Contribution of Stemness-Linked Transcription Regulators to the Progression of Breast Cancer.

Although there are currently several factors that allow measuring the risk of having breast cancer or predicting its progression, the underlying causes of this malignancy have remained unknown. Several molecular studies have described some mechanisms involved in the progress of breast cancer. These have helped in identifying new targets with therapeutic potential. However, despite the therapeutic strategies implemented from the advances achieved in breast cancer research, a large percentage of patients with breast cancer die due to the spread of malignant cells to other tissues or organs, such as bones and lungs. Therefore, determining the processes that promote the migration of malignant cells remains one of the greatest challenges for oncological research. Several research groups have reported evidence on how the dedifferentiation of tumor cells leads to the acquisition of stemness characteristics, such as invasion, metastasis, the capability to evade the immunological response, and resistance to several cytotoxic drugs. These phenotypic changes have been associated with a complex reprogramming of gene expression in tumor cells during the Epithelial- Mesenchymal Transition (EMT). Considering the determining role that the transcriptional regulation plays in the expression of the specific characteristics and attributes of breast cancer during ETM, in the present work, we reviewed and analyzed several transcriptional mechanisms that support the mesenchymal phenotype. In the same way, we established the importance of transcription factors with a therapeutic perspective in the progress of breast cancer.

Treatment of Breast Cancer With Gonadotropin-Releasing Hormone Analogs.

Although significant progress has been made in the implementation of new breast cancer treatments over the last three decades, this neoplasm annually continues to show high worldwide rates of morbidity and mortality. In consequence, the search for novel therapies with greater effectiveness and specificity has not come to a stop. Among the alternative therapeutic targets, the human gonadotropin-releasing hormone type I and type II (hGnRH-I and hGnRH-II, respectively) and its receptor, the human gonadotropin-releasing hormone receptor type I (hGnRHR-I), have shown to be powerful therapeutic targets to decrease the adverse effects of this disease. In the present review, we describe how the administration of GnRH analogs is able to reduce circulating concentrations of estrogen in premenopausal women through their action on the hypothalamus-pituitary-ovarian axis, consequently reducing the growth of breast tumors and disease recurrence. Also, it has been mentioned that, regardless of the suppression of synthesis and secretion of ovarian steroids, GnRH agonists exert direct anticancer action, such as the reduction of tumor growth and cell invasion. In addition, we discuss the effects on breast cancer of the hGnRH-I and hGnRH-II agonist and antagonist, non-peptide GnRH antagonists, and cytotoxic analogs of GnRH and their implication as novel adjuvant therapies as antitumor agents for reducing the adverse effects of breast cancer. In conclusion, we suggest that the hGnRH/hGnRHR system is a promising target for pharmaceutical development in the treatment of breast cancer, especially for the treatment of advanced states of this disease.

Activation of human gonadotropin-releasing hormone receptor promotes down regulation of ARHGAP18 and regulates the cell invasion of MDA-MB-231 cells.

The Gonadotropin-Releasing Hormone Receptor (GnRHR) is expressed mainly in the gonadotrope membrane of the adenohypophysis and its natural ligand, the Gonadotropin-Releasing Hormone (GnRH), is produced in anterior hypothalamus. Furthermore, both molecules are also present in the membrane of cells derived from other reproductive tissues such as the breast, endometrium, ovary, and prostate, as well as in tumors derived from these tissues. The functions of GnRH receptor and its hormone in malignant cells have been related with the decrease of proliferation and the invasiveness of those tumors however, little is known about the molecules associated with the signaling pathways regulated by both molecules in malignant cells. To further analyze the potential mechanisms employed by the GnRHR/GnRH system to reduce the tumorigenesis of the highly invasive breast cancer cell line MDA-MB-231, we performed microarrays experiments to evaluated changes in genes expression and validate these modifications by functional assays. We show that activation of human GnRHR is able to diminish the expression and therefore functions of the Rho GTPase-Activating Protein 18 (ARHGAP18). Decrease of this GAP following GnRHR activation, correlates to the higher of cell adhesion and also with reduction of tumor cell invasion, supporting the notion that GnRHR triggers intracellular signaling pathways that acts through ARHGAP18. On the contrary, although a decline of cellular proliferation was observed during GnRHR activation in MDA-MB-231, this was independent of ARHGAP18 showing the complex system in which is involved the signaling pathways regulated by the GnRHR/GnRH system.

