A phase I dose-escalation study of enzalutamide in combination with the AKT inhibitor AZD5363 (capivasertib) in patients with metastatic castration-resistant prostate cancer.

BACKGROUND: Activation of the PI3K/AKT/mTOR pathway through loss of phosphatase and tensin homolog (PTEN) occurs in approximately 50% of patients with metastatic castration-resistant prostate cancer (mCRPC). Recent evidence suggests that combined inhibition of the androgen receptor (AR) and AKT may be beneficial in mCRPC with PTEN loss. PATIENTS AND METHODS: mCRPC patients who previously failed abiraterone and/or enzalutamide, received escalating doses of AZD5363 (capivasertib) starting at 320 mg twice daily (b.i.d.) given 4 days on and 3 days off, in combination with enzalutamide 160 mg daily. The co-primary endpoints were safety/tolerability and determining the maximum tolerated dose and recommended phase II dose; pharmacokinetics, antitumour activity, and exploratory biomarker analysis were also evaluated. RESULTS: Sixteen patients were enrolled, 15 received study treatment and 13 were assessable for dose-limiting toxicities (DLTs). Patients were treated at 320, 400, and 480 mg b.i.d. dose levels of capivasertib. The recommended phase II dose identified for capivasertib was 400 mg b.i.d. with 1/6 patients experiencing a DLT (maculopapular rash) at this level. The most common grade ≥3 adverse events were hyperglycemia (26.7%) and rash (20%). Concomitant administration of enzalutamide significantly decreased plasma exposure of capivasertib, though this did not appear to impact pharmacodynamics. Three patients met the criteria for response (defined as prostate-specific antigen decline ≥50%, circulating tumour cell conversion, and/or radiological response). Responses were seen in patients with PTEN loss or activating mutations in AKT, low or absent AR-V7 expression, as well as those with an increase in phosphorylated extracellular signal-regulated kinase (pERK) in post-exposure samples. CONCLUSIONS: The combination of capivasertib and enzalutamide is tolerable and has antitumour activity, with all responding patients harbouring aberrations in the PI3K/AKT/mTOR pathway. CLINICAL TRIAL NUMBER: NCT02525068.

Global methylation analysis identifies PITX2 as an upstream regulator of the androgen receptor and IGF-I receptor genes in prostate cancer.

The insulin-like growth factor-I (IGF-IR) and androgen (AR) receptors are important players in prostate cancer. Functional interactions between the IGF-I and androgen signaling pathways have crucial roles in the progression of prostate cancer from early to advanced stages. DNA methylation is a major epigenetic alteration affecting gene expression. Hypermethylation of tumor suppressor promoters is a frequent event in human cancer, leading to inactivation and repression of specific genes. The aim of the present study was to identify the entire set of methylated genes ("methylome") in a cellular model that replicates prostate cancer progression. The methylation profiles of the P69 (early stage, benign) and M12 (advanced stage, metastatic) prostate cancer cell lines were established by treating cells with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza) followed by DNA microarray analysis. Comparative genome-wide methylation analyses of 5-Aza-treated versus untreated cells identified 297 genes overexpressed in P69 and 191 genes overexpressed in M12 cells. 102 genes were upregulated in both benign and metastatic cell lines. In addition, our analyses identified the PITX2 gene as a master regulator upstream of the AR and IGF-IR genes. The PITX2 promoter was semi-methylated in P69 cells but fully methylated (i. e., silenced) in M12 cells. Epigenetic regulation of PITX2 during the course of the disease may lead to orchestrated control of the AR and IGF signaling pathways. In summary, our results provide new insights into the epigenetic changes associated with progression of prostate cancer from an organ confined, androgen-sensitive disorder to an aggressive, androgen-insensitive disease.

CCL5 secreted by senescent aged fibroblasts induces proliferation of prostate epithelial cells and expression of genes that modulate angiogenesis.

