epidermal-growth-factor and Testicular-Neoplasms

Testicular cancer is the most frequent cancer in young men aged 15-40 years and accounts for 1% of all cancer diagnosed in males. Testicular germ cell tumors (TGCT) encompass a broad group of cancers, each displaying different levels of pluripotency and differentiation as well as malignancy potential. The TGCT cell of origin is thought to be a fetal germ cell that failed to correctly differentiate during development: this is known as the ‘fetal origins hypothesis’. This theory predicts that developmental pathways that control germ cell pluripotency or differentiation may be involved in the malignant transformation of these cells. Recently the Nodal/Cripto signaling pathway, known to control pluripotency and differentiation in embryonic stem (ES) cells, was implicated in regulating normal male fetal germ cell pluripotency. Although genes of this pathway are not normally expressed in germ cells during adult life, ectopic expression of this pathway was detected in several sub-groups of TGCTs. In this review, we consider the evidence for the fetal origins of TGCT and discuss the implications of Nodal/Cripto signaling in various aspects of germ cell development and cancer progression.

Topics: Animals; Cell Differentiation; Epidermal Growth Factor; Fetus; Gene Expression Regulation, Developmental; Germ Cells; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Neoplasm Proteins; Neoplasms, Germ Cell and Embryonal; Nodal Protein; Signal Transduction; Testicular Neoplasms

Type II germ cell tumors arise after puberty from a germ cell that was incorrectly programmed during fetal life. Failure of testicular germ cells to properly differentiate can lead to the formation of germ cell neoplasia in situ of the testis; this precursor cell invariably gives rise to germ cell cancer after puberty. The Nodal co-receptor Cripto is expressed transiently during normal germ cell development and is ectopically expressed in non-seminomas that arise from germ cell neoplasia in situ, suggesting that its aberrant expression may underlie germ cell dysregulation and hence germ cell cancer. Here we investigated methylation of the Cripto promoter in mouse germ cells and human germ cell cancer and correlated this with the level of CRIPTO protein expression. We found hypomethylation of the CRIPTO promoter in undifferentiated fetal germ cells, embryonal carcinoma and seminomas, but hypermethylation in differentiated fetal germ cells and the differentiated types of non-seminomas. CRIPTO protein was strongly expressed in germ cell neoplasia in situ along with embryonal carcinoma, yolk sac tumor and seminomas. Further, cleaved CRIPTO was detected in media from seminoma and embryonal carcinoma cell lines, suggesting that cleaved CRIPTO may provide diagnostic indication of germ cell cancer. Accordingly, CRIPTO was detectable in serum from 6/15 patients with embryonal carcinoma, 5/15 patients with seminoma, 4/5 patients with germ cell neoplasia in situ cells only and in 1/15 control patients. These findings suggest that CRIPTO expression may be a useful serological marker for diagnostic and/or prognostic purposes during germ cell cancer management.

Topics: Animals; Carcinoma, Embryonal; Epidermal Growth Factor; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Mice; Mice, Transgenic; Neoplasm Proteins; Testicular Neoplasms

Germ cells, the embryonic precursors of sperm or oocytes, respond to molecular cues that regulate their sex-specific development in the fetal gonads. In males in particular, the balance between continued proliferation and cell fate commitment is crucial: defects in proliferation result in insufficient spermatogonial stem cells for fertility, but escape from commitment and prolonged pluripotency can cause testicular germ cell tumors. However, the factors that regulate this balance remain unidentified. Here, we show that signaling by the TGFβ morphogen Nodal and its co-receptor Cripto is active during a crucial window of male germ cell development. The Nodal pathway is triggered when somatic signals, including FGF9, induce testicular germ cells to upregulate Cripto. Germ cells of mutant mice with compromised Nodal signaling showed premature differentiation, reduced pluripotency marker expression and a reduced ability to form embryonic germ (EG) cell colonies in vitro. Conversely, human testicular tumors showed upregulation of NODAL and CRIPTO that was proportional to invasiveness and to the number of malignant cells. Thus, Nodal signaling provides a molecular control mechanism that regulates male germ cell potency in normal development and testicular cancer.

