Modulation of [5-125I]iododeoxyuridine incorporation into tumour and normal tissue DNA by methotrexate and thymidylate synthase inhibitors.

A potentially useful method for imaging of micrometastases and in situ radiotherapy, would be the incorporation of radioactive labelled iododeoxyuridine (IdU) into tumour DNA. However, there are two main problems: incorporation of the radioactive IdU into normal cells and low incorporation into tumour cells. The aim of this study was to attempt to augment the incorporation of [5-125I]iododeoxyuridine (125IdU) into tumour DNA and to improve the tumour/normal tissue ratio by the use of inhibitors (methotrexate, 5-fluorouracil, AG337, ZD 1694, benzyloxybenzyl uracil) which would prolong the metabolic half-life of the compound. Mammary tumours were induced in GR mice, which were then treated with the inhibitors and the 125IdU. The tumours and representative normal tissue were removed following sacrifice of the animals, and radioactivity within the tissues measured. Pretreatment of mammary carcinoma-bearing GR mice with methotrexate caused approximately a 3-fold increase in the incorporation of 125IdU into tumour DNA, and approximately a > or = 10-fold increase in the tumour/small intestine ratio of incorporated radioactivity. Inhibition of thymidylate synthase, the enzyme involved in IdU dehalogenation, by 5-fluorouracil plus folic acid, or by novel inhibitors AG337 and ZD1694 led to a 3- to 5-fold increase in the 125IdU incorporation. Benzyloxybenzyl uracil, an inhibitor of dihydrouracil dehydrogenase, had little effect. Treatment of tumour-bearing mice with methotrexate plus ZD1694 significantly reduced the rate of tumour growth, but addition of 125IdU (70 microCi/mouse, three daily injections) had no additional antitumour activity. In conclusion, these results do not support the hypothesis that systemic administration of 125IdU can be used for cancer therapy or for imaging purposes unless better methods are found to boost its incorporation into tumour DNA.

Mutations in the estrogen receptor gene.

Somatically generated mutations in the estrogen receptor (ER) have been found at the mRNA/cDNA level in human breast cancer biopsies and in established breast cancer cell lines. Aberrantly spliced ER mRNA causes the appearance of truncated or internally deleted ER protein forms. Studies on the functional activity of the ER variants in expression systems have revealed dominant-positive receptors that are transcriptionally active in the absence of estrogen, and dominant-negative receptors that are themselves transcriptionally inactive but that prevent the action of the normal receptor. The ER variants are believed to confer resistance to endocrine therapy in breast cancer patients. Abnormally spliced forms of ER, similar to those in breast cancer, have been reported in human meningiomas.

Metabolism of a [18F]fluorine labeled progestin (21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone) in humans: a clue for future investigations.

Assessment of estrogen receptors and progesterone receptors (PR) with PET may allow the determination of the hormone responsiveness of tumors without the need for multiple biopsies, and the monitoring of the effect of hormonal therapy. In spite of the favourable characteristics of 21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone ([18F]FENP) found in preclinical studies, the compound failed to reveal the presence of PR in breast carcinomas and meningiomas. In view of the clinical significance of the PR assay in human breast cancer, it is worthwhile to explore mechanisms that are potentially involved in the inadequacy of [18F]FENP to image PR with PET. Our present study on the in vivo metabolism of [18F]FENP in humans demonstrates a rapid clearance and biotransformation of the compound. Results of incubation experiments suggest that the metabolic conversion of [18F]FENP is not restricted to the liver, but also occurs in blood cells (presumably the erythrocytes) and tumors (breast carcinomas and meningiomas). The predominant metabolite of [18F]FENP in plasma during the rapid distribution phase and in tumors is identified as 20-dihydro-[18F]FENP. The conversion of [18F]FENP to its 20 alpha- or 20 beta-hydroxy metabolite has a deleterious effect on the binding affinity for PR. Our findings do not justify a conclusion as to the extent of in vivo extrahepatic biotransformation of [18F]FENP, or its significance in the ineffectiveness of [18F]FENP as an imaging agent for PR. On the other hand, the ability of breast carcinomas and meningiomas to metabolize [18F]FENP avidly appears to preclude selective imaging of PR in these tumors during the time of a PET examination. It is imperative to evaluate the metabolic stability of a [18F]fluorine labeled progestin in an early stage of future screening procedures.

