Phosphorylation of the oestrogen receptor alpha at serine 305 and prediction of tamoxifen resistance in breast cancer.

Phosphorylation of oestrogen receptor alpha at serine 305 (ERalphaS305-P) induces tamoxifen resistance in experimental studies, but does not influence response to other endocrine agents, such as fulvestrant. We evaluated ERalphaS305-P using immunohistochemistry in 377 breast carcinomas from premenopausal participants of a randomized trial (n=248) and patients with advanced disease (n=129). Among the premenopausal patients, adjuvant tamoxifen improved recurrence-free survival (RFS) for ERalphaS305-P-negative tumours (multivariate HR=0.53, 95% CI 0.32-0.86, p=0.010), but not for ERalphaS305-P-positive tumours (multivariate HR=1.01, 95% CI 0.33-3.05, p=0.99) (interaction p=0.131). Notably, ERalphaS305-P was not significantly associated with RFS in patients not treated with tamoxifen (multivariate HR=0.64, 95% CI 0.30-1.37, p=0.248), indicating that ERalphaS305-P is a marker for treatment outcome rather than tumour progression. Given the direct experimental link between ERalphaS305-P and tamoxifen resistance and these first clinical data suggesting that premenopausal patients with ERalphaS305-P-positive breast cancer are resistant to adjuvant tamoxifen, further research is encouraged to study whether alternative endocrine treatment should be considered for this subgroup.

Rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in T-cell leukemias of mouse strain GR result in a novel enhancer-like structure.

Male GR mice develop T-cell leukemia at low frequency late in life. These leukemia cells invariably contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins and have extra MMTV proviruses integrated in their DNA. We show here that the extra MMTV proviruses are all derived from the endogenous MMTV provirus associated with the Mtv-2 locus and that the T-cell leukemias are clonal with respect to the acquired MMTV proviruses. The extra MMTV proviruses in six transplantable T-cell leukemia lines studied had rearranged, shortened long terminal repeats (LTRs); each T-cell leukemia, however, had a different LTR rearrangement within its extra MMTV provirus. The alteration within the extra LTRs of T-cell leukemia line 42 involved deletion of 453 nucleotides and generation of a tandem repeat region consisting of regions flanking the deletion. This alteration generated a sequence similar to the adenovirus enhancer core sequence. The viral RNAs in the T-cell leukemias contained corresponding alterations in their U3 regions. These results demonstrate that expression of MMTV in T-cell leukemias of GR mice may be the consequence of the generation of a novel enhancer, which could also stimulate expression of any adjacent cellular oncogene.

The product of the EMS1 gene, amplified and overexpressed in human carcinomas, is homologous to a v-src substrate and is located in cell-substratum contact sites.

We have previously identified two genes (EMS1 and PRAD1/cyclin D1) in the chromosome 11q13 region that are frequently coamplified and overexpressed in human breast cancer and in squamous cell carcinomas of the head and neck (E. Schuuring, E. Verhoeven, W.J. Mooi, and R.J.A.M. Michalides, Oncogene 7:355-361, 1992). We now report on the characterization of the 80/85-kDa protein that is encoded by the EMS1 gene. Amino acid sequence comparison shows a high homology (85%) to a chicken protein that was recently identified as a substrate for the src oncogene (H. Wu, A.B. Reynolds, S.B. Kanner, R.R. Vines, and J.T. Parsons, Mol. Cell. Biol. 11:5113-5124, 1991). Immunocytochemistry reveals that in epithelial cells, the human EMS1 protein is localized mainly in the cytoplasm and, to a very low extent, in protruding leading lamellae of the cell. However, in carcinoma cells that constitutively overexpress the protein as a result of amplification of the EMS1 gene, the protein, except in cytoplasm, accumulates in the podosome-like adherens junctions associated with the cell-substratum contact sites. The protein was not found in intercellular adherens junctions. Our findings, and the previously reported observations in src-transformed chicken embryo fibroblasts, suggest that the EMS1 protein is involved in regulating the interactions between components of adherens-type junctions. Since amplification of the 11q13 region has been associated with an enhanced invasive potential of these tumors, overexpression and concomitant accumulation of the EMS1 protein in the cell-substratum contact sites might, therefore, contribute to the invasive potential of these tumor cells.