Topics: Animals; Cell Differentiation; Cell Proliferation; Epidermal Growth Factor; Fibroblast Growth Factor 9; Germ Cells; Humans; Male; Membrane Glycoproteins; Mice; Neoplasm Proteins; Neoplasms, Germ Cell and Embryonal; Nodal Protein; Pluripotent Stem Cells; Signal Transduction; Spermatogenesis; Spermatogonia; Testicular Neoplasms; Testis; Transforming Growth Factor beta

Malignant germ-cell tumours arise from a neoplastic precursor, the carcinoma in situ, and develop into seminomas and/or non-seminomas (embryonal carcinomas, teratomas, yolk-sac tumours and choriocarcinomas). Based on histological and clinical findings, it has been postulated that seminomas can eventually transform into non-seminomas. Here, we used the cell line TCam-2 as model for seminomas and interrogated their differentiation potential. We demonstrate that TCam-2 cells are able to differentiate into mixed non-seminomatous lineages after supplementing the media with TGF-β1, EGF and FGF4. On a molecular level, the differentiation is initiated by repression of BMP/SMAD signalling. As a consequence, BLIMP1, a molecule known to inhibit the differentiation of murine primordial germ cells, is down-regulated and differentiation-inhibiting histone modifications are lost. The appearance of multinucleated giant cells and the expression of marker genes indicate that cells differentiate predominantly into extra-embryonic choriocarcinoma-like cells. This is most likely due to the presence of components of the Hippo pathway, TEAD4 and YAP1. These molecules have been described to trigger extra-embryonic fate determination in the murine system. This study supports the model that seminomas indeed have an intrinsic ability to transform into a non-seminoma. In addition, the data suggest that the transformation does not require an additional mutation, but can be triggered by changes in the tumour microenvironment.

Topics: Adaptor Proteins, Signal Transducing; Biomarkers; Bone Morphogenetic Protein Receptors; Cell Differentiation; Cell Line, Tumor; Choriocarcinoma; DNA-Binding Proteins; Epidermal Growth Factor; Fibroblast Growth Factor 4; Giant Cells; Histones; Humans; Male; Muscle Proteins; Neoplasms, Germ Cell and Embryonal; Polymerase Chain Reaction; Positive Regulatory Domain I-Binding Factor 1; Repressor Proteins; Seminoma; Signal Transduction; Smad Proteins; TEA Domain Transcription Factors; Testicular Neoplasms; Transcription Factors; Transforming Growth Factor beta1; Tumor Microenvironment

Steroid hormone biosynthesis in the adrenals and gonads is regulated by the steroidogenic acute regulatory (StAR) protein through its action in mediating the intramitochondrial transport of cholesterol. A role for epidermal growth factor (EGF) in modulating steroidogenesis has been previously determined, but the mechanism of its action remains unknown. The present investigation was designed to explore the potential mechanism of action of mouse EGF (mEGF) in the regulation of steroid biosynthesis and StAR protein expression in mLTC-1 mouse Leydig tumor cells. We show that treatment of mLTC-1 cells with mEGF significantly increased the levels of progesterone (P), StAR protein, and StAR mRNA in a time- and dose-dependent manner. The coordinate induction of P synthesis and StAR gene expression by mEGF was effectively inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. Also, longer exposure of mLTC-1 cells to mEGF produced a marked decrease in LH-receptor mRNA expression. These effects of mEGF were exerted through high-affinity binding sites (K(d) approximately 0.53 nmol/L) in these cells. It was also determined that the arachidonic acid (especially lipoxygenase metabolites) and mitogen-activated protein kinase pathways were also involved in the mEGF-induced steroidogenic response. However, involvement of the latter pathway was further assessed in nonsteroidogenic COS-1 cells transfected with the Elk1 trans-reporting plasmids and resulted in a significant increase in luciferase activity in response to mEGF. Furthermore, deletion and mutational analyses demonstrated a predominant involvement of activator protein-1 in addition to the multiple mEGF responsive elements found within the 5'-flanking region (-151/-1 base pairs) of the mouse StAR gene. These findings provide novel insights into the mEGF-induced regulatory cascades associated with steroid synthesis and StAR protein expression in mouse Leydig cells.

