RESUMEN
BACKGROUND: Every year approximately 74,000 women die of endometrial cancer, mainly due to recurrent or metastatic disease. The presence of tumor infiltrating lymphocytes (TILs) as well as progesterone receptor (PR) positivity has been correlated with improved prognosis. This study describes two mechanisms by which progesterone inhibits metastatic spread of endometrial cancer: by stimulating T-cell infiltration and by inhibiting epithelial-to-mesenchymal cell transition (EMT). METHODOLOGY AND PRINCIPAL FINDINGS: Paraffin sections from patients with (nâ=â9) or without (nâ=â9) progressive endometrial cancer (recurrent or metastatic disease) were assessed for the presence of CD4+ (helper), CD8+ (cytotoxic) and Foxp3+ (regulatory) T-lymphocytes and PR expression. Progressive disease was observed to be associated with significant loss of TILs and loss of PR expression. Frozen tumor samples, used for genome-wide expression analysis, showed significant regulation of pathways involved in immunesurveillance, EMT and metastasis. For a number of genes, such as CXCL14, DKK1, DKK4, PEG10 and WIF1, quantitive RT-PCR was performed to verify up- or downregulation in progressive disease. To corroborate the role of progesterone in regulating invasion, Ishikawa (IK) endometrial cancer cell lines stably transfected with PRA (IKPRA), PRB (IKPRB) and PRA+PRB (IKPRAB) were cultured in presence/absence of progesterone (MPA) and used for genome-wide expression analysis, Boyden- and wound healing migration assays, and IHC for known EMT markers. IKPRB and IKPRAB cell lines showed MPA induced inhibition of migration and loss of the mesenchymal marker vimentin at the invasive front of the wound healing assay. Furthermore, pathway analysis of significantly MPA regulated genes showed significant down regulation of important pathways involved in EMT, immunesuppression and metastasis: such as IL6-, TGF-ß and Wnt/ß-catenin signaling. CONCLUSION: Intact progesterone signaling in non-progressive endometrial cancer seems to be an important factor stimulating immunosurveilance and inhibiting transition from an epithelial to a more mesenchymal, more invasive phenotype.
Asunto(s)
Neoplasias Endometriales/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Progesterona/farmacología , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Progresión de la Enfermedad , Neoplasias Endometriales/genética , Neoplasias Endometriales/inmunología , Neoplasias Endometriales/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Genómica , Humanos , Persona de Mediana Edad , Receptores de Progesterona/metabolismo , Estudios Retrospectivos , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
Usual type VIN is a premalignant disorder caused by persistent HPV infection. High prevalence of VIN in immuno-suppressed women suggests that a good innate and adaptive immune response is important for defense against HPV. Here, we explored expression levels of chemokines and related these to the presence or absence of immuno-competent cells (dendritic and T-cells) in affected (HPV-positive VIN) and non-affected (HPV-negative) vulvar tissues from the same patients. Combining microarray data with quantitative real-time RT-PCR, it was observed that several important chemokines were differentially expressed between VIN and control samples (up-regulation of IL8, CXCL10, CCL20 and CCL22 and down-regulation of CXCL12, CCL21 and CCL14). Furthermore, an increased number of mature dendritic cells (CD208+) seemed to be bottled up in the dermis, and although a T-cell response (increased CD4+ and CD8+ cells) was observed in VIN, a much larger response is required to clear the infection. In summary, it seems that most mature dendritic cells do not receive the proper chemokine signal for migration and will stay in the dermis, not able to present viral antigen to naive T-cells in the lymph node. Consequently the adaptive immune response diminishes, resulting in a persistent HPV infection with increased risk for neoplasia.
Asunto(s)
Alphapapillomavirus , Quimiocinas/metabolismo , Células Dendríticas/inmunología , Huésped Inmunocomprometido , Infecciones por Papillomavirus/complicaciones , Linfocitos T/inmunología , Infecciones Tumorales por Virus/complicaciones , Displasia del Cuello del Útero/inmunología , Neoplasias del Cuello Uterino/inmunología , Adulto , Quimiocinas/genética , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Análisis por Micromatrices , Persona de Mediana Edad , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Infecciones Tumorales por Virus/metabolismo , Infecciones Tumorales por Virus/virología , Regulación hacia Arriba , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/virologíaRESUMEN
For the endometrium, estradiol and tamoxifen induce proliferation, and consequently, tamoxifen treatment of breast cancer results in a 2-fold to 7-fold increased risk for endometrial cancer. Here, the role of activation of growth factor receptor signaling in mediating the effects of estrogen and tamoxifen is determined. Microarray analysis of ECC-1 cells treated with estradiol or tamoxifen indicate that rapid responses to treatment (1 hour) are very distinct from long-term responses (>24 hours). Furthermore, estradiol and tamoxifen are observed to induce AKT activation. Comparing long-term estrogen- and tamoxifen-regulated genes with genes regulated by insulin-like growth factor 1 and amphiregulin reveals that the late effects of estrogen and tamoxifen signaling may partly be mediated via activation of growth factor receptor signaling pathways. It is hypothesized that both early and late effects of estrogen and tamoxifen signaling in the endometrium are partly mediated via the activation of growth factor receptor signaling, putatively at the level of AKT activation.
