3 facilitates cell proliferation and inhibits apoptosis (100,101). Via αvβ3/s2, the thyroid hormone is able to activate ERK1/2 and induce expression of fibroblast growth factor 2 (FGF or basic (b)fgf) (30) and promote angiogenesis, and it is also crucial for rapid tumor growth (30). The hormone enhances proliferation of a variety of tumor cells (31, ,117,167). These include medullary thyroid and papillary thyroid cancer cells (103), renal cell carcinoma (165), glioma cells (31, 102), breast cancer (19, 155), and lung cancer cells (117). On the other hand, the PI3K pathway which can be activated by T 3 via the S1 site of the TH receptor on integrin αvβ3 stimulates cell proliferation and survival and also inhibits apoptosis (12). However, the activation of PI3K pathwaybyt 3 can also be achieved via a TRβ-dependent pathway. When T 3 is absent, TRβ can form a complex with the p85 subunit of PI3K and Lyn, the Src family tyrosine kinase, in cytoplasm. The association depends on two canonical phosphotyrosine motifs in the second zinc finger of TRβ. When T 3 is present and binds to TRβ, TRβ dissociates from the complex and moves to the nucleus, meanwhile the product of the activated PI3K, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) increases rapidly (114). By PI3K activation, T 3 induces the expression of the α subunit of the transcription factor, hypoxia-inducible factor 1 (HIF-1) (101,118). The target genes of HIF-1α protein play key roles in cancer biology, from angiogenesis to adaptation to hypoxia and are actively expressed in rapidly growing tumors, and in invasion and metastasis (146). HIF-1α expression is a prognostic marker of renal cell carcinoma and breast and prostate cancers, among others (89). T 4 is also able to induce HIF-1α expression via the integrin αv monomer, functioning as a coactivator protein in cell nuclei (100). The complex proangiogenic activities at the integrin αvβ3 receptor include transcription of vascular growth factor genes (112), as suggested earlier, nontranscriptional modulation of the function of vascular growth factor receptors adjacent to αvβ3 (122), and stimulation of endothelial cell motility (147). Thyroid Hormone, Thyroid Hormone Receptor, and Cancer Cell Proliferation Via the integrin thyroid hormone receptor, T 4 is a proliferative factor for a number of human cancer cell lines that have been studied in vitro and in xenografts (123, 166, 167). Since circulating levels of free T 4 are far higher than those of T 3 and the affinity of the hormone receptor on αvβ3 is higher for T 4 than for T 3 (5), the importance of T 4 as a stimulator of cancer cell proliferation and angiogenesis appears to exceed that of T 3. After binding to integrin αvβ3, T 4 (medium free T 4 concentration, M), but not T 3, induces cellular internalization and nuclear translocation of the integrin αv monomer in cancer cells. The αv monomer is phosphorylated by activated ERK1/2 when it heterodimerizes with integrin β3 in vitro. The nuclear αv monomer was shown to complex with the transcriptional coactivator proteins, p300 and signal transducer and activator of transcription (STAT)1, and with the corepressor proteins, NCoR and SMRT. The nuclear αv monomer in T 4 -exposed cells is bound to promoters of specific genes that have important roles in cancer cells, including estrogen receptor (ER)-α, cyclooxygenase (COX)-2, HIF-1α, and TRβ1. Deiodinases convert T 4 to T 3 (deiodinase types 1 and 2, D1 and D2) or to reverse T 3 (rt 3 ;3,3,5-triiodothyronine) (deiodinase type 3, D3). In addition, D3 also is able to convert T 3 to T 2 and thus attenuate the action of T 3 locally. It should be noted, however, that T 2 may have important nongenomic actions on mitochondria (107). From the standpoint of nuclear thyroid hormone receptor function, D1 and D2 activate the thyroid hormone; because rt 3 is not a functional ligand of thyroid hormone receptors, increased deiodination by D3 can serve to limit the amount of active thyroid hormone (T 3 ) generated from T 4 outside of the thyroid gland. In addition to its significant role in protecting tissues from excessive thyroid hormone levels during embryogenesis, D3 is induced under conditions of hypoxia or ischemia that exist in chronic inflammation, critical illnesses, and cancer. This can be interpreted to mean that low T 3 is protective of the host for example, fosters decreased metabolic demands and protein turnover in a setting of stress or an indication of illness severity (60, 78). However, activated D3 was correlated with solid cancers in a hyperproliferative state and has 1222 Volume 6, July 2016

3 raised the possibility of a link between deiodinase-mediated thyroid hormone metabolism and carcinogenesis (20). Cell lines derived from different tumors, such as basal-cell carcinoma, hemangiomas, hepatocarcinoma, breast cancer (MCF- 7 cells), colon adenocarcinoma (Caco2, SW280, and HCT116 cells), thyroid cancer, endometrial cancer (ECC-1 cells), and neuroblastoma (SH-SY5Y cells), express elevated D3 levels (42, 43, 79, 87). This would certainly indicate that T 3 is not essential to the function of cancer cells and is consistent with important roles for the T 4 -responsive hormone receptor on integrin αvβ3 in tumors. It should also be noted that rt 3 does have functional activity in supporting the integrity of the actin cytoskeleton (52) that is critical to normal cell and tumor cell structures. While D3 expression is significantly higher in benign adenomas and in colon carcinomas than in normal tissues, it