Epigenetics: unforeseen regulators in cancer

The past several years have seen a tremendous advance in the understanding of the basic mechanisms of epigenetic regulation. A large number of studies have not only linked epigenetics with cell cycle regulation but also partially unravelled how epigenetics may regulate gene expression. The aim of this review is to provide an overview of the latest findings and current ideas on epigenetics with a focus on emphasizing the emerging influence epigenetics has on the onset and progression of cancer.     

Epigenetics: unforeseen regulators in cancer.

The past several years have seen a tremendous advance in the understanding of the basic mechanisms of epigenetic regulation. A large number of studies have not only linked epigenetics with cell cycle regulation but also partially unravelled how epigenetics may regulate gene expression. The aim of this review is to provide an overview of the latest findings and current ideas on epigenetics with a focus on emphasizing the emerging influence epigenetics has on the onset and progression of cancer.     

Radiogenic initiation of thyroid cancer: a common cellular event

A transplantation system for clonogenic cells in rat thyroid was used, which allows quantitative evaluation of both the acute cytotoxicity and the late neoplastic effects of ionizing radiation at the cellular level in vivo. We have obtained direct experimental evidence that radiogenic initiation of neoplasia in vivo is a common cellular event, and that cell number influences the expression of initiation. Ten per cent of those graft sites which had received 26 clonogens surviving 5 Gy developed carcinomas, while 4 per cent of those which received 26 unirradiated clonogens developed carcinomas. By comparison, 26 per cent of the sites that were inoculated with 411 surviving irradiated clonogens developed carcinomas while none of the 38 transplant sites that received 411 unirradiated clonogens developed carcinomas. Total tumour incidence (carcinomas plus adenomas) followed the same pattern.     

Cell surface involvement in cancer metastasis: an NMR study

NMR spectroscopy is one of the few techniques which has the sensitivity to detect subtle changes to the surface chemistry of cells. It has previously been demonstrated that high resolution ¹H NMR methods can distinguish tumour cells with the capacity to metastasise and this information appears to arise from a type of proteolipid in or attached to the plasma membrane. Here we report that the ¹H NMR signal, which we have used to identify metastatic cells in rat tumours, is significantly reduced in intensity after cultured cells are treated with trypsin/EDTA. The long T₂ relaxation value (⪢ 350 ms) observed in metastatic cells is absent after enzyme treatment. 2D scalar correlated NMR (COSY) spectra of these treated cells show that a cross peak normally associated with malignancy and metastatic disease is markedly reduced. These findings indicate that the plasma membrane lipid particle which generates the high resolution spectrum is directly affected by trypsin/EDTA. Alterations to the cell surface properties were also demonstrated in vivo since reduced numbers of metastases were observed in animals injected with enzyme-treated cells. The correlation between the absence of a long T₂ relaxation value and the diminished numbers of metastases in animals suggests that the plasma membrane particle is involved in the metastatic process.     

Point mutations of ras oncogenes are an early event in thyroid tumorigenesis

Identifying the nature of the genetic mutations in thyroid neoplasms and their prevalence in the various tumor phenotypes is critical to understanding their pathogenesis. Mutational activation of ras oncogenes in human tumors occurs predominantly through point mutations in two functional regions of the molecules, codons 12, 13 (GTP-binding domain) or codon 61 (GTPase domain). We examined the prevalence of point mutations in codons 12, 13, and 61 of the oncogenes K-ras, N-ras, and H-ras in benign and malignant human thyroid tumors by hybridization of PCR-amplified tumor DNA with synthetic oligodeoxynucleotide probes. None of the eight normal thyroid tissues harbored point mutations. Four of nineteen nodules from multinodular goiters (21%), 6/24 microfollicular adenomas (25%), 3/14 papillary carcinomas (21%), and 0/3 follicular carcinomas contained ras point mutations. The predominant mutation was a valine for glycine substitution in codon 12 of H-ras. None of the multinodular goiter tumors known to be polyclonal (and thus due to hyperplasia) had point mutations, whereas one of the two monoclonal adenomas arising in nodular glands contained in H-ras codon 12 valine substitution, which was confirmed by sequencing the tumor DNA. These data show that ras activation is about equally prevalent in benign and malignant thyroid neoplasms, and thus may be an early event in the tumorigenic process.     