Topics: Animals; Arachidonic Acid; Base Sequence; Bucladesine; Chorionic Gonadotropin; Epidermal Growth Factor; Iodine Radioisotopes; Leydig Cell Tumor; Male; MAP Kinase Signaling System; Mice; Molecular Sequence Data; Phosphoproteins; Progesterone; Receptors, LH; RNA, Messenger; Signal Transduction; Steroids; Testicular Neoplasms; Tumor Cells, Cultured

The expression of cripto, a novel transforming gene of the epidermal growth factor superfamily, was immunohistochemically examined in 20 cases of urinary bladder, one ureter, three renal pelvic, 18 kidney, nine prostate, three adrenal and four testicular tumors. Three cases of urinary bladder carcinomas, two of grade 2 and one of grade 3 transitional cell carcinoma which were T1a and T3b, and INF beta and INF gamma, respectively, showed positive binding of specific antibody, along with one cortical carcinoma of the adrenal gland positive staining. None of the ureter, renal pelvic, kidney, prostate or testicular tumors showed positive staining. These results indicate that cripto is not frequently expressed in urological tumors.

Topics: Adrenal Gland Neoplasms; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Epidermal Growth Factor; Female; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Prostatic Neoplasms; Testicular Neoplasms; Urologic Neoplasms

The teratocarcinoma-derived growth factor-1 (TDGF-1) gene codes for a 188-aminoacid glycoprotein that shares structural homology with the epidermal growth factor (EGF) family of growth factors. TDGF-1 is highly expressed in the undifferentiated embryonal carcinoma stem cell line NTERA2 clone D1 (NT2/D1) and its expression is downregulated in response to differentiating agents such as retinoic acid (RA) and hexamethylen-bisacetamide (HMBA). To assess the role of TDGF-1 in the onset and/or progression of human germ cell tumors, we analysed TDGF-1 expression by Northern blot and immunostaining in a panel of 59 human germ cell tumors of different histological origins. We show that TDGF-1 expression is markedly elevated in a subset of human testicular germ cell tumors as compared to normal testes. TDGF-1 overexpression occurs in about 100% of tumors with non-seminomatous phenotype, such as embryonal carcinomas and malignant undifferentiated teratocarcinomas. To address the questions of how TDGF-1 (previously called CRIPTO) may affect the growth and/or the differentiation of embryonal carcinoma cells, we have characterized the effects of exogenous recombinant TDGF-1 protein on the proliferation rate and differentiation 'potential of NT2/D1. Exogenous TDGF-1 protein stimulated DNA synthesis and cell proliferation in both undifferentiated and differentiated NT2/D1 cells. However, TDGF-1 protein treatment was unable to block differentiation induced by both RA and HMBA. These results suggest that TDGF-1 growth factor may represent an autocrine growth factor that may be involved in the process of development of testicular neoplasms.