is negatively correlated with the histologic grade of the lesions. Those observations suggest that D3 could be a marker of the early stages of tumorigenesis (20). Hepatic hemangiomas show high D3 activity resulting in an accelerated rate of thyroid hormone degradation (75, 140). Finally, decreased nuclear thyroid hormone availability due to elevated levels of D3 has also been shown in skin basal cell carcinomas, compared to normal keratinocytes. D3 is the physiological inactivator of thyroid hormone, except for certain hormonal actions attributable to T 2. Overexpression of D3 increases accumulation of rt 3 and T 2 and increases the T 4 /T 3 ratio, which creates hypothyroidism locally in terms of the genomic effects of TH, which are T 3 dependent. The consequence is the reduction of available nuclear T 3 for TRβ1. Thus, the increased D3 probably enhances the proliferation rate (20). The genomic actions of thyroid hormone require expression of genes coding for the thyroid hormone nuclear receptors (TRα1, TRβ1, and TRβ2), the distribution of which is tissue-specific. In the cardiac ventricle (153) and in brain (6), TRα1 is primarily expressed; TRβ is more evenly distributed in other organs. TRβ is the most potent regulator of the production of thyroid-stimulating hormone (TSH, thyrotropin) (59). TRs have been shown to be involved in developmental processes of biological functions (55) and in adult brain function (6). The genomic actions of thyroid hormones are mediated through nuclear thyroid hormone receptors and regulation of gene expression (169). TR genes can function as tumor-suppressor genes (161). Studies of the expression pattern of TRβ1 in the colorectal mucosa and in colorectal tumors and its relationship to the tumor growth type were conducted by Hörkkö et al. Nuclear TRβ1 is almost always present in the normal epithelium (96%), but less frequently in adenomas (83%) and in cancer (68%; P < and P < 0.001, respectively). The patterns of TRβ1 s location in cells are associated with polypoid growth, suggesting that abnormalities in thyroid hormone signaling involving TRβ1 may play a role in the development of colorectal adenocarcinomas (74). On the other hand, a convincing case was made by Cheng and co-workers for wild-type TRβ1 being a tumor-suppressor gene, notably in the case of thyroid cancer (88, 110, 111), and perhaps in other tumors as well (106, 130). Thus, aberrant expression or mutation of the TRβ1 may play a role in carcinogenesis (86). Wild type TRβ shows a positive prognostic factor for 5 years (P = 0.007) in BRCA1 associated breast cancer cases and overall survival (P = 0.026). On the other hand, wild type TRα is positively correlated with the reduction of 5-year survival (73). Activation of TRβ results in downmodulation of CTNNB1 (β-catenin) in breast cancers (73). Wild type TRα can play an important role in cancer proliferation (73) and its mutations are also associated with the development of distant (hematogenous) metastasis (P = ) and high expression levels of the Nm23 protein (P = 0.020); this suggests that TRα might be involved in tumorigenesis and that interactions between the TRα and nm23 genes might take part in hematogenous metastasis of gastric cancer mutation (161). The TRα locus also undergoes frequent loss of heterozygosity in breast cancer, and rearrangement of the TRα gene occurs in leukemia, breast cancer, and gastric cancer (1). Additionally, the interaction of thyroid hormone (T 3 ) and TRα is able to activate β-catenin signal transduction pathway, which plays an important role in the development of gastrointestinal cancers (133). The cytosolic location of TRα presents a pathological sign in breast cancer (22). The probable roles of thyrotropin, TH, integrin αvβ3, TR, and deiodinases in cancer proliferation are shown in Figure 1. Altered thyroid hormone status has been implicated in cancer cell proliferation/tumor growth. An increased risk for colon, lung, prostate, and breast cancers has been associated with lower circulating thyrotropin (69, 116) defining a state of subclinical hyperthyroidism suggesting that thyroid hormone levels affect the occurrence of cancer. Furthermore, higher thyroid hormone levels are associated with an advanced clinical stage of breast and prostate cancers. In rodent models, thyroid hormone stimulates growth and metastasis of tumor transplants, whereas hypothyroidism has opposite effects (119). Further, in a clinical experience involving pharmacologic induction of a euthyroid hypothyroxinemic state in advanced cancer patients, maintenance of circulating T 3 levels and euthyroidism with substantial reductions in circulating T 4 levels was recently associated with stabilization of tumors or reductions in tumor sizes (70). Tyrosine kinase inhibitors (TKIs) such as sunitinib are relatively new targeted therapy drugs used in chemotherapy, and hypothyroidism is often a side effect of this treatment (170). Evidence from several clinical centers indicates that TKIinduced hypothyroidism may beneficially affect the tumor course, for example, in renal cell carcinoma (144). Therefore, future cancer therapy studies with substances that might induce hypothyroidism may need to be conducted in a way that allows for an analysis of the thyroid function status and its contribution to treatment outcomes. We should also point out that spontaneous hypothyroidism may beneficially modify the course and aggressiveness of breast cancer (27). In the following sections, we discuss the effects of thyroid hormones, specific deiodinase activities, and thyroid hormone receptors on Volume 6, July

