Molecular Changes

The Natural Thyroid Diet

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Experimental evidence has allowed the delineation of a few crucial pathways in human primary cell transformation. Although there are many important cellular capabilities, allowing a normal cell to bypass cell cycle arrest checkpoints, escape apoptosis, guard against crisis, and provide its own mitogenic signals, may be sufficient to allow for transformation to the oncogenic phenotype. These basic genetic elements may be generalized to most human cancers, however, specific alterations that contribute to oncogenesis are found in some cancers, such as thyroid cancer. These well-defined specific molecular alterations involved both in thyroid-specific and general malignant transformation are described below.

The P53 tumor suppressor gene

Perhaps one of the most common alterations in human cancers is mutation of the P53 pathway, found altered in most, if not all, human cancers (54). Loss of wild-type p53 protein expression, in conjunction with gain-of-function from mutant proteins (55), contribute to acquisition of specialized cell properties, such as proliferative and survival advantages. p53 performs these tasks by acting as a transcription factor induced in response to DNA damage, hypoxia, or oncogene activation (54,56). This, in turn, initiates a program of gene regulation leading down at least two major separate pathways, one for cell cycle arrest to allow time to repair damaged DNA and another for apoptosis to trigger the cell to euthanize (54,57).

Wild-type p53 protein may act as a cellular defense mechanism through its effects on cell cycle arrest and apoptosis, both major obstacles to tumor formation. Cells that are unable to arrest and correct DNA damage have increased potential to develop genetic instability with ongoing replication. At the same time, survival of a neoplastic cell, also includes evasion of apoptosis, preventing the cell-suicide program from taking an antitumor effect. Thus, abrogation of wild-type p53 function, may be sufficient in some tumor types to dismantle the apoptotic machinery (58). However, in other tumors, specific components of the apoptotic cascade, such as bcl-2 (59), Akt (60), or caspases (61), must also be inactivated.

p53 regulates a number of genes involved in the cell cycle. One of these proteins, p21CIl>1, is upregulated by p53 and inhibits the cyclin dependent kinases, resulting in G1 cell cycle checkpoint arrest. Another is Hdm2, a negative regulator of p53, which is also positively regulated by p53 protein itself (Figure 3). Hdm2 physically binds p53

Figure 3. The P53 and RB tumor suppressor pathways. These are both central molecular pathways that are often dysregulated in cancer. Each of these tumor suppressors are regulated by multiple proteins, and disruption can occur at any of these points in human cancer. The role of p53 in apoptosis entails a complex pathway that is not shown on this diagram. Arrows signify activation of the target while blunt lines act in an inhibitory fashion.

Figure 3. The P53 and RB tumor suppressor pathways. These are both central molecular pathways that are often dysregulated in cancer. Each of these tumor suppressors are regulated by multiple proteins, and disruption can occur at any of these points in human cancer. The role of p53 in apoptosis entails a complex pathway that is not shown on this diagram. Arrows signify activation of the target while blunt lines act in an inhibitory fashion.

protein, inhibiting its activity as a transcriptional factor, meanwhile catalyzing p53 ubiquitination which marks it for proteasomal degradation (62) Hdm2 is itselfregulated by P14arf , another tumor suppressor whose protein product binds to and inactivates Hdm2 (63).

While P53 may be directly mutated in over half of all human cancers, in some tumors no P53 mutation is observed, yet other genes in the pathway are altered. For example, Hdm2 can be overexpressed and antagonize p53 protein function in a variety of cancers, including B-cell lymphomas (64), melanomas (65), and breast cancers (66). Other tumors harbor deletions or suppression by methylation, permitting

Hdm2 to remain active and drive the degredation of p53 (63,67). Thus, a various array of genetic and biochemical alterations can converge to enforce a common resultant phenotype, aiding in tumor development and progression.

As will be described in greater detail elsewhere, in thyroid carcinoma, P53 alterations have been found more frequently in both poorly differentiated and undifferentiated thyroid carcinomas (68). Thus, p53 may have a role in the dedifferentiation process. A combination of mutation (69), loss of heterozygosity (70), and overexpression (71,72), presumably from decreased degredation, have all been found in thyroid cancer, again declaring the importance of this critical pathway. (See Chapter 8).