Effects of the lifestyle habits in breast cancer transcriptional regulation.

Through research carried out in the last 25 years about the breast cancer etiology, it has been possible to estimate that less than 10 % of patients who are diagnosed with the condition are carriers of some germline or somatic mutation. The clinical reports of breast cancer patients with healthy twins and the development of disease in women without high penetrance mutations detected, warn the participation more factors in the transformation process. The high incidence of mammary adenocarcinoma in the modern woman and the urgent need for new methods of prevention and early detection have demanded more information about the role that environment and lifestyle have on the transformation of mammary gland epithelial cells. Obesity, alcoholism and smoking are factors that have shown a close correlation with the risk of developing breast cancer. And although these conditions affect different cell regulation levels, the study of its effects in the mechanisms of transcriptional and epigenetic regulation is considered critical for a better understanding of the loss of identity of epithelial cells during carcinogenesis of this tissue. The main objective of this review was to establish the importance of changes occurring to transcriptional level in the mammary gland as a consequence of acute or chronic exposure to harmful products such as obesity-causing foods, ethanol and cigarette smoke components. At analyze the main studies related to topic, it has concluded that the understanding of effects caused by the lifestyle factors in performance of the transcriptional mechanisms that determine gene expression of the mammary gland epithelial cells, may help explain the development of this disease in women without genetic propensity and different phenotypic manifestations of this cancer type.

A través de la investigación realizada en los últimos 25 años en torno a la etiología del cáncer de mama, ha sido posible estimar que menos del 10 % de las pacientes que son diagnosticadas con la enfermedad son portadoras de alguna mutación de línea germinal o somática. Los informes clínicos de pacientes de cáncer de mama con gemelas saludables y el desarrollo de la enfermedad en las mujeres sin mutaciones de alta penetrancia detectadas, advierten la participación de otros factores en el proceso de transformación. La alta incidencia de adenocarcinoma de mama en la mujer moderna y la necesidad urgente de nuevos métodos para la prevención y detección temprana han exigido una mayor información en relación al papel que el medio ambiente y el estilo de vida tienen en la transformación de las células epiteliales de la glándula mamaria. La obesidad, el alcoholismo y el tabaquismo son factores que han demostrado una estrecha correlación con el riesgo de desarrollar cáncer de mama. Y aunque estas condiciones pueden afectar distintos niveles de regulación celular, el estudio de sus efectos en los mecanismos de regulación transcripcional y epigenética, es considerado fundamental para un mayor entendimiento de la pérdida de identidad de las células epiteliales durante la carcinogénesis de este tejido. El objetivo principal de esta revisión fue establecer la importancia de los cambios que ocurren a nivel transcripcional en la glándula mamaria, como consecuencia de una exposición aguda o crónica a productos nocivos, tales como los alimentos que favorecen la obesidad, el etanol y los componentes del humo del cigarro. Al analizar los principales estudios relacionados con el tema, se ha llegado a la conclusión de que la comprensión de los efectos causados por los factores de estilo de vida sobre el desempeño de los mecanismos de regulación transcripcional responsables de la expresión génica de las células epiteliales de la glándula mamaria, pueden ayudar a explicar el desarrollo de esta enfermedad en las mujeres que no son genéticamente propensas así como las diferentes manifestaciones fenotípicas de este tipo de cáncer.

The gonadotropin-releasing hormone system: Perspectives from reproduction to cancer (Review).