4 T 3 T 4 T 4 αvβ3 D3 T 4 D1/D2 T 3 PI3K ERK1/2 rt 3 TRα1 T 2 D3 Akt TRβ1 β-catenin D3 Gene expressions involved in biological activities T 3 TRβ1 Cell membrane TRα1 Cytosol Cell proliferation Metastasis HIF-1α Nucleus Figure 1 Schematic diagram of the proposed mechanisms by which thyroid hormone regulates cancer cell proliferation. In one mechanism, T 4 penetrates cell membrane via active transporters and is converted to T 3 by a deiodinase (D1 or D2). When T 3 binds to a nuclear thyroid hormone receptor α1 (TRα1), β-catenin abundance is enhanced and the protein translocates to the nucleus, where it stimulates cell proliferation. When T 3 binds to TRβ1, the genomic consequences are normal thyroid hormonedependent biological activities, which also shows antiproliferative effect in cancer cells. However, the antiproliferative effect of TRβ1 is reduced by overexpression of D3 induced by TRα-enhanced β- catenin-dependent mechanisms. In a mechanism that begins at the cell surface, T 4 and, less avidly, T 3 bind to integrin αvβ3 and activate ERK1/2 phosphorylation, and nongenomically stimulate cancer cell proliferation. When T 3 binds to integrin αvβ3, it activates not only ERK1/2 by phosphorylation, but also activates Akt by triggering PI3K phosphorylation. Thus, Akt enhances HIF-1α expression and then stimulates cancer cell metastasis. The opened-boxes represent the normal signal pathways, however, colored-boxes represent signals involved in the progression of cancers. the proliferation and metastasis of different types of cancers (Table 1). Breast cancer It was shown that hypothyroidism is associated with a lower incidence of breast cancer, while hyperthyroidism is associated with an increased incidence and aggressiveness of breast cancer (62). Chen and co-workers also reported an increased risk of breast cancer in a Graves disease patient population (16). A prospective study of 2696 women showed an association between circulating T 3 levels at the baseline (intake into the study) and breast cancer risk during a mean followup of 19.3 years (156). The risk of breast cancer occurrence increased as the concentrations of T 3 increased. The relative risk increased from 1.00 to 3.26, 5.53, and 6.87, respectively from the lowest to highest T 3 quartiles. A direct relation between the T 3 quartile and breast cancer occurrence was observed. Studies revealed that the direct relation between the T 3 quartile and breast cancer occurrence was revered when only tumors diagnosed 3 years after the baseline were considered. The overall risk of breast cancer in the women in the higher T 3 quartile was significantly higher than that of women in the lower T 3 quartile. Since all 1,322 participants were peri-/postmenopausal, the direct relationship between T 3 levels and breast cancer occurrence was significant. However, there was no direct relationship between T 3 and breast cancer occurrence in premenopausal women. Measurement of thyroid function in menopausal breast cancer patients showed that serum thyrotropin levels in breast cancer patients did not statistically differ from those of a control group, but that serum free T 4 levels of patients were significantly higher than those of the controls (24). A study conducted by Saraiva et al. in Brazil also showed that subclinical hyperthyroidism increased mean serum T 3 and T 4 levels associated with a lower mean thyrotropin was the most frequent endocrine disorder encountered in breast cancer patients (31%) (143). The condition was most common in postmenopausal patients. A role for circulating thyrotropin in breast cancer development has not yet been consistently defined. As indicated earlier, TSH levels are either not significantly changed as compared to control (23, 68) or deceased in postmenopausal patients (143), and NM Villa et al. also concluded that there is no association between thyrotropin level and markers of breast cancer invasiveness (159). Szychta et al. found that TSH receptor (TSHR) antibodies could be increased in breast cancer patients (154); this linked autoimmune hyperthyroidism 1224 Volume 6, July 2016

5 Table 1 The Effects of Thyroid Hormones, Deiodinase Activities, and Thyroid Hormone Receptors on the Proliferation and Metastasis in Cancers Binding receptor Deiodinase activity Thyrotropin Thyroxine Integrin αvβ3 TRα TRβ1 D1/D2 D3 Breast cancer No statistical difference compared to control (119, 143, 156, 159) No association between thyrotropin level and invasive markers of breast cancer (159) Lower thyrotropin in postmenopausal patients (33) Increased TSHR antibodies in breast cancer patients (154) Increasing proliferation (155) Hyperthyroidism (16, 156) Positive related to cancer growth (19, 32, 38) Crosstalk with ERα (117) 74% highly expressed TRα1 (11) and 40% highly expressed TRα2 and correlated with survival (85) T 3 inhibits TRβ pathway through STAT5 (64) TRβ Activation resulted in down-modulation of β-catenin (73) Low (non-cancerous breast tissue); high (cancer, well-differentiated cancer) (41) More than 56% of breast cancers, largely grades G1 and G2, expressed abundant D1 activity and/or a high mrna level and showed a positive correlation between the presence of D1 and the differentiation of breast cancers. (20, 41) Stimulating cell proliferation in MCF-7 cells (41, 87) Brain tumors No relevant activity Increasing proliferation (31, 101, 102) Positive related to cancer growth (31) TRα1 andtrα2 decrease with grade (80) Correlation with malignancy and grade (80) D2 activity higher than found in nontumoral tissues of the human brain (150) The highest D2 activity found in gliosarcomas (127) D3 activity increased in neuroblastoma (SH-SY5Y cells) (87), gliomas malignant lymphomas (150, 157) gliosarcomas and GBM (127) Lung cancer Decrease in patients (69, 116, 119) Significant reduction of circulating T 3 and increase of T 4 /T 3 ratio and modest lowering of rt 3 (135) Hyperthyroidism (25) Crosstalk with ERα (117) No information available Loss or inactivated (84) D1 expression reduced in lung cancer (20) Lower D1 activity than normal tissues (61, 162) Similar D2 activities observed in lung cancer and in peripheral tissue (162) No information available (continued) Volume 6, July