The retinoblastoma (RB) protein

Regulation of passage through the checkpoint of the cell cycle is one of the most important roles of the retinoblastoma protein (73). In its hypophosphorylated form, this protein inhibits cellular commitment to mitosis by blocking cell cycle entry into S-phase. In that state, it is bound to various members of the E2F family of proteins (74). These RB-E2F complexes can inhibit gene transcription by multiple methods: (1) Interfering the ability of free E2Fs' to act as transcriptional factors for cyclin E, cyclin A, and multiple other genes necessary for DNA replication (75) (2) Actively recruiting histone deacetylases (HDACs) (76) and other chromatin remodeling factors to E2F responsive promoters (77).

RB inactivation is a crucial step in allowing a cell to pass the checkpoint and continue through the cell cycle (Figure 3). Normally, one of the cyclin D subtypes (D1, D2, or D3) assembles with one of the cyclin-dependent kinases, CDK4 or CDK6, and cyclin E binds to CDK2. These active holoenzymes phosphorylate RB proteins. Once in a hyperphosphorylated state, RB is unable to bind E2F or HDACs, and releases the repression on genes required for S-phase entry.

Several other tumor suppressor genes also contribute to the phosphorylation status of pRB. For instance, pl6INK4A inhibits the activity of cyclin D-dependent kinases to prevent RB phosphorylation and halt cell division (78). The cyclin E-CDK2 complex is inhibited by both (79) and (80). However, when a strong mitogenic stimulus is present, increased cyclin D1 tends to complex with CDK4, and this combination sequesters This leaves cyclin E-CDK2 free from inhibition to phosphorylate and inactivate RB. E2F, as a result, dissociates from hyper-phosphorylated RB and acts as a transcription factor for a number of responder genes, including cyclin E. The transcription of these responder genes are required for cell cycle progression through the G1 restriction checkpoint, facilitating cellular division.

Like P53, mutations in RB or its associated tumor suppressor genes occur frequently, and disabling this pathway may be required for the formation of human cancer cells (81,82). For example, loss of function mutations of RB also can be found in osteosarcomas and lung cancers, particularly small cell tumors (81). Although RB mutations do occur in non-small cell lung carcinomas, they appear to be present in approximately 20-30% of cases as compared to 80% of the small cell subtype (75). However, p16INK4A loss is evident in over half of all non-small cell lung cancers. Inactivation of P16LS<K4A, by genetic lesions or by methylation, disrupts the RB pathway in a large array of other cancers, including pancreatic, breast, glioblastoma multiforme, and T cell ALL (67,75). Cyclin D1 overexpression drives the cell cycle forward and can also substitute for RB inactivation, as noted in breast cancers (83) and mantle cell lymphomas, where there is juxtaposition of the cyclin D1 gene with the immunoglobulin heavy chain promoter enhancer via a t(11:14) translocation (75). Cyclin E overexpression in breast cancers have also been noted and may help drive past the RB inhibition checkpoint in G1 (84). Finally, in many cervical cancers, human papillomaviruses (HPV) E7 oncopro-tein sequesters and tags RB for degredation (85). Even in those cervical carcinomas that do not express HPV E7, RB somatic mutation is detectable. Alterations in the RB pathway seem to be mutually exclusive, as usually only one component of the pathway is mutated or lost; nonetheless, convergence on the loss of growth suppression by RB does seem to exist in the majority of human cancers (81).

However, the role of the RB in human thyroid cancer remains unclear. Although there are several human immunohistochemical studies (86-88) that remain inconclusive as well as studies evaluating E2f and Rb in rodents (89-91), definitive molecular evidence for the role of RB in human thyroid cancers is lacking. (See Chapter 8).