Hormone resistance in cancer: the role of abnormal steroid receptors.

Breast tumors that have become resistant to endocrine therapy have been found to contain estrogen receptor (ER) variants due to aberrant splicing mechanisms of the ER gene. Exon skipping can give rise to dominant-positive receptors that are transcriptionally active in the absence of estrogen, or dominant-negative receptors that are themselves transcriptionally inactive but prevent the action of the normal receptor. ER splice variants similar to those in breast cancer have also been reported in human meningiomas. Androgen receptor (AR) variants have been detected in some prostate cancers that exhibit resistance to androgen therapy. In leukemia and lymphoma, mutations in the glucocorticoid receptor (GR) cause resistance to cell lysis by dexamethasone. Thus, there is increasing evidence that mutations in the genes of steroid receptors can cause loss of hormone dependency in different cancer types.

Epidermal growth factor receptor levels increase but epidermal growth factor receptor ligand levels decrease in mouse mammary tumors during progression from hormone dependence to hormone independence.

Twenty-six serially transplanted Grunder (GR) strain mouse mammary tumors were analyzed for epidermal growth factor receptor (EGFR) and EGFR-ligand levels, in addition to steroid hormone receptors (estrogen receptor, ER, progesterone receptor, PgR). In concordance with earlier studies, hormone dependent (HD) and hormone responsive (HR) tumors were found to be positive for both ER and PgR, whereas hormone independent (HI) tumors contained only 30% of the ER concentration that was found in the HD tumors. PgR was undetectable in HI tumors. HI tumors contained 2.5 to 3-fold higher EGFR levels than HD/HR tumors, an observation which shows remarkable concordance with studies on EGFR in human breast cancer. On the other hand, the level of EGFR-ligand(s) was positively associated with ER levels and was three-fold higher in HD/HR tumors than in HI tumors. The low EGFR in HD/HR tumors relative to HI tumors may be the result of downregulation by EGFR ligands produced under ER control. During progression to hormone independence this downregulation of EGFR is then abolished in absence of ER. The increase in EGFR may therefore be a secondary effect rather than a key event in the progression to hormone independence in this mouse mammary tumor model.

Role of estrogen receptor variants in the development of hormone resistance in breast cancer.

Recent evidence suggests that the progression to hormone resistance in some breast tumors is due to mutations in the estrogen receptor (ER). Various types of ER variants have been found in breast cancer biopsies and breast cancer cell lines. The ER variants include dominant-positive receptors that are transcriptionally active in the absence of estrogen, and dominant-negative receptors that are themselves transcriptionally inactive but prevent the action of the normal receptor. The mechanisms by which these variants cause loss of hormonal control is becoming clear. ER variants may be prognostic factors for breast cancer. By modifying the action of ER variants, it should be possible to develop new strategies for treatment of malignant breast disease.

Phosphorylation and estradiol binding of estrogen receptor in hormone-dependent and hormone-independent GR mouse mammary tumors.

The effect of phosphorylation on the hormone-binding capacity of the estrogen receptor (ER) was investigated in hormone-dependent (HD) and hormone-independent (HI) mammary carcinomas of GR mice. Tumor cytosols were incubated with ATP under conditions previously used to study the tyrosine kinase which confers hormone binding to phosphatase-treated or in vitro-synthesized ER. The ATP-dependent increases in hormone-binding capacity of 8 out of 20 HI tumors ranged from values of 23 to 124 fmol/mg cytosol protein. The enhancement by ATP of hormone binding to ER was significantly less marked in HD and HR tumors than in HI tumors. In only 3 out of 13 HD and HR tumors was an increase ranging from 15 to 20 fmol/mg protein detected. Analysis by Scatchard plot of estradiol binding to ER showed that cytosol incubation of HI tumors with ATP markedly increased the hormone binding without any change in affinity. The data suggest that ER of HI tumors is less phosphorylated in vivo than the ER of HD/HR tumors, so that the receptor of HI tumors is more susceptible to gamma-32P-ATP phosphorylation and ATP-induced hormone binding in vitro. Western blot of ER with antiphosphotyrosine antibody showed that, in HI tumors, the large ATP-induced increase in hormone binding to ER was associated with phosphorylation on tyrosine of the receptor itself. Our findings indicate that the process of activation-inactivation of binding through tyrosine-phosphorylation/phosphotyrosine-dephosphorylation of ER observed in estrogen target tissues is altered in some HI mammary tumors.