6 Table 1 (Continued) Binding receptor Deiodinase activity Thyrotropin Thyroxine Integrin αvβ3 TRα TRβ1 D1/D2 D3 Thyroid cancer Physiologic levels of thyrotropin supporting DTC growth (34, 159) Hyperthyroidism (65, 128) Positive related to cancer growth (102, 103) No information available Mutation increases in follicular thyroid cancer (109) Induction by the retinoid of D1 as a functional differentiation parameter of FTC cells (28) D1 expression reduced in thyroid cancer (41) Upregulated in papillary thyroid cancer (94) and positively correlated with the tumor size and disease spread (138) Liver cancer No relevant activity Hypothyroidism (>10 years) in HCC (68) Consumptive hypothyroidism with low serum T 3 and elevated serum rt 3 (75) No information available Mutant TRα (13, 104) Mutant TRβ (13, 104) No significant difference of D1 activity between the focal nodular hyperplasia and HCCs and the matched normal liver parenchyma control (92) Decreased D1 activity in hepatic adenocarcinoma (141) High D3 activity in hepatocarcinomas (87) and hepatic hemangiomas (75) Colorectal cancer Decrease in patients (69, 119) Decreased risk of cancer with the use of L-thyroxine for 5 years (54) and Graves disease (8) Induced colon cancer proliferation (18, 63, 98) Positively related to cancer growth (26) Correlation with tumor progression and metastasis in APC+/1638N mice (54) Increasing cancer growth (120) TRβ1 involved in abnormalities in thyroid hormone signaling (74). Loss of TRβ1 accompanied malignant transformation of human colon tumors (26) No information available D3 expression increase (43) Stimulating cancer growth in vitro, xenograft (43) and in vivo (21, 139) TSHR, TSH receptor; HCC, hepatocellular carcinoma; ER, estrogen receptor; TR, thyroid hormone receptor; STAT, Signal Transducer and Activator of Transcription Volume 6, July 2016

7 (Graves disease) with breast cancer. The observation is unrelated to TSH (which is suppressed in Graves disease) and presumably reflects increased circulating levels of T 4. Decreased serum T 3 levels are found in many breast cancer patients during chemotherapy, and this usually reflects the presence of the nonthyroidal illness syndrome (NTIS). Serum thyrotropin is not elevated, and the free T 4 concentration may be normal or elevated in NTIS. Huang et al. prospectively evaluated the effects of chemotherapy on thyroid function in breast cancer patients. Significant decreases in serum T 3,T 4, free T 3, and thyrotropin were found, compared to prechemotherapy measurements (P < 0.05) (76) and could be interpreted as NTIS. Thyroid function test results began to recover postchemotherapy, and there was no significant difference between two consecutive prechemotherapy sets of thyroid function tests. Retrospective studies conducted by Cristofanilli et al. (27) investigated the prevalence of hypothyroidism in patients with breast cancer, and 74 hypothyroid women among 884 breast cancer patients were identified. The average age of the hypothyroid patients was older than that of the euthyroid patients (58.8 vs years, P < 0.001). Their breast cancer symptoms were diagnosed at an earlier stage than that of the euthyroid counterparts (pathologic stage I/II: 95% vs. 85.9%, P = 0.025), tumor sizes were smaller (T1 ( 2 cm): 72.5% vs. 55%, P = 0.002), and no pathologic lymph nodes were involved (63.9% vs. 55.9%). Hypothyroidism alters the body composition, breast morphology, leptin secretion, and serum 17β-estradiol (E 2 ); certain of these changes may retard mammary carcinogenesis in rats (108). Autoimmune thyroid gland failure as a cause of hypothyroidism may spontaneously develop and be unrelated to the cancer in cancer patient cohorts, but in the latter, it is possible that tumor-related increases in secretion of interferon-γ, tumor necrosis factor (TNF), and interleukin-1 may also contribute to inhibition of thyroid hormone secretion (129, 134). Hypothyroidism can decrease inherent chemoresistance mechanisms (76), e.g., by decreasing expression of the P-glycoprotein (P-gp) export pump (see later) (35). Hypothyroidism is a common adverse event in patients treated with antivascular endothelial growth factor receptor (VEGFR)-2 antibody targeting agents such as famitinib and may be a valuable predictive factor of drug efficacy (9). Experimentally, there is evidence in breast cancer cells of overlapping nongenomic and genomic actions of thyroid hormones, estrogens, and testosterone. For example, estrogen and thyroid hormones have similar ERK1/2-dependent proliferative effects on estrogen receptor (ER)-α-positive human breast cancer cells (36). Thus, E 2 and T 3 caused cell proliferation in dose-dependent manners in both MCF-7 and T47-D human breast cancer cell lines. Tang et al. showed