Mitogenic stimuli and oncogenic RAS

Normal and cancer cells differ in their innate ability to proliferate in the absence of mitogenic stimulation. The presence of surrounding growth factors are crucial for the continued proliferation of normal human cells. Cancer cells, in contrast, have reduced their dependence on external stimuli due to the activation of oncogenic mutations that generate constitutively active mitogenic signals (92). For example, alterations in growth-factor receptors, such as HER2/NEU amplification in breast cancer (93,94) or epidermal growth factor receptor mutation in most carcinomas (95), function as autonomous growth stimuli.

In human thyroid cancer, multiple activating receptors have been implicated in disease pathogenesis. Characteristic chromosomal rearrangements linking the promoter and amino-terminus domains of unrelated gene(s) to the carboxy-terminus of the RET gene result in a constitutively active chimeric receptor, termed (RET/PTC). This event may initiate papillary thyroid cancers (96). Constitutive activation of this mutant kinase promotes interaction with SHC adaptor proteins, intermediates in the RAS signaling pathway (97). Although rare, another early event in papillary thyroid cancers, may involve rearrangements of specific TRK tyrosine kinase receptors (98).

Both epidermal growth factor receptor (EGFR) and its ligands, epidermal growth factor (EGF) and transforming growth factor alpha (TGF-a), are also widely expressed in both normal thyroid and thyroid neoplastic tissue (99,100); however, EGF has a higher binding affinity for neoplastic thyroid tissue when compared to normal tissue (101). EGF and its receptor stimulate proliferation of thyroid cancer cells and enhance invasion (102), suggesting their potential role in malignant progression.

Multiple intracellular protein networks exist downstream of growth factor receptors that can become constitutively active in a mutated state, conferring a growth-inducing effect. As discussed above, introduction of one of these aberrant signals, H-RAS, turns an activating switch on and facilitates malignant transformation to previously immortalized human and rodent primary cells. (See Chapter 7).

Various RAS proteins, members of a large superfamily of low-molecular-weight GTP-binding proteins, control several crucial signaling pathways that regulate cell proliferation. Their ability to effect downstream intracellular signaling proteins first rely on post-translational farnesylation to localize the RAS protein to the cell membrane. Then the ratio of biologically active RAS-GTP to inactive RAS-GDP depends upon the presence and activity of various guanine nucleotide exchange factors (GEFs) and their antagonists, GTPase activating proteins (GAPs) (103).

Multiple effector pathways lay immediately downstream of RAS (Figure 4). The RAF family of proteins, which can trigger a cascade of phosphorylating events through the mitogen-activated protein kinase (MAPK) pathway, leads to cell cycle progression. There is resultant ERK-mediated transcriptional upregulation of angiogenic factors, and increased capability for invasiveness through expression of matrix metallopro-teinases. Through RAS stimulation of phosphatidylinositol 3-kinases (PI3Ks), RAC, which is a Rho family protein, can also increase invasiveness through its effects on the actin cytoskeleton. PI3K also triggers a strong anti-apoptotic survival signal through Akt/protein kinase B (PKB). Much like Akt, RALGDS, which is activated by RAS, inhibits the Forkhead transcription factors ofthe FoxO family which have a role in cell cycle arrest through induction of and apoptosis through the expression of BIM

and FAS ligand (104). Finally, phospholipase C (PLC) is another RAS effector which promotes activation of protein kinase C and calcium mobilization (105). Alterations in the RAS proteins or their downstream effectors can therefore have the potential to lead to constitutively active signals, aiding the oncogenic phenotype. (See Chapter 7).

Activating point mutations of RAS occur in approximately 20% of human tumors, most frequently in pancreatic, thyroid, colorectal, and lung carcinomas, obviating the requirement for the neoplastic cells to encounter external growth stimuli (106,107). In general human cancer and thyroid cancer cells, somatic RAS mutations seem to be an early event. These activating mutations are frequently found in follicular thyroid carcinomas and occasionally papillary thyroid carcinoma (108).