'New' active steroids and an unforeseen mechanism of action

Steroid hormones mostly act via nuclear receptors. Dehydroepiandrosterone (DHEA), pregnenolone (PREG) and their sulphate(s), classically known only as hormonal precursors, are also neurosteroidal efficacious signals, modulating neurotransmitter receptor function at the membrane level. An additional unforeseen mechanism of steroid action is reported here: PREG binds to neural microtubule-associated protein MAP2 and increases both the rate and extent of tubulin polymerization studied in vitro with purified tubulin and MAP2, forming microtubules of normal electron microscopic appearance. Progesterone and PREGS also bind to MAP2, but counteract PREG activity. In cultured neurons, PREG specifically increased immunostaining by an antiMAP2 monoclonal antibody and its extension into neurites. This novel mechanism may play a role in regulating microtubule formation and dynamics, which are altered in brain ageing and diseases.     

Photorefractoriness in Japanese quail: possible involvement of the thyroid gland

Quail have a form of refractoriness to long daylengths that is relative and not absolute. The properties of "relative refractoriness" are compared with the more typical "absolute" form seen in starlings and the question is posed as to whether the two types represent the extremes of a single underlying process or are quite separate phenomena. A model is offered that assumes that a single process is involved, and some tests of the hypothesis, notably the involvement of the thyroid gland, are described.     

Parathyroid hormone and plasma calcium control: an editorial

The purpose of this report is to examine the various processes by which parathyroid hormone might control the ionic calcium concentration of plasma and extracellular fluid, and to emphasize the need for study of the maintenance of plasma calcium in the absence of the parathyroid glands. The report discusses mechanisms to explain the control of extracellular calcium and proposes new approaches to the study of calcium homeostasis.     

Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site

Silencer elements, by analogy to enhancer elements, function independently of their position and orientation. We show that the chicken lysozyme silencer S-2.4 kb has many other characteristics in common with enhancer elements. The silencer is comprised of modules that independently repress gene activity--repression being increased synergistically when different or identical modules are combined. Repression is effective both on a complete and on a minimal promoter consisting of a TATA box only. One silencer module is bound in vitro by a 75-93 kd protein, termed NeP1; the other can be bound either by the product of the oncogene v-erbA or by the thyroid hormone receptor. This erbA binding site is unusual in that the palindromic sequence is inverted.     

Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and enhances prostate tumor cell growth both in vivo and in vitro. PTHrP expression also plays a role in the development of bone metastasis, which is a frequent complication in patients with prostate carcinoma. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. We previously showed that PTHrP overexpression increases adhesion of the human prostate cancer cell line PC-3 to the ECM molecules collagen type I, fibronectin, and laminin. Increased adhesion is accompanied by upregulation in the expression of alpha1, alpha5, alpha6, and beta4 integrin subunits. We used the same cell line to study the mechanism via which PTHrP upregulates integrin expression. Clonal PC-3 cells were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Mutation of the NLS negated the effects of PTHrP on alpha1, alpha5, alpha6, and beta4 integrin expression, indicating that these effects are mediated via an intracrine pathway requiring nuclear localization. Expression of the alpha2, alpha3, alphav, and beta1 integrin subunits were comparable in wild-type and NLS-mutated PTHrP transfectants. These findings indicate that PTHrP may play a role in prostate tumor invasion and metastasis by upregulating the expression of specific integrin subunits via an intracrine pathway.     

Parathyroid autotransplantation: Use of cryopreserved glands in an investigation of hypocalcemia

Parathyroid glands were removed from 12 dogs, cryopreserved, and autotransplanted: one-half of these dogs were made eucalcemic with 1,25-(OH)₂-cholecalciferol during the peritransplant period while the other dogs were allowed to become hypocalcemic. The interval of hypocalcemia did not aid in autograft function success. Cryopreserved autotransplanted parathyroid glands can maintain calcium homeostasis in the dog.     