that a physiological concentration of T 4 (total T 4,10 7 M, free T 4, <10 10 M in the bovine serum-supplemented medium used) promoted MCF-7 cell proliferation. The hormone also induced phosphorylation of ER-α at Ser-118 via activated ERK1/2 (155); this was comparable to the action of estradiol. These actions of T 4 were blocked by ICI 182,780 (faslodex), a specific ERα antagonist. Similar results were obtained by Moudgil et al. in T47D cells (45). T 3 (10 9 M) was also shown to enhance the effect of E 2 on cell proliferation in the MCF-7 (67, 148) and T47D cell lines (67). Experimental hyperthyroidism and hypothyroidism are, respectively, associated with increased and decreased incidences and aggressiveness of breast cancer in tumor-prone female mice (62). Experimental hypothyroidism was shown to increase apoptosis in female rats in which breast cancer was induced by dimethylbenzanthracene (108). There are also reports in which the thyroid hormone appears to have had an anticancer effect. For example, T 3 downregulated SMP30 gene expression and induced apoptosis in human breast cancer MCF-7 cells (142). In that study, however, pharmacological (micromolar) levels of T 3 were used. T 3 was also shown to downregulate the expression of T1, a gene that is overexpressed in human breast adenocarcinomas and is induced by mitogens, serum, and several oncogenes and cytokines (63). Inhibition of the T1 gene by high concentrations of T 3 (150 nmol/l, threefold to fourfold higher than the physiological free hormone) required both de novo messenger (m)rna and protein synthesis and may interfere with the activation protein 1 transcription factor (63). T 3 represses STAT5 signaling by decreasing STAT5-mediated transcription activity and target gene expression (64). This is a complex model that appears to involve TRβ1 as a tumor-suppressing protein (see later). In examining such studies, one should distinguish between T 3 and T 4. As noted earlier, our own studies of cancer cells and thyroid hormones concluded that T 4 at physiological free hormone concentrations is a proliferative factor for cancer and endothelial cells, but that supraphysiological levels of T 3 are required to induce cancer cell division (30-32, 103, 117). A recent clinical study by Hercbergs and co-workers indicated that induction of a hypothyroxinemic state maintaining eumetabolism with T 3 administration slows or arrests the growth of a variety of advanced cancers (70). In this clinical report, it is clear that physiologic levels of T 3 do not stimulate tumor cell proliferation. We should also point out that the proliferative effects of T 4 appear to be initiated exclusively at the hormone s cell surface receptor, whereas T 3 acts genomically at thyroid hormone receptors in cell nuclei, as well as at the thyroid hormone receptor on integrin αvβ3 (17). A clinical study by Huang and co-workers concluded that T 3 may enhance the chemosensitivity of breast cancer MCF- 7 and MDA-MB-231 cells to 5-fluorouracil (5-FU) and taxol (76). On the other hand, there is evidence that the thyroid hormone can support chemoresistance (35). This may occur via induction of expression of the P-gp (ABCB1) gene, the product of which in plasma membranes exports cancer chemotherapeutic agents from tumor cells. Taxols are exported by this mechanism and, to some extent, 5-FU. We know that the activity of P-gp can be controlled by the plasma membrane thyroid hormone receptor described on integrin αvβ3 (136); Volume 6, July

8 this receptor in cancer cells is more responsive to T 4 than it is to T 3. The euthyroid hypothyroxinemic state, described by Hercbergs (70) to be a cancer-slowing state, depends upon T 3 administration and suppression of endogenous (patient) T 4. We therefore raise the possibility that the observations of Huang et al. on chemosensitization may depend upon suppression of patient T 4 levels via T 3 administration and a reduction of the effect of T 4 on P-gp s function. Thyroid hormone deiodinases D1 and D2 convert T 4 in breast cancer cells and tissues to intracellularly active T 3 (11). On the other hand, estrogens and progesterone independently increase D3 expression in the uterus (4). High enzymatic activity of D1 can potentially lead to an increase in the production of T 3, which may affect target gene transcription, including genes responsible for energy expenditure, growth, differentiation, and proliferation (41). Debski et al. examined the expression and activity of D1 in tissues collected from 36 breast cancer patients undergoing a radical mastectomy or local tumor resection (41). In all noncancerous breast tissues, D1 activity was found to be at a very low or immeasurable level, and D1 mrna was detected in only 2 of 36 samples. In contrast, more than 56% of breast cancers, largely grades G1 and G2, expressed abundant type-1 5 -deiodinase activity and/or a high mrna level and showed a positive correlation between the presence of D1 and the differentiation of breast cancers. TRβ may function as a tumor suppressor in breast cancer development. In a xenograft mouse model, large tumors rapidly developed after inoculation of athymic mice with MCF-7-Neo cells. In contrast, much smaller tumors were found when MCF-7-TRβ cells were inoculated into athymic mice, indicating that TRβ inhibited the E 2 -dependent tumor growth of MCF-7 cells (64). T 3 inhibited STAT5 signaling in TRβ-expressing cells, whereas sustained STAT5 signaling was observed in TRβPV-expressing cells (64). Thus, a TRβ mutation promoted