Cyclin D1 triggers autonomous growth of breast cancer cells by governing cell cycle exit.

Cyclin D1 controls G1-associated processes, including G0-to-G1 and G1-to-S transitions. This study demonstrates a novel aspect of cyclin D1 as a regulator of the transition between G1 and G0. Overexpression of cyclin D1 in MCF7 breast tumor cells resulted in a continued proliferation under low-serum conditions, whereas nonoverexpressing cells ceased to grow. This difference in growth was due to a reduced exit from G1 to G0 in cyclin D1-overexpressing cells. Our data therefore suggest a model in which cyclin D1 overexpression in tumor cells is responsible for hyperproliferation under growth factor-deprived conditions.

Autogenous antibodies against the murine mammary tumor virus in strains of mice with low incidences of mammary tumors.

The radioimmunoprecipitation assay for the murine mammary tumor virus (MuMTV) was used to detect naturally occurring antibodies against MuMTV in 3 groups of highly inbred mouse strains. 1) Some strains had high incidences of mammary tumors, such as strains GR and C3H. Antibodies against MuMTV were detected in the sera of females of these strains at early ages. 2) Some mouse strains had low incidences of mammary tumors with an intermediate MuMTV expression, such as strains C3Hf, RIIIf, and BALB/c. Some females of these strains developed antibodies against MuMTV. Hormone treatment of these mice resulted in an increase in the proportion of mice carrying antibodies against MuMTV. 3) Some mouse strains were MuMTV-free, such as strains O20, C57BL, and Gr-Mtv2-. No antibodies against MuMTV were detected in the sera of these mice. However, antibodies against MuMTV appeared in the sera of these animals after hormone treatment. The presence of a natural humoral immunity toward MuMTV appeared to be related to the expression of MuMTV in the animals.

A new locus (Mtv-4) for endogenous mammary tumor virus expression and early mammary tumor development in the SHN mouse strain.

The SHN mouse strain, which has a high incidence of mammary cancer, developed by inbreeding and selection from Swiss stock mice by Dr. H. Nagasawa and co-workers (Meiji University, Tokyo, Japan), harbored an endogenous mammary tumor virus (MTV) responsible for a high frequency of mammary tumors early in life. The locus was called "Mtv-4" and was only comparable with Mtv-2 of the GR mouse strain in its inducing capacity of mammary cancer. Molecular hybridization with 32P-labeled MTV complementary DNA showed that the characteristic Mtv-2 bands of the GR strain were absent in the SHN strain.

Characterization of mouse mammary tumor viruses from primary tumor cell cultures. II. Biochemical and biophysical studies.

Primary mammary tumor cultures of RIII, GR, DD, BALB/c, and BALB/cfC3H mice were examined for mouse mammary tumor virus (MuMTV) production. Levels of production of 12-32 mug virus protein/day/75-cm2 culture flask could be maintained for 30-50 days with daily virus harvests. The viruses from tumor cell cultures of these mouse strains contained DNA polymerase with a strong preference for Mg++ over Mn++ as the divalent cation, a characteristic of DNA polymerase of MuMTV from mouse milk. These viruses from tumor cell cultures were excellent sources of MuMTV 3H-complementary DNA (complexed to 60-70S RNA) and radioactive 60-70S RNA, sufficiently free of contaminating murine leukemia virus nucleic acids, that can be used in molecular hybridization experiments. The effects of several culture parameters on MuMTV production were also studied.

Biochemical characterization of putative subviral particulates from human malignant breast tumors.