There is increased interest in the effects of secretory products from aged cells on promoting both benign and malignant cell growth. We identified a human fibroblast line, AG04382, from an aged donor that naturally demonstrated senescence-associated features and whose conditioned media significantly induced proliferation of benign prostatic hyperplasia (BPH1) cells. Candidate cytokines mediating this effect were identified with protein arrays and validated by ELISA. We found that the AG04382 fibroblast line secreted high levels of CXCL5, CCL5, and CCL2, but relative to the other lines, its conditioned media was unique in its increased expression of CCL5. Blocking studies using specific antibodies against CXCL5, CCL5, and CCL2 in the conditioned media of AG04382 showed that only CCL5 contributed significantly to BPH1 proliferation. Stimulation of BPH1 cells with rhuCCL5 resulted in increased proliferation and migration, as well as significant changes in the expression of genes that influence angiogenesis. These data suggest that CCL5 is a candidate chemokine secreted by aged cells that promotes prostate growth and regulates angiogenesis.

Systematic and functional characterization of novel androgen receptor variants arising from alternative splicing in the ligand-binding domain.

The presence of intact ligand-binding domain (LBD) ensures the strict androgen-dependent regulation of androgen receptor (AR): binding of androgen induces structural reorganization of LBD resulting in release of AR from HSP90, suppression of nuclear export which otherwise dominates over import and nuclear translocation of AR as a transcription factor. Thus, loss or defects of the LBD abolish constraint from un-liganded LBD as exemplified by constitutively active AR variants (AR-Vs), which are associated with emerging resistance mechanism to anti-AR therapy in castration-resistant prostate cancer (mCRPC). Recent analysis of the AR splicing landscapes revealed mCRPC harboring multiple AR-Vs with diverse patterns of inclusion/exclusion of exons (exons 4-8) corresponding to LBD to produce namely exon-skipping variants. In silico construction for these AR-Vs revealed four novel AR-Vs having unique features: Exclusion of specified exons introduces a frameshift in variants v5es, v6es and v7es. ARv56es maintains the reading frame resulting in the inclusion of the C-terminal half of the LBD. We systematically characterized these AR-Vs regarding their subcellular localization, affinity for HSP90 and transactivation capability. Notably, ARv5es was free from HSP90, exclusively nuclear, and constitutively active similarly as previously reported for v567es. In contrast, v6es and v7es were similar in that they are cytoplasmic, transcriptionally inactive and bind HSP90, ARv56es was present in both nucleus and cytoplasm, does not bind HSP90 and is transcriptionally inactive. Converting these transcriptionally inactive AR-Vs into active forms, we identified the two separate elements that allosterically suppress otherwise constitutively active AR-Vs; one in exon 5 for v6es and v7es and the other in exon 8 for v56es. Our findings identify a novel constitutively active AR-V, ARv5es and establish a method to predict potential activities of AR-Vs carrying impaired LBD.

Effects of intranasal insulin on cognition in memory-impaired older adults: modulation by APOE genotype.

Raising insulin acutely in the periphery and in brain improves verbal memory. Intranasal insulin administration, which raises insulin acutely in the CNS without raising plasma insulin levels, provides an opportunity to determine whether these effects are mediated by central insulin or peripheral processes. Based on prior research with intravenous insulin, we predicted that the treatment response would differ between subjects with (epsilon4+) and without (epsilon4-) the APOE-epsilon4 allele. On separate mornings, 26 memory-impaired subjects (13 with early Alzheimer's disease and 13 with amnestic mild cognitive impairment) and 35 normal controls each underwent three intranasal treatment conditions consisting of saline (placebo) or insulin (20 or 40 IU). Cognition was tested 15 min post-treatment, and blood was acquired at baseline and 45 min after treatment. Intranasal insulin treatment did not change plasma insulin or glucose levels. Insulin treatment facilitated recall on two measures of verbal memory in memory-impaired epsilon4- adults. These effects were stronger for memory-impaired epsilon4- subjects than for memory-impaired epsilon4+ subjects and normal adults. Unexpectedly, memory-impaired epsilon4+ subjects showed poorer recall following insulin administration on one test of memory. These findings suggest that intranasal insulin administration may have therapeutic benefit without the risk of peripheral hypoglycemia and provide further evidence for apolipoprotein E (APOE) related differences in insulin metabolism.

High IGFBP-3 levels in marrow plasma in early-stage MDS: effects on apoptosis and hemopoiesis.