the development of mammary hyperplasia via aberrant activation of STAT5 (64). T 3 induces the inhibition of the estrogen (E 2 )-dependent proliferation of MCF-7 cells via TRβ expression. T 3 reduced the proliferation of mammary epithelial cells and inhibited expressions of the cyclin D1 and T1 genes (64). Both TRα and TRβ are expressed in nuclei of most breast cancer cells. Recent breast cancer studies conducted by Jerzak indicated that 74% of tumors had high expression of TRα1 (Allred score 6), and 40% had high expression of TRα2. The expression of TRα2 was positively correlated with the expression of the ER (P < 0.001) and progesterone receptor (PR; P < 0.001), but negatively with human epidermal growth factor (EGF) receptor (HER2) (P = 0.018). Patients with low TRα2 expression had a reduced 5-year overall survival (75.3%) compared to those with high expression (91.7%; P = 0.06). Many breast tumors express TRα2 at high levels, and those patients experience improved survival (85). Significant correlations of the expression of thyroid hormone receptors especially TRα2 were found with other prognostic histopathological parameters such as tumor size, axillary lymph node involvement, tumor grading, and hormone receptor status (46). A multivariate analysis showed a trend for TRα2 as an independent predictor of disease-free and overall survival (85). Brain tumors Glioblastoma multiforme (GBM) is the most common malignant brain tumor. It has an aggressive growth pattern and poor prognosis despite multiple treatment interventions. The survival rate of patients with GBM is low. Recent preclinical evidence suggests that T 4 stimulates the growth of glioblastomas via a plasma membrane receptor on integrin αvβ3 (31,101,102). Contributions from this receptor and receptorbased stimulation of cellular ERK1/2 activity enhance cell proliferation. In addition, T 3 induces Src kinase or PI3K activation in glioma cells. The thyroid hormones, T 3 and T 4, are equipotent stimulators in vitro of proliferating cell nuclear antigen (PCNA) accumulation in C6, F98, GL261, and U87 cells, but the physiologic concentration of T 3 is 50- fold lower than that of T 4. Although both T 3 and T 4 stimulate ERK1/2, activated ERK1/2 does not contribute to T 3 -induced Src kinase or PI3K activation. Thus, the PI3K, Src kinase, and ERK1/2 signaling cascades are parallel pathways in T 3 - treated U-87MG cells. Both T 3 and T 4 cause proliferation of U-87MG cells, a commonly studied human glioblastoma cell line. PI3K-dependent actions of T 3 in these cells include shuttling of nuclear TRβ from the cytoplasm to nuclei (101) and accumulation of HIF-1α mrna. In clinical studies of glioblastomas, hypothyroid patients exhibited longer survival than euthyroid patients (119). Glioblastoma cell proliferation is stimulated by the thyroid hormone, and this provides a molecular basis for recent clinical observations that induction of mild hypothyroidism, as in other solid tumor patients, may improve the duration of survival (31, 70). Optic gliomas represent 0.6% 1.2% of all brain tumors, and in isolated cases have responded clinically to induction of hypothyroidism with prolonged survival (2). Patients treated for recurrent high-grade gliomas with highdose tamoxifen had significantly longer survival when chemical hypothyroidism was induced with propylthiouracil (PTU) (71). In human astrocytomas, the frequency of TRα1 ortrα2 expression significantly decreases with the grade of the malignancy (P = and P = 0.043, respectively), but the frequency of TRβ1 expression significantly increases with the malignancy grade (P = 0.017) (80). Studies of a human medulloblastoma cell line (HTB-185) revealed that transcription of TRα2 increased and TRβ2 was not expressed (120). Thus, a variety of patterns of thyroid hormones actions are exhibited in brain tumor cells with regard to expression of nuclear thyroid hormone receptors. Mori et al. studied activities of deiodinase 3 (D3) in 20 human brain tumor biopsies obtained intraoperatively. D3 activity was found in 6 of 10 astrocytomas, 2 oligodendrogliomas, 1 of 2 glioblastomas, and 1 malignant lymphoma Volume 6, July 2016

9 Deiodinase was not affected by 1 mmol/l PTU, but was inhibited by iopanoic acid and aurothioglucose in dose-dependent manners. These data suggest that human gliomas and probably malignant lymphomas contain D3 activity that may decrease the intracellular concentration of T 3 in gliomas (121). Observations of Nauman et al. further support this conclusion. In those studies, D3 activity was increased in 17 of 25 tumors (8 of 8 cases of gliosarcomas and 9 of 10 cases of GBM) or decreased in 8 of 8 astrocytomas compared to the mean activity of this enzyme found in nontumoral brain tissues (127). Those studies also showed that the activity of D2 which is responsible for converting T 4 to T 3 in gliomas was present in most astrocytomas (5 of 8 patients), gliosarcomas (8 of 8 patients), and GBM (10 of 10 patients). In general, the mean D2 enzyme activity in tumor tissues was significantly higher than that found in non-tumoral tissues of the human brain (21.79 fmol of newly generated T 3 /h/mg of protein vs fmol of T 3 /h/mg protein, respectively). The highest D2 activity was found in gliosarcomas. rt 3 generated by D3 supports stability of the actin cytoskeleton in brain cells (52), but is otherwise inactive within cells. Lung cancer Lung cancer still has one of the leading mortality rates in the United States, and about 60% of lung cancer patients have advanced