Three members of the RAS family, K-RAS (around 85% of total), which is ubiquitously expressed, N-RAS (about 15%), and H-RAS (less than 1%), are commonly found to be activated by mutation in human tumors (109). These point mutations all prevent GAP induced GTPase activity, leaving RAS in its active, GTP-bound form. GAP deletion also leads to a similar resultant RAS activation; NFI or neurofibromin

Growl^ Factors

Growl^ Factors

Figure 4. Downstream Mediators of RAS. The RAS family of proteins lead down multiple signal transduction networks to not only effect a mitogenic stimulus, but also to provide other important cellular capabilities important for cancer cells. These signaling pathways can lead to cell survival, angiogenic potential, and invasion.

loss is an example of this phenomenon and leads to benign and occasionally malignant tumors of neural crest origin (110). These single point mutations in RAS contribute to many of the "acquired capabilities" of cancer cells, including dysregulated growth, inappropriate survival, invasiveness, and angiogenesis (111).

In many cancers that lack RAS mutations, downstream effectors of RAS signaling are frequently altered, leading to acquisition of a similar set of neoplastic attributes (105). Mutations of the BRAF gene were initially found to be present in around 66% of melanomas and also approximately 12% of colon cancers (112). Recently, two unique somatic mutations of the BRAF gene have been identified in papillary thyroid carcinoma (113,114), and they offer genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway (113). Amplification of the PllOa gene results in PI3K activation in 40% of ovarian tumors, while one of its downstream targets, AKT2, can also be amplified in breast and ovarian carcinomas (115). Finally, the PTEN tumor suppressor gene, acts as a phosphatase on specific downstream targets of PI3K, such as AKT, inactivating that pathway; PTEN deletions occurs in 30-40% of human cancers (116). Altogether, in human cancers the RAS proteins are not only central mediators of both upstream growth factor receptors, but also their downstream targets play critical cellular roles, bestowing constitutively active mitogenic signals as well as multiple other important functions for oncogenesis.

Telomeres and telomerase

Telomeres are terminal structures at the ends of each eukaryotic chromosome and are composed of guanine rich, DNA 5'-TTAGGG-3' repeats, as well as multiple DNA-binding proteins (117,118). At the end of each telomere is a single stranded 3' overhang (119-121) that forms a large secondary loop structure, termed a T-loop (122). Telom-eric DNA is maintained by telomerase, a RNA-dependent, DNA polymerase (123). Telomerase is composed of multiple subunits, two of which are crucial for enzymatic function, the RNA component (hTERC) and catalytic component (hTERT) (124) hTERT is the rate limiting component of the holoenzyme, as hTERT expression is restricted solely to cells that demonstrate telomerase enzymatic activity (125).

One of the main functions of telomeres are to protect the ends of chromosomes from forming illegitimate fusions, which would lead to genetic instability (126-128). Many DNA damage-associated proteins, such as the MRE11 complex (129) and Ku 70/80 (130,131) bind to telomere associated proteins. Thus, it has also been hypothesized that the telomere may serve as a cap, guarding the chromosome end from recognition as damaged DNA (132,133).

Both telomere length and maintenance are associated with human cell lifespan, genetic instability, senescence, immortalization, and transformation. In approximately 90% of human tumors, telomere maintenance and replicative immortality may be achieved through activation of telomerase; the remaining tumors may be maintained through "alternative lengthening of telomeres" (ALT), a telomerase-independent mechanism (134). Interestingly, studies examining malignant transformation in ALT cells lacking P53 and RB function, but expressing oncogenic RAS, confirm that malignant transformation is impossible even with stable telomere lengths unless hTERT is ectopically introduced (135). Thus, overhang and T-loop maintenance by hTERT may have a role in the mechanism of transformation (136). Additionally, hTERT itself may serve some physical capping function that may be important for malignant transformation. Finally, it remains possible that hTERT has some either direct or indirect role in regulation of other important gene(s) that are critical for transformation.

In thyroid cancer, the correlation of telomere length to telomerase activity is poor, implying that there are other mechanisms that regulate telomere dynamics (137). However, most thyroid cancer cells do have sustained telomere length and have assayable telomerase activity while telomerase negative cell telomeres are likely maintained through ALT (138). Thus, similar to other types of cancers, telomere length is also important for thyroid cancer; although hTERT function and telomerase activity in thyroid cancer require further delineation of their mechanism(s) in cancer pathogenesis.

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