The pathophysiology of the myelodysplastic syndromes (MDS) is incompletely understood. Tumor necrosis factor (TNF)alpha levels are elevated, particularly in early-stage MDS, and apoptosis in marrow cells is upregulated. Observations in other models have shown a role for insulin-like growth factor binding protein 3 (IGFBP-3) in TNFalpha-mediated apoptosis. We observed increased levels of IGFBP-3 in the marrow plasma of patients with MDS (P = 0.005) and hypothesized that altered IGFBP-3 levels contribute to the dysregulation of hemopoiesis in MDS by affecting proliferation and apoptosis. Western analysis of marrow plasma from MDS patients revealed an increase in the ratio of intact vs fragmented IGFBP-3 in early-stage MDS (relative to controls) that decreased with MDS disease progression, suggesting increased proteolysis with more advanced disease. Thus, these results provide evidence for dysregulation of IGFBP-3 in patients with MDS. While the data are complex, they are consistent with a modulatory effect of IGFBP-3 on hemopoiesis in MDS. Conceivably, understanding these mechanisms may allow for the development of novel therapeutic strategies.

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a potential tumor suppressor protein for prostate cancer.

Insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1) has been shown to have decreased expression in the progression from benign to malignant prostate epithelial cells (V. Hwa et al., J. Clin Endocrinol. Metab., 83: 4355-4362, 1998). The present study was undertaken to determine the effects of the re-expression of IGFBP-rP1 in a cell line from a model of human prostate cancer, M12, in which IGFBP-rP1 expression had been demonstrated to decrease from the parent epithelial cell, P69, to the malignant subline, M12. An IGFBP-rP1 cDNA encoding the protein was transfected into M12 cells in a plasmid that resulted in constitutive-expression of IGFBP-rP1. Clones of transfected M12 cells were selected for low (L) and high (H) levels of expression, and the plasmid vector alone was transfected into M12 as a control. After transfection, there was a marked alteration in the morphology of the M12 cells such that the H clones had an elongated appearance when compared with the M12 control cells. The M12 clones overexpressing IGFBP-rP1 had a dose-related increase in population doubling time, decreased colony formation in soft agar, an increased propensity to undergo apoptosis in response to 6-hydroxyurea, and decreased tumor formation in male athymic, nude mice. These data suggest that IGFBP-rP1 may have a suppressive effect on prostate cancer development.

Hevin, an antiadhesive extracellular matrix protein, is down-regulated in metastatic prostate adenocarcinoma.

Hevin, a gene closely related to the extracellular matrix protein SPARC, is an acidic cysteine-rich glycoprotein shown to be important for the adhesion and trafficking of cells through the endothelium. Through the use of differential display and differential EST analysis, we identified Hevin as a gene whose transcription is down-regulated in transformed prostate epithelial cell lines and metastatic prostate adenocarcinoma. These results were confirmed by comparing expression levels between normal and neoplastic human prostate tissues using Northern analysis. In situ hybridization with an 35S-labeled antisense riboprobe demonstrated the loss of Hevin expression in metastatic prostate carcinoma. The expression pattern of Hevin in transformed and metastatic epithelium may provide further insights into the complex cell adhesion events involved in the metastatic progression of prostate carcinoma.

Relationship between sex hormone binding globulin and estrogen receptors in breast cancer.

It has been suggested that the level of sex hormone binding globulin (SHBG) is a better predictor of response of breast cancer to hormone treatment than the measurement of estrogen receptor (ER). However, no correlation of SHBG with ER status has been shown. To define the relationship between SHBG and the ER, the following study was undertaken. Fifty women with breast cancer and known ER status had SHBG measured by dextran-coated charcoal saturation analysis. The mean SHBG in all ER-positive patients was 20.7 +/- 2.4 (ng DHT bound/mL) and in all ER-negative patients was 11.5 +/- 2.0 (p less than 0.01). The mean premenopausal level of SHBG was 22.2 +/- 3.8 ng/mL and postmenopausal level was 20.0 +/- 5.7 ng/mL. There was no significant difference between these groups. ER-positive patients on tamoxifen had a SHBG of 29.8 +/- 9 ng/mL and ER-negative subjects on tamoxifen had a mean SHBG level of 8.3 +/- 3.4 ng/mL, p less than 0.01. ER-positive patients have a higher SHBG level than ER-negative patients; furthermore, this difference between ER-positive and ER-negative subjects was further demarcated by changes in SHBG after the subjects had been placed on tamoxifen.

Enhancement of memory in Alzheimer disease with insulin and somatostatin, but not glucose.