Recently, an increasing amount of evidence indicates that human gonadotropin-releasing hormone (hGnRH) and its receptor (hGnRHR) are important regulatory components not only to the reproduction process but also in the regulation of some cancer cell functions such as cell proliferation, in both hormone-dependent and -independent types of tumors. The hGnRHR is a naturally misfolded protein that is retained mostly in the endoplasmic reticulum; however, this mechanism can be overcome by treatment with several pharmacoperones, therefore, increasing the amount of receptors in the cell membrane. In addition, several reports indicate that the expression level of hGnRHR in tumor cells is even lower than in pituitary or gonadotrope cells. The signal transduction pathways activated by hGnRH in both gonadotrope and different cancer cell types are described in the present review. We also discuss how the rescue of misfolded receptors in tumor cells could be a promising strategy for cancer therapy.

Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion. METHODS: Actin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay. RESULTS: Agonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers. CONCLUSIONS: We here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer.

Pharmacological chaperones correct misfolded GPCRs and rescue function: protein trafficking as a therapeutic target.

G-protein-coupled receptors (GPCRs) are a large superfamily of plasma membrane proteins that play central roles in transducing endocrine, neural and -sensory signals. In humans, more than 30 disorders are associated with mutations in GPCRs and these proteins are common drug development targets, with 30-50% of drugs targeting them. GPCR mutants are frequently misfolded, recognized as defective by the cellular quality control system, retained in the endoplasmic reticulum and do not traffic to the plasma membrane. The use of small molecules chaperones (pharmacological chaperones or "pharmacoperones") to rescue misfolded GPCRs has provided a new approach for treatment of human diseases caused by misfolding and misrouting. This chapter provides an overview of the molecular basis of this approach using the human gonadotropin-releasing hormone receptor (hGnRHR) as model for treatment of conformational diseases provoked by -misfolded GPCRs.

Biochemical mechanism of pathogenesis of human gonadotropin-releasing hormone receptor mutants Thr104Ile and Tyr108Cys associated with familial hypogonadotropic hypogonadism.

The pathogenic mechanisms whereby the Thr104Ile and Tyr108Cys mutations in the gonadotropin-releasing hormone receptor (GnRHR) gene cause hypogonadotropic hypogonadism in humans are unknown. Transient expression of Thr104Ile and Tyr108Cys mutants in COS-7 cells revealed that both GnRHR mutants neither bind nor respond to agonist. Removal of Lys191 rescued function of both mutants, while addition of a carboxyl-terminal targeting sequence only rescued function of the Thr104Ile mutant. Exposure to the pharmacoperone In3 rescued almost completely Thr104Ile mutant function to wild-type levels, whereas rescue was partial for the Tyr108Cys GnRHR. Additional mutations that block formation of bridges involving Cys108 showed that a Cys108-Cys200 disulfide bridge is the predominant moiety formed in the Tyr108Cys mutant. Thr104Ile and Tyr108Cys GnRHRs are misfolded structures whose function is rescuable by genetic and/or pharmacological strategies. The Tyr108Cys mutant forms an aberrant disulfide bridge that prevents formation of the required Cys14-Cys200 bridge essential for GnRHR plasma membrane expression.

Dominant negative effects of human follicle-stimulating hormone receptor expression-deficient mutants on wild-type receptor cell surface expression. Rescue of oligomerization-dependent defective receptor expression by using cognate decoys.