Topics: Amino Acid Sequence; Biomarkers, Tumor; Blotting, Northern; Carcinoma, Embryonal; Cell Differentiation; Cell Division; Epidermal Growth Factor; Gene Expression; Germinoma; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Proteins; Recombinant Proteins; Teratocarcinoma; Testicular Neoplasms; Tumor Cells, Cultured

We analyzed expression of transforming growth factor (TGF)-alpha, epidermal growth factor (EGF) and their receptor, EGF receptor (EGFR), by immunohistochemistry in the human testis to determine the possible roles of these growth factors in human testicular function. Specimens were obtained from 17 patients including 9 patients with infertility, 4 patients with prostatic carcinoma and 4 patients with contralateral testicular tumor. EGF immunoreactivity was positive in the hyperplasic Leydig cells of one patient but negative in the other cases. On the other hand, strong TGF-alpha immunoreactivity was observed in Leydig cells, with weak staining in Sertoli cells and germ cells in cases with normal spermatogenesis. EGFR immunoreactivity was observed in the Leydig and peritubular cells, appearing as membrane staining. Marked immunoreactivity for TGF-alpha was observed in the Sertoli cells in testes with decreased spermatogenesis, especially in the Sertoli-cell-only syndrome. This finding may indicate a compensatory increase of TGF-alpha expression in the Sertoli cells accompanying a decrease in spermatogenesis. No significant correlation was found between the degrees of spermatogenesis and immunolocalization of the EGF receptor. These findings suggest that TGF-alpha is a locally produced growth factor that is involved in spermatogenesis in the human testis via an autocrine and/or paracrine mechanism.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Infertility, Male; Male; Middle Aged; Orchiectomy; Prostatic Neoplasms; Spermatogenesis; Testicular Neoplasms; Testis; Transforming Growth Factor alpha

A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na2SeO3 (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lentil lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linked N-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium. alpha 1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9 +/- 1.5 pmol.h-1.mg-1 protein) than cultured with FBS-containing media (18.2 +/- 1.2 pmol.h-1.mg-1 protein). These results have indicated that the selective increase of alpha 1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.

Topics: alpha-Fetoproteins; beta 2-Microglobulin; Carcinoembryonic Antigen; Cell Division; Culture Media; Epidermal Growth Factor; Fucosyltransferases; Glycosyltransferases; Humans; Immunohistochemistry; Lectins; Male; Mesonephroma; Phosphopyruvate Hydratase; Platelet-Derived Growth Factor; Polyamines; Selenium; Serum Albumin, Bovine; Testicular Neoplasms; Transferrin; Tumor Cells, Cultured

We measured epidermal growth factor (EGF) in the urine of 54 untreated patients with malignant tumors (7 prostatic cancer, 11 renal tumor, 31 bladder cancer, 3 renal pelvic tumor, 2 ureter tumor) in the Toyama Medical & Pharmaceutical University Hospital. We also measured EGF in the urine of 77 normal subjects (43 males, 34 females). Urinary concentration of EGF in normal subjects decreased with increasing age. It was significantly higher in females than males (p less than 0.05). Urinary concentration of EGF in patients with prostatic cancer or renal tumor was similar to that in normal subjects. The patients with prostatic cancer controlled by estrogen showed a slightly high level of EGF in the urine. In patients with renal tumor, urinary concentration of EGF decreased after nephrectomy. Patients with bladder cancer showed a significant decrease of EGF in the urine compared with normal subjects (p less than 0.05), and urinary concentration of EGF in the patients with bladder cancer of high stage was remarkably low. Low concentration of EGF in the urine was recognized in patients with renal pelvic tumor or ureter tumor. However, the relationship between the decrease of urinary concentration of EGF in these urothelial tumors and the growth of these tumors remains to be elucidated.

Topics: Adult; Age Factors; Aged; Creatinine; Epidermal Growth Factor; Female; Humans; Kidney Neoplasms; Male; Middle Aged; Nephrectomy; Radioimmunoassay; Sex Factors; Testicular Neoplasms; Urologic Neoplasms