Asunto(s)
Endometrio/metabolismo , Estradiol/farmacología , Estrógenos/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Tamoxifeno/farmacología , Línea Celular Tumoral , Endometrio/efectos de los fármacos , Receptores ErbB/metabolismo , Estrógenos/genética , Estrógenos/farmacología , Femenino , Perfilación de la Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-akt/genética , Receptores de Factores de Crecimiento/genética , Transducción de SeñalRESUMEN
OBJECTIVES: In different tissues, estrogens, selective estrogen receptor modulators (SERMs), and anti-estrogens exert different biologic activities. For the endometrium, estradiol and tamoxifen induce proliferation, and because of this, tamoxifen treatment of breast cancer patients results in a two- to sevenfold increased risk for development of endometrial cancer. Use of raloxifene, or the anti-estrogen ICI182780, does not result in such an increased risk. The objective of the current study was to generate and analyze gene expression profiles that reflect the transcriptional response of the human endometrium to estradiol, SERMs like tamoxifen and raloxifene, and anti-estrogens like ICI182780. METHODS: Transient transfections were performed to analyze the transcriptional response of ECC-1 cells to estradiol, tamoxifen, raloxifene, and ICI182780. Subsequently, to reveal the molecular mechanism of action, gene expression profiles were generated and some of the observed regulated genes were confirmed by Northern blotting. Biostatistical methods were employed to analyze the expression profile results further, and amphiregulin effects on ECC-1 cell signaling were investigated using Northern and Western blotting, and 3H-thymidine incorporation. RESULTS: Analysis of the profiles revealed that estradiol, tamoxifen, raloxifene, and ICI182780 influence the same biologic processes, but they do so via regulation of different sets of genes. Upon construction of a genetic network it was observed that the largest possible network centered on epidermal growth factor (EGF) receptor signaling. Furthermore, the EGF receptor ligand amphiregulin was differentially regulated by all four ligands. Next it was shown that amphiregulin indeed could stimulate EGF receptor signaling in ECC-1 cells. Based on these results, it was hypothesized that EGF receptor signaling could differentially be affected by estrogen, tamoxifen, raloxifene, and ICI182780 because these four compounds differentially regulate the EGF receptor ligand amphiregulin. CONCLUSIONS: Regulation of amphiregulin coincides with the described in vivo effect of the four ligands on the endometrium. Therefore, it is possible that modulation of EGF receptor signaling is a significant player in estrogen-agonistic growth of the endometrium and needs to be investigated further.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Neoplasias Endometriales/patología , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Glicoproteínas/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Clorhidrato de Raloxifeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Anfirregulina , Familia de Proteínas EGF , Estradiol/farmacología , Estradiol/fisiología , Femenino , Fulvestrant , Perfilación de la Expresión Génica , Humanos , Transcripción Genética , Células Tumorales CultivadasRESUMEN
OBJECTIVE: Progesterone plays an important role in controlling proliferation and differentiation of the human endometrium. Because there are two progesterone receptor isoforms (PRA and PRB), it was important to generate tools to be able to study the role of these two progesterone receptors separately. METHODS: Using stable transfection techniques, both human progesterone receptor isoforms (hPRA and hPRB) were reintroduced into a hPR-negative subclone of the well-differentiated endometrial cancer cell line Ishikawa. Several Ishikawa subcell lines were constructed, each expressing different levels of hPRA, hPRB, or hPRA and hPRB, respectively. RESULTS: These Ishikawa subcell lines showed a marked progesterone-induced growth inhibition with induction of apoptosis after long-term culture in the presence of hormone. Upon measuring gene regulation, a clear difference in regulation of expression of the selected genes by progesterone treatment was observed between the PRA-, PRB-, or PRA/B-expressing cell lines. Integrin beta4 (ITGB4) was only regulated in PRA-expressing cells; amphiregulin was highly regulated in PRB-expressing cells; insulin-like growth factor binding protein 3 (IGFBP3) was only regulated in PRB- and PRA/B-expressing cells; and metallothionein 1L (MT1L) was highly regulated in PRA/B-expressing cells. Interestingly, based on literature data, these genes can be implicated in induction of apoptosis, but are modulated here in such a way that suggests induction of resistance against apoptosis. CONCLUSION: Reintroduction of PRs into Ishikawa cells rescued progesterone responsiveness in these cells. Furthermore, using these human endometrial cancer subcell lines, clear and distinct functional differences between the PR isoforms were observed.