Current evidence indicates that G protein-coupled receptors form dimers that may affect biogenesis and membrane targeting of the complexed receptors. We here analyzed whether expression-deficient follicle-stimulating hormone receptor (FSHR) mutants exert dominant negative actions on wild-type FSHR cell surface membrane expression. Co-transfection of constant amounts of wild-type receptor cDNA and increasing quantities of mutant (R556A or R618A) FSHR cDNAs progressively decreased agonist-stimulated cAMP accumulation, [(125)I]-FSH binding, and plasma membrane expression of the mature wild-type FSHR species. Co-transfection of wild-type FSHR fragments involving transmembrane domains 5-6, or transmembrane domain 7 and/or the carboxyl-terminus specifically rescued wild-type FSHR expression from the transdominant inhibition by the mutants. Mutant FSHRs also inhibited function of the luteinizing hormone receptor but not that of the thyrotropin receptor or non-related receptors. Defective intracellular transport and/or interference with proper maturation due to formation of misfolded mutant:wild-type receptor complexes may explain the negative effects provoked by the altered FSHRs.

Conformational effects of Lys191 in the human GnRH receptor: mutagenesis and molecular dynamics simulations studies.

In the present study, we analyzed the role of Lys191 on function, structure, and dynamic behavior of the human GnRH receptor (hGnRHR) and the formation of the Cys14-Cys200 bridge, which is essential for receptor trafficking to the plasma membrane. Several mutants were studied; mutants lacked either the Cys14-Cys200 bridge, Lys191 or both. The markedly reduced expression and function of a Cys14Ser mutant lacking the 14-200 bridge, was nearly restored to wild-type/DeltaLys191 levels upon deletion of Lys191. Lys191 removal resulted in changes in the dynamic behavior of the mutants as disclosed by molecular dynamics simulations: the distance between the sulfur- (or oxygen-) sulfur groups of Cys (or Ser)14 and Cys200 was shorter and more constant, and the conformation of the NH(2)-terminus and the exoloop 2 exhibited fewer fluctuations than when Lys191 was present. These data provide novel information on the role of Lys191 in defining an optimal configuration for the hGnRHR intracellular trafficking and function.

Increased plasma membrane expression of human follicle-stimulating hormone receptor by a small molecule thienopyr(im)idine.

A thienopyr(im)idine (Org41841) activates the luteinizing hormone (LH) receptor but does not compete with the natural ligand binding site and does not show agonistic action on the follicle-stimulating hormone receptor (hFSHR) at sub-millimolar concentrations. When this drug is preincubated at sub-micromolar concentrations with host cells expressing the hFSHR, and then washed out, binding analysis and assessment of receptor-effector coupling show that it increases plasma membrane expression of the hFSHR. Real-time PCR shows that this effect did not result from increased hFSHR mRNA accumulation. It is possible that Org41841 behaves as a pharmacoperone, a drug which increases the percentage of newly synthesized receptor routing to the membrane. Like pharmacoperones for other receptors, this drug was able to rescue a particular mutant hFSHR (A(189)V) associated with misrouting and endoplasmic reticulum retention, although other mutants could not be rescued. This is potentially the first member of the pharmacoperone drug class which binds at a site that is distinctive from the ligand binding site.

Beyond the signal sequence: protein routing in health and disease.

Receptors, hormones, enzymes, ion channels, and structural components of the cell are created by the act of protein synthesis. Synthesis alone is insufficient for proper function, of course; for a cell to operate effectively, its components must be correctly compartmentalized. The mechanism by which proteins maintain the fidelity of localization warrants attention in light of the large number of different molecules that must be routed to distinct subcellular loci, the potential for error, and resultant disease. This review summarizes diseases known to have etiologies based on defective protein folding or failure of the cell's quality control apparatus and presents approaches for therapeutic intervention.

Transcriptional regulation of the GnRH receptor gene by glucocorticoids.

Expression of the gonadotropin-releasing hormone (GnRH) receptor gene is stimulated by dexamethasone in GnRH-deficient rodents. In this study we identify a 1226 bp sequence at the 5'-flanking region of the mouse GnRH-R gene that confers dexamethasone responsiveness when expressed in host cells. Further, a glucocorticoid antagonist blocks transcriptional activity of the mGnRHR promoter. Progressive 5'-deletion of the mGnRHR promoter localized the response sequence between the -331/-255 region. Analysis of this region revealed binding sites for the AP-1 transcription factor. Mutation in AP-1 modified the functional activity of the mGnRHR promoter following GnRH agonist or dexamethasone-stimulation. Using an electrophoretic mobility shift assay, a protein complex is shown to bind to the AP-1 site. These results suggest that AP-1 proteins and glucocorticoid receptor regulate transcription of the GnRH-R promoter in a heterologous system.