Influence of estrogen receptor variants in mammary carcinomas on the prognostic reliability of the receptor assay.

We have used GR mouse mammary carcinomas as a model for evaluating the effect of estrogen receptor (ER) variants, present in the tumor cytosols, on the prognostic reliability of the receptor assay. Rapid, high resolution procedures were used for the assessment of wild-type and variant estrogen receptors in the cytosols of hormone-responsive and nonresponsive mammary tumors of GR mice. Two main ER types were resolved by high performance ion-exchange and size-exclusion chromatography: the wild-type receptor (II), and a fraction representing low molecular weight ER (I). ER types I and II both bound estradiol and both reacted with the anti-ER monoclonal antibodies H222 and D547 whose epitopes are in the C-terminal part of ER. The level of ER type II was higher in hormone-responsive than in hormone-nonresponsive tumors, whereas ER type I was about equally low in both types of tumor groups. ER types I and II both influence the standard ligand binding and antibody binding (Abbott EIA) test, but only the wild-type ER causes hormone-responsive growth of the tumor. These data suggest that prognostic clinical tests, currently in use for estimating ER in tumors of breast cancer patients, may be impaired by the presence of low molecular weight estrogen receptor variants in the tumor samples.

Comparison of estrogen receptors in hormone-dependent and hormone-independent Grunder strain mouse mammary tumors.

Hormone-dependent (HD) Grunder strain mouse mammary carcinomas contain a 65-kDa estrogen receptor (ER) with minor amounts of 50- and 35-kDa components which apparently still contain the intact hormone-binding (COOH-terminal) domain. When the HD tumors lose their hormonal dependence during serial transplantation, the hormone-independent (HI) transplants show an increase in 50- and 35-kDa components relative to 65-kDa ER. In HI transplants of three of five tumor lines studied (TSl 85, 86, and 106), the 65-kDa receptor was entirely replaced by 50- and 35-kDa receptors, whereas in the two other lines (TSl 101 and 104) there usually were about equal amounts of 65- and 50-kDa ERs. No difference was found between ERs of HD and HI tumors in affinity for estradiol, steroid specificity, or immunoreactivity for the monoclonal antibody JS34/32. Estrogen stimulation of HI tumors did not increase the concentration of progesterone receptor in the tumor tissue, indicating that ER in these tumors was not functional in enhancing progesterone receptor. Incubation of 65-kDa ER with HI tumor cytosol or combined homogenization of HD and HI tumor tissue did not cause degradation of 65-kDa ER. alpha-Chymotrypsin-like protease activity generally was lower in HI than in HD tumor cytosols, indicating that the lower molecular size of ER in HI tumors cannot be attributed to the increased level of this protease activity.

A fluorine-18 labeled progestin as an imaging agent for progestin receptor positive tumors with positron emission tomography.

The potential of the fluorine-18 labeled progestin 21-[18F]fluoro- 16 alpha-ethyl-19-norpregn-4-ene-3,20-dione [( 18F]FENP) as an imaging agent for the in vivo assessment of progestin receptor (PR) positive neoplasms with positron emission tomography has been investigated. Tissue distribution studies in immature estrogen primed female rats revealed high uptake of radioactivity, expressed as the differential absorption ratio, by uterine tissue. After simultaneous administration with unlabeled FENP, a significant decrease (83%) in uterine uptake was observed 60 min after injection. Uterine uptake was highly selective. The ratio of uptake of radioactivity by uterine tissue to that by blood was 39 at 180 min. In mice bearing transplanted Grunder strain mammary carcinomas tissue, distribution studies demonstrated a selective uptake of [18F]FENP by PR positive tumors. Pretreatment with unlabeled FENP caused a significant decrease (66%) in tumor uptake. Uptake by other tissues was not affected by the presence of unlabeled progestin. The ratio of uptake of radioactivity by tumor tissue to that by blood was 4.7 at 180 min. For FENP pretreated mice and mice bearing PR negative tumors, this ratio was 1.7 and 1.1, respectively. It is concluded that the uptake of [18F]FENP by uterine and by PR positive mammary tumor tissue in vivo is primarily receptor related, presumably to the PR. Furthermore, [18F]FENP appears to be suitable for imaging of PR positive human neoplasms with positron emission tomography.