disease at the time of diagnosis. Most recently diagnosed lung cancers are already too late for surgery. And the progress of chemotherapy for lung cancer is very limited with a 5-year survival rate of only 15% for all combined stages of lung cancer patients (93). Small-cell lung carcinoma patients may present with symptoms suggestive of hyperthyroidism, such as weight loss without anorexia (49). Almost four decades ago, Ratcliffe reported abnormalities in thyroid function in 67 of 204 (33%) lung cancer patients. The findings included a reduced concentration of circulating T 3 consistent with what later was known as the NTIS (51) and a significantly increased T 4 /T 3 ratio and modest lowering of rt 3 (135). Wawrzynska et al. studied 44 patients undergoing a thoracotomy for lung cancer. Twenty-three patients had squamous cell cancer, and 21 patients had an adenocarcinoma. D1 activity was significantly lower in lung cancer tissues than that in the peripheral lung (13.3 ± 9.5 vs ± 13.4 pmol/min/mg protein, respectively, P < 0.001) (162). D1 expression was found by others to be reduced in lung cancer, renal clear cell carcinoma, and thyroid cancer (41). Studies by Cornelli examined the correlation between the prevalence of lung cancer in 18 regions of Italy and sales of levothyroxine, the most commonly prescribed thyroid hormone replacement for hypothyroid patients. The cancer prevalence was analyzed in women aged years. This age range included more than 80% of the consumers of the drug and accounted for about 99% of all cancers. There was a correlation between lung cancer and T 4 treatment (P < 0.05). The authors proposed that T 4 may contribute to the incidence of lung cancer (25). Hyperthyroidism was examined as a cancer risk factor in animal models, for example, its effects on growth and development of spontaneous pulmonary metastases of Lewis lung carcinoma (3LL) cells (91). A subcutaneous (s.c.) injection of T 4 to induce a hyperthyroid state with elevated T 3 and T 4 levels resulted in primary tumor growth and development of pulmonary metastases of 3LL cells. Treatment with methimazole to induce hypothyroidism in tumor-bearing animals suppressed primary and metastatic tumor growth and prolonged survival. We showed that at physiologic free hormone concentrations, T 4 significantly increased PCNA abundance in lung cancer cells, as did T 3 at a supraphysiologic concentration (117). The effects were mediated by the thyroid hormone receptor on integrin αvβ3. In ERα-positive human lung cancer cells studied in vitro, thyroid hormones (T 4 > T 3 ) caused phosphorylation of ERα. The specific ERα antagonist, ICI 182,780 (faslodex), blocked T 4 -induced, but not T 3 -induced, ERK1/2 activation, ERα phosphorylation, expression of PCNA, and hormone-dependent thymidine uptake by tumor cells. Thus, in ERα-positive human lung cancer cells, the proliferative action of the thyroid hormone initiated at a plasma membrane integrin is at least in part mediated by ERα (117). TRβ1 expression was absent in 61% of small-cell lung cancers (SCLCs) and 48% of non-sclcs. Although TRβ1 mutations are not common in lung cancer, epigenetic inactivation of TRβ1 through aberrant methylation in lung cancer has been described. TRβ1 methylation status was significantly associated with loss of TRβ1 expression. Sixty-seven percent of SCLCs and 45% of non-sclcs carried TRβ1 promoter methylation, while no somatic mutations were found in any cell lines (84). Thyroid cancer Thyroid cancer accounts for a large portion of endocrinerelated cancer deaths each year. The origin of the vast majority of these cancers is the follicular epithelium. The three morphological subtypes are papillary thyroid carcinoma, follicular thyroid carcinoma (FTC), and anaplastic carcinoma (132). Differentiated thyroid cancers, papillary thyroid carcinoma, and FTC constitute 90% of all thyroid cancers (132). Several studies concluded that thyroid carcinoma is common in hyperthyroidism (65, 128). In addition, in a 5-year study of 177 patients who underwent surgery for hyperthyroidism, Ocak et al. found thyroid cancer in 13 (7.3%) on retrospective postoperative histopathologic examination (128). Acting via the thyrotropin receptor on papillary and follicular thyroid cancer cells, TSH is a growth factor for these differentiated thyroid carcinomas (DTCs). Pituitary TSH synthesis/release in this clinical setting is reduced by administration of T 4 as part of management (34). The risks and benefits of this management approach in DTC patients have been addressed by Biondi and Cooper (7). Exposure of human papillary and follicular thyroid cancer cell lines in vitro to the thyroid hormone enhances cell proliferation. The thyroid hormone also induced activation of Volume 6, July