Somatic and germinal mosaicism for the steroid sulfatase gene deletion in a steroid sulfatase deficiency carrier.

Steroid sulfatase deficiency results in X-linked ichthyosis, an inborn error of metabolism in which the principal molecular defect is the complete deletion of the steroid sulfatase gene and flanking markers. Mosaicism for the steroid sulfatase gene has not yet been reported in X-linked ichthyosis. In this study we describe an X-linked ichthyosis patient with complete deletion of the steroid sulfatase gene and his mother with somatic and germinal mosaicism for this molecular defect. The family (X-linked ichthyosis patient, grandmother, mother, and sister) was analyzed through steroid sulfatase enzyme assay, polymerase chain reaction, DNA markers, and fluorescence in situ hybridization of the steroid sulfatase gene. Steroid sulfatase activity was undetectable in the X-linked ichthyosis patient, very low in the mother, and normal in the grandmother and sister. The X-linked ichthyosis patient showed a 2 Mb deletion of the steroid sulfatase gene and flanking regions from 5'DXS1139 to 3'DXF22S1. The mother showed one copy of the steroid sulfatase gene in 98.5% of oral cells and in 80% of leukocytes. The grandmother and sister showed two copies of the steroid sulfatase gene. The origin of the X chromosome with the deletion of the steroid sulfatase gene corresponded to the grandfather of the proband. We report the first case of somatic and germinal mosaicism of the steroid sulfatase gene in an X-linked ichthyosis carrier and propose DNA slippage as the most plausible mechanism in the genesis of this mosaicism.

Rescue of hypogonadotropic hypogonadism-causing and manufactured GnRH receptor mutants by a specific protein-folding template: misrouted proteins as a novel disease etiology and therapeutic target.

In the present study, we demonstrate pharmacological rescue (assessed by ligand binding and restoration of receptor coupling to effector) of five naturally occurring GnRH receptor (GnRHR) mutants (T(32)I, E(90)K, C(200)Y, C(279)Y, and L(266)R), identified from patients with hypogonadotropic hypogonadism, as well as rescue of other defective receptors intentionally manufactured with internal or terminal deletions or substitutions at sites expected to be involved in establishment of tertiary receptor structure. The pharmacological agent used is a small, membrane-permeant molecule, originally designed as an orally active, nonpeptide receptor antagonist, but is believed to function as a folding template, capable of correcting the structural defects caused by the mutations and thereby restoring function. After rescue, this agent can be demonstrably removed. The rescued receptor, now stabilized in the plasma membrane, couples ligand binding to activation of the appropriate effector system. For comparison, low-, intermediate-, or high-affinity peptide antagonists of GnRHR (that do not penetrate the cell) were unable to effect rescue, as was a nonbinding peptidomimetic congener of the rescue agent; this latter effect demonstrates specificity of the rescue agent. Our findings, taken in concert with an earlier study showing rescue of a mutant by modifications to the receptor structure that enhance plasma membrane expression of the GnRHR, suggest that mutant GnRHRs have frequently not lost intrinsic functionality and are subject to rescue by techniques that enhance membrane expression. The present findings demonstrate the efficacy of an approach based on pharmacological rescue and suggest the basis of new approaches for intervention in this and similar diseases.

Molecular basis of hypogonadotropic hypogonadism: restoration of mutant (E(90)K) GnRH receptor function by a deletion at a distant site.