Photorefractoriness in the black-headed buntingEmberiza melanocephala: Possible involvement of the thyroid glands

In the male black-headed bunting,Emberiza melanocephala, photostimulated testicular and/or body weight growth was followed by the regression. Transfer of photorefractory birds from 20L/4D to 23L/1D or from natural lighting (12–13 h) to 20L/4D failed to evoke testicular and/or body weight recrudescence. Thyroidectomy suppressed light-induced increase in the testes and resulted in early regression. Fully developed testes of breeding birds also regressed following thyroidectomy; an effect which was reversed by daily injections of 1 μg/bird of L-T₄. Treatment with L-T₄ at doses from 0·5-2·0 μ/bird/day/ 30 days had no effect on the testes of birds maintained on 12L/12D or following a shift from 12L/12D to 20L/4D. Photoinduced increase in body weight was inhibited by thyroidectomy; an effect which was reversed by treatment with L-T₄ at dose level 1 μg/bird/day. The extent to which thyroidectomy decreased body weight of birds depended upon the lipid reserves at the time of operation. It is suggested that in the male black-headed bunting (i) breeding is terminated by development of absolute-gonadal and metabolic-photorefractoriness and (ii) thyroid hormones are necessary for sustaining light-induced increase in the gonads and/or body weight and for their maintenance, but not for the development of photorefractory state.     

Everolimus is an active agent in medullary thyroid cancer: a clinical and in vitro study

Everolimus, an mTOR inhibitor, which has been demonstrated to induce anti-tumour effects in different types of neuroendocrine tumours, has never been evaluated in patients with medullary thyroid cancer (MTC). The aim of this study was to evaluate the in vitro and in vivo effects of everolimus in combination with octreotide in MTC. Two patients with progressive metastatic MTC and high calcitonin levels were treated with everolimus 5-10 mg/day. Both patients were under treatment with octreotide LAR at the study entry. An in vitro study was also performed to assess everolimus effects on MTC cell lines (TT and MZ-CRC-1 cells). A tumour response was observed in both patients. Serum calcitonin decreased by 86% in patient 1 and by 42% in patient 2. In TT and MZ-CRC-1 cells, everolimus induced a significant dose-dependent inhibition in cell proliferation. This effect seems to be related to a cell cycle arrest in G(0) /G(1) phase in both cell lines and to the induction of cellular senescence in TT cells. Everolimus in combination with octreotide may be active as anti-tumour therapy in patients with progressive metastatic MTC, suggesting to further evaluate this agent in MTC patients in a large prospective study.     

The role of kallikrein-related peptidases in prostate cancer: potential involvement in an epithelial to mesenchymal transition

Several members of the kallikrein-related peptidase family of serine proteases have proteolytic activities that may affect cancer progression; however, the in vivo significance of these activities remains uncertain. We have demonstrated that expression of PSA or KLK4, but not KLK2, in PC-3 prostate cancer cells changed the cellular morphology from epithelial to spindle-shaped, markedly reduced E-cadherin expression, increased vimentin expression and increased cellular migration. These changes are indicative of an epithelial to mesenchymal transition (EMT), a process important in embryonic development and cancer progression. The potential novel role of kallikrein-related peptidases in this process is the focus of this brief review.     

Parathyroid adenoma diagnosed as papillary carcinoma of thyroid on needle aspiration smears

Needle aspiration of a neck mass was performed on a 55-year-old white female with hypercalcemia. The cytologic material showed cohesive clusters of monomorphic hyperchromatic cells resembling follicular cells. Papillary formation and follicle-like arrangements suggested papillary carcinoma of the thyroid. The histopathologic section of the excised mass, however, revealed a parathyroid adenoma. The diagnostic difficulties in differentiating parathyroid adenoma from well-differentiated thyroid carcinoma are discussed. Data published in the literature emphasize similar difficulties, and it is concluded that a final diagnosis can be established only on histopathologic sections.     

Parathyroid hormone related protein and hypercalcaemia in breast cancer.