BACKGROUND: Increasing plasma glucose levels improves memory in patients with Alzheimer disease (AD). Increasing plasma glucose levels also increases endogenous insulin levels, raising the question of whether memory improvement is due to changes in insulin, independent of hyperglycemia. We address this question by examining memory and counterregulatory hormone response during hyperglycemia when endogenous insulin was suppressed by concomitant infusion of the somatostatin analogue octreotide (Sandostatin). METHODS: Twenty-three patients with AD and 14 similarly aged healthy adults participated in 4 metabolic conditions on separate days: (1) hyperinsulinemia (538 pmol/L) with fasting glucose (5.6 mmol/L [100 mg/dL]), achieved by insulin and variable dextrose infusion; (2) hyperglycemia (12.5 mmol/L [225 mg/dL]) with fasting insulin (57 pmol/L), achieved by dextrose and somatostatin (octreotide) infusion (150 mg/h); (3) placebo with isotonic sodium chloride solution (saline) infusion (fasting insulin and glucose); and (4) an active control condition in which somatostatin alone was infused (150 mg/h). Declarative memory (story recall) and selective attention (Stroop interference test) were measured during steady metabolic states. RESULTS: Patients with AD showed improved memory during hyperinsulinemia relative to placebo (P = .05) and relative to hyperglycemia (P<.005). Memory did not improve during hyperglycemia when insulin was suppressed. Somatostatin analogue infusion alone also improved memory for patients with AD (P<.05). Hyperinsulinemia increased cortisol levels in subjects with AD, whereas somatostatin alone lowered cortisol concentrations. CONCLUSIONS: These results confirm that elevated insulin without hyperglycemia enhances memory in adults with AD, and indicate that insulin is essential for hyperglycemic memory facilitation. These results also suggest a potential therapeutic role for somatostatin in AD.

Thyroid uptake of 123I in a normal population.

Iodine 131 for thyroidal uptake and scan exposes the thyroid to potentially carcinogenic doses of radiation. Iodine 123, on the other hand, compares equally with 131I for uptake studies yet reduces thyroid radiation exposure substantially. Reported fluctuations in normal thyroidal iodine uptake over the past decade, as well as possible radiation injury with 131I, prompted examination of uptake values in a normal population using 123I. The normal range defined in 100 euthyroid subjects was 1% to 8.8% at two hours, and 4% to 27% for the 24-hour uptake. These results are significantly lower than observed eight years ago in this area. No relationship was noted between uptake values and thyroid indices, age, sex, ponderal index, estrogen ingestion, or urinary iodine excretion. Purified 123I appeared to provide clinically reliable results with a substantial reduction in potential radiation injury.

Altered growth and insulin-like growth factor-binding protein-3 production in PC3 prostate carcinoma cells stably transfected with a constitutively active androgen receptor complementary deoxyribonucleic acid.

The network of androgen-dependent growth factors regulating the growth and function of normal or neoplastic prostate epithelium is largely unknown. To facilitate studies directed at investigating this issue, androgen receptor-negative (AR-) PC3 prostate carcinoma cells were stably transfected with the expression plasmid CMV3 containing a constitutively active AR construct that is truncated at its hormone-binding domain (CMV-ARCA). The major characteristic of the resulting cell line (PC3-ARCA) was a growth rate approximately 35% slower than that of a control mock-transfected cell line (PC3-Neo). Of the several growth factors known to be present in the prostate, the current studies focused on the insulin-like growth factor (IGF) axis, specifically the IGF-binding proteins (IGFBPs), several of which are known to be abnormally produced by prostate cancer. Northern analysis showed that IGFBP-1 and -5 are not expressed by PC3-ARCA and -Neo cells. Western ligand and immunoblot analysis of medium conditioned by PC3-Neo and PC3-ARCA cells revealed that equal amounts of IGFBP-2, -4, and -6 were secreted. In contrast, IGFBP-3 was undetectable in the conditioned medium of PC3-ARCA cells, but normally produced by the AR- cell line PC3-Neo. IGFBP-3 disappearance from the conditioned medium of PC3-ARCA cells was transcriptionally regulated, as a marked decrement in IGFBP-3 messenger RNA was detected by S1 protection analysis. We investigated the responses of these cells to exogenously added IGF-I, IGF-II, or IGFBP-3. IGF-I and IGF-II stimulated the proliferation of PC3-ARCA cells, but not of PC3-Neo cells. IGFBP-3 had no effect when given alone. When IGFBP-3 was administered together with IGF-I or IGF-II, it further increased the mitogenic response observed in PC3-ARCA cells, but no effect on PC3-Neo cells was observed. In conclusion, our studies suggest that the presence of an active AR modulates the proliferation of transfected PC3 prostate cancer cells, and that this phenomenon occurs at least in part through the regulation of IGFBP-3 production.