In a recent publication we showed that addition of mouse epidermal growth factor (mEGF) to MA-10 Leydig tumor cells rapidly leads to an increase in the incorporation of [3H]inositol-derived radioactivity into an unusual lipid that was identified as phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2). Other ligands that are known to bind to MA-10 cells, such as hCG and arginine vasopressin, however, did not elicit this effect. Inasmuch as mEGF modulates the differentiated functions of MA-10 cells in a number of ways, our findings raised the possibility that PI-3,4-P2 may be an intracellular mediator of these actions of mEGF. In an attempt to answer this question, we set out to determine if other ligands increase the labeling of PI-3,4-P2 in MA-10 cells prelabeled with [3H]inositol, and if such ligands mimic the diverse biological actions of mEGF on these cells. The experiments presented herein show that insulin, insulin-like growth factor-I, and transforming growth factor-alpha increase the labeling of PI-3,4-P2 in MA-10 cells, but only transforming growth factor-alpha mimics the actions of mEGF on the differentiated functions of MA-10 cells. We conclude that an increase in the labeling of PI-3,4-P2 is not sufficient to elicit these actions of mEGF.

Topics: Animals; Epidermal Growth Factor; Insulin; Insulin-Like Growth Factor I; Leydig Cell Tumor; Male; Phosphatidylinositol Phosphates; Phosphatidylinositols; Testicular Neoplasms; Tumor Cells, Cultured

We have previously reported that the cloned cell line (B-1-A-2) derived from an estrogen-responsive mouse Leydig cell tumor shows an estrogen-dependent enhancement of cell proliferation in medium supplemented with charcoal-dextran-stripped fetal bovine serum. To avoid the involvement of unknown factors present in the serum in the pathway for estrogen-dependent cell growth, the present study was designed to establish a serum-free culture system to which growth factors could be added. To this end, we subcloned B-1 cells from the parental tumor cell line. The proliferation of B-1 cells was markedly stimulated by the addition of 10(-11)-10(-8) M estradiol into the serum-free medium [Eagle's Minimum Essential Medium-Ham's F-12 (1:1, vol/vol) containing 0.2% (wt/vol) BSA]. Epidermal growth factor (0.1-50 ng/ml) or insulin (0.1-50 micrograms/ml) alone or in combination with 10(-8) M estradiol did not affect the proliferation rate of B-1 cells. In contrast, a greater than 10-fold molar excess of 4-hydroxytamoxifen blocked estradiol-induced cell proliferation, while 4-hydroxytamoxifen alone failed to show a stimulatory effect on cell multiplication. Additionally, the conditioned medium collected from estradiol-stimulated cells was found to contain a growth-promoting factor(s) whose activity was not antagonized by 4-hydroxytamoxifen. Nonstimulated cells secreted a significant but low level of the growth-promoting factor. Finally, B-1 cells were found to be estrogen dependent for cell proliferation in BALB/c mice. Their growth was markedly inhibited by the administration of tamoxifen to the host mice. These results indicate that the serum-free culture system presented here is suitable for studying the autocrine mechanisms of cell growth regulated by estrogens as well as triphenylethylene compounds.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Insulin; Kinetics; Leydig Cell Tumor; Male; Mice; Mice, Inbred BALB C; Receptors, Estrogen; Tamoxifen; Testicular Neoplasms

The M5480A murine Leydig cell tumor was used to investigate the effects of three hormones, which produce distinct biochemical actions, on cytoplasmic protein synthesis. Human choriogonadotropin treatment of tumor-bearing mice induced the synthesis of six proteins with relative molecular weights (Mr) of 135K, 82K, 60K, 19K, 18.2K and 17.3K (K = kilodaltons). Diethylstilbestrol induced one protein peak in common with the gonadotropin Mr = 135K) and three additional proteins with Mr's of 120K, 50K and 36K. Epidermal growth factor induced one major protein with Mr = 33K, which is similar to that of a protein induced in murine epidermal cells by tumor promoters. These studies demonstrate the induction of specific gene products in a hormone-responsive tumor.

Topics: Animals; Chorionic Gonadotropin; Cytoplasm; Diethylstilbestrol; Epidermal Growth Factor; Leydig Cell Tumor; Male; Mice; Mice, Inbred C57BL; Molecular Weight; Protein Biosynthesis; Testicular Neoplasms