GnRH regulates the synthesis and release of pituitary gonadotropins. Mutations in the human GnRH receptor (hGnRHR) gene have been reported in families with hypogonadotropic hypogonadism. Our group recently described a novel homozygous E(90)K mutation of the hGnRHR in two siblings with the complete form of hypogonadotropic hypogonadism. In the present study, mutational analysis of the E(90)K substitution was performed to assess the functional role of this particular residue, which is located in the second transmembrane helix of the hGnRHR. Although E(90) is highly conserved in all other known mammalian GnRH receptors, this residue has not been previously implicated in GnRH binding and/or GnRHR activation. Transient expression of the mutant E(90)K receptor in COS-7 cells resulted in a virtual abolition of GnRH agonist binding and agonist-stimulated phosphoinositide turnover, initially suggesting that E(90) may be essential for GnRH binding. Furthermore, incubation with 1 microM of different GnRH agonists (D-Trp(6)-GnRH, GnRH, leuprolide, Catfish-1 GnRH, Catfish-2 GnRH, D-Lys(6)-Pro(9)-EA-GnRH, DesGly(10)-GnRH, D-Trp(6)-Pro(9)-EA-GnRH, Buserelin, and D-Lys(6)-GnRH) or antagonists (Antide and "Nal-Arg") did not result in elevated inositol phosphate production from cells expressing the E(90)K mutant. To examine the role of a site known to suppress hGnRHR function, mutants with deletion of K(191) (DeltaK(191)) from the hGnRHR and/or addition of catfish GnRHR intracellular carboxyl-terminal tail (cfCtail) to hGnRHR were prepared. Exposure to the GnRH analog Buserelin resulted in a significant increase in total inositol phosphate production in cells expressing the hGnRHR-cfCtail, hGnRHR(DeltaK(191)) and hGnRHR(DeltaK(191))-cfCtail. Activation of intracellular signaling in response to Buserelin was restored by deletion of K(191) from the E(90)K mutant receptor but minimally by addition of the catfish GnRHR carboxyl-terminal tail. There were no significant differences in total inositol phosphate production between the chimeric receptors bearing the DeltaK(191) or the E(90)K/DeltaK(191) modifications. All but the (E(90)K) and (E(90)K)-cfCtail altered receptors were membrane expressed as disclosed by Western blot analysis of epitope-tagged receptors. This study provides evidence that the E(90)K mutation impairs hGnRHR-effector coupling. The observation that sequence modifications that enhance surface expression of the receptor restore function, presents the possibility that loss of surface expression may underlie the severe phenotype exhibited by hypogonadotropic hypogonadism patients bearing this mutational defect.

Conserved mammalian gonadotropin-releasing hormone receptor carboxyl terminal amino acids regulate ligand binding, effector coupling and internalization.

The mammalian gonadotropin-releasing hormone receptor (GnRHR), with 327 amino acids, is among the smallest G protein coupled receptors identified. Absent from this receptor is the cytoplasmic tail, characteristic of other members of this superfamily, which frequently mediates desensitization and down-regulation. The fifteen carboxyl terminal residues in the mammalian GnRHR are absolutely conserved, suggesting important roles for these residues. In the current study, mutations of the mammalian GnRHR were made to study the carboxyl terminus. The receptor mutant GnRHR(Ser(326)Ala) was reduced in ligand affinity (117% reduction compared to wild type (wt)), while receptor numbers and internalization remained unchanged. GnRHR(Ser(326)Tyr) was decreased in effector coupling, while ligand affinity remained unchanged compared to wt. These studies also show that, while mutation of Ser(326) caused a change in ligand binding and effector coupling, truncation at this residue (GnRHR[des(326-327)]) had no measurable effect on GnRHR ligand binding, effector coupling or internalization, functions which appear to require different structural determinants than expression and routing. Removal of all three carboxyl terminal residues (Phe(325), Ser(326) and Leu(327)) or mutation of the receptor (GnRHR[Phe(325)Ala]) caused a complete loss of measurable ligand binding and effector coupling, clearly suggesting an unexplained role for Phe(325).