Overexpression of an inhibitory insulin-like growth factor binding protein (IGFBP), IGFBP-4, delays onset of prostate tumor formation.

Insulin-like growth factor (IGF) binding proteins (IGFBPs) have been shown to either inhibit or enhance the action of IGF, or act in an IGF-independent manner in the prostate. We have overexpressed the IGF-inhibitory IGFBP-4 in the malignant M12 prostate epithelial cell line to determine the effects on tumor formation and apoptosis. Overexpression was determined by Northern, Western immunoblot and Western radioligand blot analysis. IGF-induced proliferation was reduced in the IGFBP-4 transfected cells compared with control cells (P < or = 0.01). Colony formation in soft agar was significantly inhibited up to 14 days after plating in the IGFBP-4 transfected cells when compared with the M12 controls (P < or = 0.01): however, in the presence of des(1-3)IGF-I, there was no significant difference between the control and IGFBP-4 transfectants in colony formation in soft agar. Apoptosis in an IGFBP-4 transfected cell line was significantly increased in response to induction by 6-hydroxyurea compared with the control line. When injected s.c. into male athymic/nude mice, a marked delay was noted in tumor formation in animals receiving IGFBP-4 transfected cells (P < or = 0.01). Interestingly, IGFBP-2 protein levels were reduced in the conditioned media of all IGFBP-4 transfected cell cultures. These data indicate that an inhibitory IGFBP may significantly delay the growth of malignant prostate epithelial cells and enhance the sensitivity of these cells to apoptosis.

Inhibition of growth and increased expression of insulin-like growth factor-binding protein-3 (IGFBP-3) and -6 in prostate cancer cells stably transfected with antisense IGFBP-4 complementary deoxyribonucleic acid.

Insulin-like growth factor-binding proteins (IGFBPs) both stimulate and inhibit IGF activity, and in the M12 prostate cancer cell line, overexpression of IGFBP-4 was shown to delay tumorigenesis while decreasing the production of IGFBP-2. We have performed the reverse experiment, inhibition of IGFBP-4 expression with antisense complementary DNA, in two prostate tumor cell lines, ALVA-31 and M12. Expression of antisense messenger RNA transcripts was verified by RNase protection assays, and inhibition of mature IGFBP-4 in cell medium was demonstrated by Western blotting. Both transfected lines (ALVA-31asBP4 and M12asBP4) proliferated more slowly in monolayer culture than parental controls. Colony formation in soft agar was strongly inhibited in both cases, and the rate of tumor formation and growth in male athymic nude mice injected with M12asBP4 was markedly reduced relative to that in mice receiving M12 control cells. Apoptosis induced by the topoisomerase inhibitor etoposide was also enhanced in transfected cells. The effects on colony formation in soft agar and tumor formation in mice were maintained for the duration of the experiments, in contrast to the delayed growth observed in the previous study of IGFBP-4 overexpression. A significant difference was found in the patterns of IGFBP expression; production of both messenger RNA and protein for IGFBP-3 and IGFBP-6 was greatly increased in the M12asBP4 and ALVA31asBP4 cell lines. Up-regulation of these binding proteins has been observed in association with actions of 1,25-dihydroxyvitamin D(3) in prostate cancer cells, and the data suggest a role for IGFBP-3 and IGFBP-6 in the suppression of prostate tumor cell growth.

Reexpression of the type 1 insulin-like growth factor receptor inhibits the malignant phenotype of simian virus 40 T antigen immortalized human prostate epithelial cells.