Nuclear hormone receptor variants: their role in malignancy and progression to hormone resistance in cancer.

Structural variants of nuclear hormone receptors have been found in tumour tissues. Experimental evidence suggests two ways in which these variants may have oncogenic potential: 1. by suppressing the action of the normal hormone receptor, thereby acting as dominant negative oncogenes; 2. by activating hormone-responsive genes in a hormone-independent manner. These mechanisms may not only contribute to oncogenesis but also to the development of hormone resistance in tumours, e.g. breast and prostate cancer.

Nuclear hormone receptor variants: their role in malignancy and progression to hormone resistance in cancer.

Mutations in functional domains of steroid and thyroid hormone receptors cause these molecules to become constitutive (hormone independent) transactivators of gene transcription. Mutations in, for example, the estrogen receptor (ER) may contribute to the loss of estrogen control of some breast carcinomas. The crucial factor in determining estrogen requirement for the growth of tumors may not be the presence or absence of estrogen-regulated ER, but whether the ERs present require estrogen to be able to cause transcription activation of growth regulatory genes. This also has implications for the therapy of breast cancer because it may be necessary to design effective anticancer agents to suppress the malignant effects of ER mutants.

Steroid/thyroid receptor-like proteins with oncogenic potential: a review.

Mutated or truncated forms of certain members of the steroid/thyroid receptor superfamily have oncogenic potential. The aberrant forms compete with the normal receptor for binding to the responsive element on the DNA and thus interfere negatively with the normal transcription control mechanism. Oncogenes that arise from dominant negative mutations may therefore be called "dononcs," to distinguish them from recessive types such as that causing retinoblastoma ("renoncs"). It is possible that dononcs are also responsible for the loss of hormonal responsiveness of some tumors during progression.

Assignment of estradiol receptor gene to mouse chromosome 10.

Differences in restriction fragment lengths were detected with murine estrogen receptor cDNA (clone MOR-100) between Chinese hamster and mouse. These were used to determine the chromosomal location of the estrogen receptor in the mouse by Southern blot analysis of DNAs obtained from a panel of mouse-Chinese hamster somatic cell hybrids. The mouse estrogen receptor gene was localized on mouse chromosome 10.

Breast carcinomas detected by thallium-201 scintigraphy.

Thallium-201 chloride scintigrams were made of GR mice bearing transplanted mammary tumours, and of human patients with breast cancer. The studies on the experimental system using GR mouse mammary tumours showed that the oestrogen-responsive tumours incorporated significantly more Tl-201 chloride than the oestrogen non-responsive tumours. In order to investigate the usefulness of Tl-201 chloride scintigraphy for human patients, a study was carried out on 15 women with primary or advanced breast cancer in which Tl-201 chloride scintigraphy was compared with bone scintigraphy using Technetium-99m. The results indicated that Tl-201, chloride scintigraphy was a very useful method for detection of primary breast cancer and metastases, and in some cases it revealed tumour structures not detected by Technetium-99m. We propose that Tl-201 chloride scintigraphy can be a useful method for early diagnosis of breast cancer.

Different int-1 region DNA rearrangements within different zones of a single mouse mammary tumor.

Fragments were taken from separate parts of hormone-dependent (HD) primary GR mouse mammary tumors and serially transplanted in estrone plus progesterone treated or hormonally untreated castrated mice. The transplants were examined with respect to int-1 DNA rearrangement, proviral integrations of the murine mammary tumor virus (MMTV), and estrogen and progesterone receptor content. One of the fragments (b) taken from the primary tumor of line TSI 96 produced transplants that showed int-1 rearrangement in one allele and also MMTV proviral integrations not at the int-1 gene, whereas transplants from another fragment (a) only had the normal germ-line int-1 arrangement and no extra MMTV provirus. These respective genotypes were retained when the tumors became hormonally independent during further transplantations. The results indicate that int-1 rearrangement was not present in the originally transformed cell but occurred in a HD cell during growth of the tumor. Furthermore they indicate that loss of hormonal dependence in GR mammary tumors is due to a mutational event, unrelated to int-1 rearrangement.