10 the Ras/mitogen-activated protein kinase (MAPK) (ERK1/2) signal transduction pathway. An ERK1/2 cascade inhibitor at mitogen-activated protein kinase kinase (MEK), PD 98059, blocked hormone-induced cell proliferation. Induction of gene expression and apoptosis by resveratrol was inhibited by >50% by physiological concentrations of T 4.Thus,plasma membrane integrin αvβ3-initiated activation of the MAPK cascade by the thyroid hormone supports papillary and follicular thyroid cancer cell proliferation in vitro and may be antiapoptotic (103). In addition, the PI3K-Akt pathway is activated in both primary and metastatic lesions of thyroid carcinoma (57). Inhibition of PI3K activation by the specific inhibitor, LY , blocks Akt-mammalian target of rapamycin-p70(s6k) signaling, decreases cyclin D1, and increases p27(kip1) expression to inhibit thyroid tumor growth and reduce tumor cell proliferation. Treatment with LY compound also increased caspase-3 expression and decreased phosphorylated-bad to induce apoptosis. In addition, LY reduces Akt-matrix metalloproteinase 2 signaling to decrease cell motility and oppose metastatic spread of thyroid tumors (58). D3 generates rt 3 from T 4, as noted above. D3 is upregulated in papillary thyroid cancer, with tumors harboring the BRAFV600E mutation having the highest levels of D3 activity (138). In those cancers, increased D3 activity was positively correlated with the tumor size and disease spread (138). Interestingly, retinoic acid (RA) was shown to induce D1 activity in human thyroid carcinoma cell lines. RA transcriptionally increased the abundance of the p27 D1 subunit. RA stimulated D1 activity to a lesser extent in FTC-238 cells than it did in FTC-133 cells, whereas there was no effect of RA in anaplastic thyroid carcinoma. Interestingly, T 4 and T 3 had no effect on basal or RA-stimulated D1 activity. Induction by the retinoid of D1 may serve as a functional differentiation parameter of FTC cells (145). Thyroid hormone receptors mediate critical activities of thyroid hormones on growth, development, and differentiation. Decreased expression and/or somatic mutations of thyroid hormone receptors are associated with several types of human cancers including those of breast, kidney, lung, liver and thyroid (86, 105, 106, 110). Spontaneous development of FTC in a knock-in mouse model harboring a mutated TRβ (denoted as PV; Thrb(PV/PV) mice) a dominant negative mutation identified in a patient with resistance to the thyroid hormone was used to demonstrate that TRβ mutants may function as oncogenes. The oncogenic activity of PV is mediated by both nucleus-initiated transcription and extranuclear actions that alter gene expressions and signal transduction activities in Thrb(PV/PV) mice (109). This indicates that mutant TRβ can play an important role in thyroid cancer development. However, hypothyroidism reduces PI3K activation in Thrb(PV/PV) mice in which T 3 is unable to bind with mutant TRβ(PV); this suggests that thyroid hormone may promote the thyroid gland carcinogenesis of Thrb(PV/PV) mice via membrane signaling events, possibly binding to integrin αvβ3 (111). Liver cancer and colorectal cancer A high dominance of truncating and point mutations for both TRα and TRβ genes are observed in human hepatocellular carcinoma (HCC) specimens (13, 104, 105). Mutant thyroid hormone receptors with dominant negative activity are often found in human HCC cells (157). The viral oncoprotein, v-erba, also has a dominant-negative effect on the expression of a panel of T 3 -responsive genes involved in carcinogenesis. This raises the possibility that mutant forms of thyroid hormone receptors found in HCC are involved in the pathogenesis of this tumor (157), such as that reported twenty years ago in male mice (3). Mutant TRs have been shown to exert dominant-negative interference with wild type TR function or alter in transcriptional regulation in HCC (13). These alternations appear likely to contribute to oncogenesis in HCC. Overexpression of aldoketo reductase family 1, member B1 (AKR1B1) (99) plays an important role in the development of HCC. The regulation of the expression of AKR1B1 is T 3 -TR dependent. T 3 regulates AKR1B1 gene expression via a TRE-dependant mechanism. A long history of hypothyroidism (>10 years) is associated with a statistically significant increased risk of HCC in women (68). However, in a study such as this, long histories of T 4 -replacement therapy are coincident and whether dosage of T 4 was excessive is not analyzed. A case report by Howard et al. suggests that high D3 activity in hepatic hemangioma patients results in an accelerated rate of TH degradation and clinically relevant hypothyroidism ( consumptive hypothyroidism ) with a low serum T 3 concentration and elevated serum rt 3 (75). However, there is no significant difference of D1 activity between the focal nodular hyperplasia and HCCs and the matched normal liver parenchyma control (92), although decreased D1 activity is reported in hepatic adenocarcinoma (141). Hellevik and co-workers stratified more than 29,000 participants according to circulating thyrotropin levels and prospectively studied cancer risks in the Nord-Trondelag Health Study in Norway (69). Colon cancer was among a number of tumors including breast, prostate, and lung the incidence of which was found by this group of investigators to be increased by a thyrotropin level below the reference range (= subclinical hyperthyroidism). In apparent contrast, the Molecular Epidemiology of Colorectal Cancer (MECC) study conducted in Israel found a decreased risk of largebowel cancer with the use of L-thyroxine for 5 years (137). Interpretation of such findings should take into account the duration of hypothyroidism prior to institution of T 4 replacement, but this may be difficult to estimate. The importance of this factor was emphasized by Cristofanilli and co-workers who found that hypothyroidism delayed the age of appearance (onset) of breast cancer (27). No measurements of circulating thyrotropin levels were available in the MECC study to provide insights into the level of serum thyroid hormone achieved with the amounts of T 4 used for hypothyroidism treatment. Thus, the population surveyed was incompletely 1230 Volume 6, July 2016