OBJECTIVE--To see whether parathyroid hormone related protein has a humoral role in breast cancer. DESIGN--Plasma concentrations and tumour expression of parathyroid hormone related protein were determined (by two site immunoradiometric assay and immunohistochemistry respectively) in women with breast cancer and related to the presence of bone metastases and serum calcium concentrations. SUBJECTS--Plasma concentrations of parathyroid hormone related protein were measured in 57 women with early breast cancer without apparent bone metastases, 28 women with bone metastases, and 13 women with bone metastases and hypercalcaemia. Tissue positivity for parathyroid hormone related protein was determined retrospectively in 106 primary breast tumours from women without apparent bone metastases and 72 tumours from women with bone metastases, 25 of whom subsequently developed hypercalcaemia. RESULTS--Plasma parathyroid hormone related protein concentrations were detectable (greater than 0.23 pmol/l) in 12 (92%) of the 13 hypercalcaemic patients with bone metastases compared with 10 (36%) of the 28 normocalcaemic patients with bone metastases and five (9%) of the 57 normocalcaemic patients without bone metastases. Parathyroid hormone related protein concentrations were significantly higher in hypercalcaemic than normocalcaemic patients with bone metastases. Tumour staining was positive for parathyroid hormone related protein in 22 (88%) of the 25 primary breast cancers from patients with bone metastases. Tumour staining was positive for parathyroid hormone related protein in 22 (88%) of the 25 primary breast cancers from patients with bone metastases who later developed hypercalcaemia compared with 25 (53%) of the 47 from women in this group who remained normocalcaemic and 55 (52%) of the 106 early breast cancers from women without known metastases. CONCLUSION--Tumour derived parathyroid hormone related protein may have an important humoral role in hypercalcaemia associated with metastatic breast cancer.     

Parathyroid hormone-related protein regulates tumor-relevant genes in breast cancer cells

The effect of endogenous parathyroid hormone-related protein (PTHrP) on gene expression in breast cancer cells was studied. We suppressed PTHrP expression in MDA-MB-231 cells by RNA interference and analyzed changes in gene expression by microarray analysis. More than 200 genes showed altered expression in response to a PTHrP-specific small interfering (si) RNA (siPTHrP). Cell cycle-regulating gene CDC2 and genes (CDC25B and Tome-1) that control CDC2 activity showed increased expression in the presence of siPTHrP. CDC2 activity was also found to be higher in siPTHrP-treated cells. Studies with PTHrP peptides 1-34 and 67-86, forskolin, and a PTH1 receptor (PTH1R)-specific siRNA showed that PTHrP regulates CDC2 and CDC25B, at least in part, via PTH1R in a cAMP-independent manner. Other siPTHrP-responsive genes included integrin alpha6 (ITGA6), KISS-1, and PAI-1. When combined, siRNAs against ITGA6, PAI-1, and KISS-1 could mimic the negative effect of siPTHrP on migration, whereas siKISS-1 and siPTHrP similarly reduced the proliferative activity of the cells. Comparative expression analyses with 50 primary breast carcinomas revealed that the RNA level of ITGA6 correlates with that of PTHrP, and higher CDC2 and CDC25B values are found at low PTHrP expression. Our data suggest that PTHrP has a profound effect on gene expression in breast cancer cells and, as a consequence, contributes to the regulation of important cellular activities, such as migration and proliferation.     

Tubercular involvement of the thyroid gland: a report of two cases

Thyroid tuberculosis is rare. In the last decade, however, the incidence of extrapulmonary forms of tuberculosis has increased. We report on 2 cases of thyroid tuberculosis. In case 1, a tubercular abscess mimicking acute thyroiditis was found which was correctly diagnosed by fine-needle aspiration biopsy (FNAb). No evidence of active disease was noticed. Pleural thickening on chest X-ray was the only sign compatible with a previous infection. In case 2, tubercular thyroiditis with lymph node enlargement was also diagnosed by FNAb in a reevaluation setting. In both cases treatment with antitubercular drugs resulted in complete recovery. Thyroid tuberculosis should be kept in mind in the differential diagnosis of thyroid nodules, notably in patients with a history of tuberculous disease. FNAb represents the main approach to making the diagnosis.