Type 1 insulin-like growth factor receptor (IGF-1R) expression is decreased in prostate cancer compared to that in noncancerous prostate epithelium. We have demonstrated that as the simian virus 40 T antigen (SV40T) immortalized human prostate epithelial cell line, P69SV40T, undergoes transformation from a poorly tumorigenic to a malignant phenotype, the M12 subline, there is a significant decrease in IGF-1R expression. In the present study, we examine the effects of reexpression of the IGF-1R on the malignant phenotype of M12 cells. The IGF-1R was reexpressed in M12 cells using a retroviral vector containing a 7-kilobase coding sequence for the IGF-1R, LISN, to create several clones of the M12-LISN cell line. As a control, M12 cells were also infected with a retroviral vector (LNL6) without the 7-kilobase IGF-1R insert (M12-LNL6 clones). Functional assays were performed with two separate clones each of M12-LNL6 and M12-LISN cells. Each clone of M12-LISN cells regained the proliferative response to IGF that was lost in the transition from P69SV40T cells to M12 cells. In addition, M12-LISN clones had a significantly decreased growth rate compared to the M12-LNL6 cells when injected s.c. in athymic/nude mice (P < 0.001). Tumorigenicity, as assessed by anchorage-independent growth of colonies in soft agar, was also decreased by 75% in the M12-LISN clones compared to that in the M12-LNL6 control cells. These data demonstrate that reexpression of the IGF-1R in a malignant human prostate epithelial cell line results in decreased tumor growth and decreased anchorage-independent colony formation independent of an increased proliferative response to IGF. Reexpression of the IGF-1R may be associated with reacquisition of the regulation of cellular proliferative and differentiation functions mediated by the IGF-1R that are lost as prostate epithelial cells undergo conversion to a malignant phenotype.

Characterization of insulin-like growth factor-binding protein-related proteins (IGFBP-rPs) 1, 2, and 3 in human prostate epithelial cells: potential roles for IGFBP-rP1 and 2 in senescence of the prostatic epithelium.

Insulin-like growth factor (IGF)-binding protein (IGFBP)-related proteins (IGFBP-rPs) are newly described cysteine-rich proteins that share significant aminoterminal structural similarity with the conventional IGFBPs and are involved in a diversity of biological functions, including growth regulation. IGFBP-rP1 (MAC25/Angiomodulin/prostacyclin-stimulating factor) is a potential tumor-suppressor gene that is differentially expressed in meningiomas, mammary and prostatic cancers, compared with their malignant counterparts. We have previously shown that IGFBP-rP1 is preferentially produced by primary cultures of human prostate epithelial cells (HPECs) and by poorly tumorigenic P69SV40T cells, compared with the cancerous prostatic LNCaP, DU145, PC-3, and M12 cells. We now show that IGFBP-rP1 increases during senescence of HPEC. IGFBP-rP2 (also known as connective tissue growth factor), a downstream effector of transforming growth factor (TGF)-beta and modulator of growth for both fibroblasts and endothelial cells, was detected in most of the normal and malignant prostatic epithelial cells tested, with a marked up-regulation of IGFBP-rP2 during senescence of HPEC. Moreover, IGFBP-rP2 noticeably increased in response to TGF-beta1 and all-trans retinoic acid (atRA) in HPEC and PC-3 cells, and it decreased in response to IGF-I in HPEC. IGFBP-rP3 [nephroblastoma overexpressed (NOV)], the protein product of the NOV protooncogene, was not detected in HPEC but was expressed in the tumorigenic DU145 and PC-3 cells. It was also synthesized by the SV40-T antigen-transformed P69 and malignant M12 cells, where it was down-regulated by atRA. These observations suggest biological roles of IGFBP-rPs in the human prostate. IGFBP-rP1 and IGFBP-rP2 are likely to negatively regulate growth, because they seem to increase during senescence of the prostate epithelium and in response to growth inhibitors (TGF-beta1 and atRA). Although the data collected on IGFBP-rP3 in prostate are modest, its role as a growth stimulator and/or protooncogene is supported by its preferential expression in cancerous cells and its down-regulation by atRA.

Transcriptional regulation of insulin-like growth factor-I receptor gene expression in prostate cancer cells.