Asunto(s)
Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Progesterona/fisiología , Receptores de Progesterona/biosíntesis , Apoptosis , Diferenciación Celular , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Isoformas de Proteínas , Transfección , Células Tumorales CultivadasRESUMEN
In advanced prostate cancer, cellular changes occur leading to a transition from androgen-dependent to androgen-independent growth. During this transition, proliferation of androgen-dependent prostate cancer cells becomes more and more dependent on growth factors, like the epidermal growth factor (EGF). Endocytosis of growth factor receptors, one of the mechanisms that controls growth factor signalling, was observed to be markedly changed in advanced metastatic prostate cancer. Internalisation and signalling of EGF receptors was examined in different prostate cancer cell lines, in relation to the expression level of the endocytosis-related REPS2 gene. It was observed that a high level of REPS2 correlates with reduced EGF-internalisation. To investigate this more thoroughly, prostate cancer cells with inducible REPS2 expression were generated. Using these cells, it was found that REPS2-induction indeed results in reduction of EGF-internalisation. Furthermore, when EGF receptor signalling was evaluated, by examination of mRNA expression for several EGF-responsive genes (EGF receptor, EGR-1, Fos and Jun), it was observed that induced expression of REPS2 exerts an inhibiting effect on this signalling. From these experiments, it is concluded that increased REPS2 expression negatively affects EGF receptor internalisation and subsequent signalling. Therefore, decreased REPS2 expression during prostate cancer progression, observed in earlier work, may result in enhanced EGF receptor expression and signalling, which could add to the androgen-independent state of advanced prostate cancer.
Asunto(s)
Endocitosis , Factor de Crecimiento Epidérmico/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias de la Próstata/metabolismo , Andrógenos/farmacología , Proteínas de Unión al Calcio , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Neoplasias Hormono-Dependientes/genética , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
PURPOSE: In endometrial cancer, loss of progesterone receptors (PR) is associated with more advanced disease. This study aimed to investigate the mechanism of action of progesterone and the loss of its receptors (PRA and PRB) in development of endometrial cancer. EXPERIMENTAL DESIGN: A 9600-cDNA microarray analysis was performed to study regulation of gene expression in the human endometrial cancer subcell line Ishikawa PRAB-36 by the progestagen medroxy progesterone acetate (MPA). Five MPA-regulated genes were selected for additional investigation. Expression of these genes was studied by Northern blot and by immunohistochemistry in Ishikawa subcell lines expressing different PR isoforms. Additionally, endometrial cancer tissue samples were immunohistochemically stained to study the in vivo protein expression of the selected genes. RESULTS: In the PRAB-36 cell line, MPA was found to regulate the expression of a number of invasion- and metastasis-related genes. On additional investigation of five of these genes (CD44, CSPG/Versican, Tenascin-C, Fibronectin-1, and Integrin-beta 1), it was observed that expression and progesterone regulation of expression of these genes varied in subcell lines expressing different PR isoforms. Furthermore, in advanced endometrial cancer, it was shown that loss of expression of both PR and E-cadherin was associated with increased expression CD44 and CSPG/Versican. CONCLUSION: The present study shows that progestagens exert a modulatory effect on the expression of genes involved in tumor cell invasion. As a consequence, loss of PR expression in human endometrial cancer may lead to development of a more invasive phenotype of the respective tumor.
Asunto(s)
Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Regulación Neoplásica de la Expresión Génica/genética , Receptores de Progesterona/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Línea Celular Tumoral , Proteoglicanos Tipo Condroitín Sulfato/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Receptores de Hialuranos/genética , Lectinas Tipo C , Acetato de Medroxiprogesterona/farmacología , Invasividad Neoplásica/genética , Proteínas de Neoplasias/genética , Tenascina/genética , VersicanosRESUMEN
To study the functional differences between the two progesterone receptor isoforms (hPRA and hPRB) in human endometrial cancer, two new endometrial carcinoma cell lines were created-one expressing hPRA and one expressing hPRB.A well-differentiated, hPR-negative Ishikawa cell line was stably transfected with either hPRA or hPRB cDNA. Transfected cells were selected, and two cell lines expressing approximately equal amounts of receptor were isolated-one expressing hPRA (PRA-14) and one expressing hPRB (PRB-59). Cell growth experiments revealed a growth-inhibitory response to progestins (MPA and R5020) in the PRB-59 cells but not in the PRA-14 cells. Differences in expression of genes targeted by the two isoforms were studied using a cDNA expression array technique. A different set of genes appeared to be progesterone regulated in the PRA-14 cells than in the PRB-59 cells. None of the genes were regulated by both hPRA and hPRB. Insulin-like growth factor binding protein 3 expression was studied in more detail as an example of a gene regulated in PRB-59 cells but not in PRA-14 cells. We established a new model to study functional differences between the two hPR isoforms in human endometrial carcinoma cells. This model revealed distinctive differences in target gene regulation between the two hPR isoforms. Moreover, antiproliferative actions of progesterone on human endometrial cancer cells could be observed only in the PRB-expressing cell line.