A marked decrease in the type 1 insulin-like growth factor (IGF) receptor (IGF-IR) occurs in prostate epithelial cells during transformation from the benign to the metastatic state. One of the principal regulators of IGF-IR gene expression, the WT1 tumor suppressor, is expressed in prostate cancer and in prostate cancer cell lines. The purpose of this study was to determine whether the decrease in IGF-IR expression was transcriptionally regulated, and whether WT1 action may be involved in the repression of the IGF-IR gene in prostate cancer cells. The P69 cell line was derived by immortalization of human primary prostate epithelial cells with simian virus-40 T antigen and is rarely tumorigenic. The M12 line was derived from the P69 line by selection for tumor formation in nude mice and is tumorigeneic and metastatic. P69 cells express 20,000 IGF-IR/cell, whereas M12 cells express 3,500 IGF-IR/cell. These differences in receptor number are reflected in proportional differences in IGF-IR mRNA levels. To assess IGF-IR promoter activity in these cell lines, each was transiently transfected with luciferase reporter vectors containing the IGF-IR gene transcription start site and 476 bp of 5'-flanking sequence, 640 bp of 5'-untranslated region sequence, or both regions. The promoter activity of the full-length construct was 50% lower (P < 0.01) in M12 cells compared with P69 cells, the activity of the 5'-flanking region construct was 53% lower (P < 0.0001), and that of the 5'-untranslated region construct was 36% lower (P = 0.01). P69 clones stably transfected with a WT1 expression vector exhibited decreased expression of the endogenous IGF-IR gene and decreased promoter activity in transient transfection assays with IGF-IR promoter constructs containing multiple WT1 binding sites. The observed reduction in endogenous IGF-IR expression was sufficient to inhibit IGF-I-stimulated cell proliferation. These data suggest that most of the decreased expression of the IGF-IR seen in malignant prostate epithelium is the result of transcriptional repression of the IGF-IR gene, and that this repression may be due in part to the increased expression of the WT1 tumor suppressor in metastatic prostate cancer.

Relationship of serum inhibin levels to serum follicle stimulating hormone and sperm production in normal men and men with varicoceles.

The purpose of this study was to examine the relationships between serum inhibin levels as measured by RIA and serum FSH and sperm concentration. Three groups of men were used for this study: group I, normal fertile men (n = 67); group II, fertile men with a varicocele (n = 57); and group III, infertile men with a varicocele (n = 21). There were no differences in mean serum inhibin levels between the three groups. The two groups of men with varicoceles exhibited higher serum FSH levels and FSH responses to GnRH than the normal men. Sperm counts in both groups II and III were significantly lower than group I. In the normal men there was an inverse correlation between baseline serum inhibin and serum FSH levels and GnRH stimulated FSH levels, r = -0.415 and 0.422, P less than 0.005, respectively. Furthermore, the normal men exhibited a positive correlation between serum inhibin measurements and sperm concentration and testicular volume, r = 0.35 and 0.26, P less than 0.01 and less than 0.05, respectively. In neither group of men with a varicocele were these relationships found. These data demonstrate that serum inhibin does correlate with FSH in a negative fashion, when the reproductive system is normal, as would be expected for a negative feedback factor. Finally, the relationship of serum inhibin levels to testicular size and sperm count in the normal men suggests that serum inhibin levels reflect to some extent the integrity of seminiferous tubule function.

Obesity and its role in polycystic ovary syndrome.

To examine the mechanism by which obesity influences ovulation, 55 patients with oligo- or anovulation were studied. Parameters measured in serum were sex steroid-binding globulin (SSBG), testosterone (T), PRL, LH, FSH, and estradiol (E2). The women were divided into 2 groups: an obese group (group 1), greater than 145% of ideal body weight, and a normal weight group, less than 120% ideal body weight. SSBG was measured by saturation analysis T, LH, FSH, PRL, and E2 were measured by RIA. SSBG group 1 levels were 7.14 ng dihydrotestosterone bound/ml compared to 14.7 ng dihydrotestosterone bound/ml in group 2 (P less than 0.05). There were no significant differences in FSH, T, or E2. The correlation of body weight vs. SSBG in all patients was r = -0.62. In these 2 groups, the SSBG was significantly lower in the obese patients compared to that in the normal weight patients, independent of T or E2 levels. SSBG correlated negatively with body weight, suggesting that obesity has an influence on SSBG levels independent of hormonal status. When SSBG is lowered, there may be an increase in free T which, by inhibiting follicular maturation, may begin the sequence of events seen in polycystic ovary syndrome.