Particulates with the properties of cores and/or ribonucleoproteins of RNA tumor viruses have been isolated from Sterox-SL-treated fractions of murine and human mammary adenocarcinomas. These particulates have an RNA-directed DNA polymerase, a 60 to 70 S RNA, and a density of 1.26 g/ml or greater in sucrose equilibrium density gradients. Their uniquely higher densities lead to banding in regions comparatively free of cellular contaminants. These circumstances minimize some of the technical complications of performing the simultaneous detection assay in the presence of cell debris.

Mammary tumor virus DNA as a marker for genotypic variance within hormone-responsive GR mouse mammary tumors.

Mammary tumors of GR mice acquire extra mammary tumor virus (MMTV) DNA information within their DNA during tumor growth and development. These extra MMTV genes have been used by us as genotypic markers to investigate the heterogeneity of GR mammary tumors and their loss of hormone dependence during serial transplantation. Our studies reveal that the various subpopulations of cells within individual GR mammary tumors are characterized by differences in number and location of acquired extra MMTV DNA fragments. Losses of certain of these extra MMTV DNA fragments occur when mammary tumors become hormone independent, indicating a loss of hormone-dependent cells. The study of MMTV DNA markers also reveals that low levels of autonomous cells are already present in some hormone-dependent mammary tumors at an early stage of their development. The genotypic analysis strongly indicates that mammary tumor progression is not due to phenotypic adaptation but to clonal selection of the more aggressive sublines.

Cyclin D1 overexpression enhances radiation-induced apoptosis and radiosensitivity in a breast tumor cell line.

Overexpression of cyclin D1, a G1 cell cycle regulator, is often found in many different tumor types, such as breast carcinoma and squamous cell carcinoma of the head and neck. The overexpression of this protein is, in several cases, associated with a poor prognosis. In this study, the effect of cyclin D1 on radiosensitivity was investigated in a breast tumor cell line, MCF7, containing a cyclin D1 gene construct under the control of a tetracycline-sensitive regulator. MCF7 cells cultured without tetracycline resulted in a 6-fold increase in the cyclin D1 protein. Cyclin D1-overexpressing MCF7 cells were more sensitive to ionizing radiation than the nonoverexpressing counterparts. The cyclin D1-overexpressing cells also exhibited a higher induction of apoptosis. Treatment with a dose of 5 Gy resulted in a rapid increase of p53 and p21 in the cyclin D1-overexpressing cells. Nonoverexpressing cells showed a more transient expression of these proteins after ionizing radiation. A pronounced G2-M block was observed in both cell lines. The cyclin D1-overexpressing cells were, however, released earlier from the block than the control cells. These data suggest that overexpression of cyclin D1 alters sensitivity toward ionizing radiation by modulating gamma-radiation-induced G2-M transition.

Overexpression of cyclin D1 correlates with recurrence in a group of forty-seven operable squamous cell carcinomas of the head and neck.

We evaluated the prognostic significance of overexpression of cyclin D1 in 47 patients with surgically resected squamous cell carcinomas of the head and neck. Overexpression of cyclin D1 was detected immunohistochemically using an affinity-purified polyclonal antibody directed against the carboxyl-terminal part of the cyclin D1 protein, applied to formalin-fixed, paraffin-embedded tissue sections. Overexpression of cyclin D1 was found in 30 of 47 head and neck squamous cell carcinoma (HNSCC) cases and was associated with a more rapid and frequent recurrence of disease (P = 0.027). There was a 5-year disease-free interval of 47% for HNSCC patients with a strong overexpression of cyclin D1 and of 80% for cyclin D1-negative HNSCC patients. Overexpression of cyclin D1 was also associated with a shortened overall survival of these patients (P = 0.0095), with a 5-year survival of 60% for the cyclin D1 strongly positive cases and of 83% for cyclin D1-negative cases. Overexpression of cyclin D1 appears to indicate poor prognosis in operable HNSCC.

Constitutive overexpression of cyclin D1 does not prevent inhibition of hormone-responsive human breast cancer cell growth by antiestrogens.

Cyclin D1 is a target for positive regulation by estrogens in growth-responsive cells, in which it mediates their mitogenic effects. Amplification and overexpression of the cyclin D1 gene (CCND1) might thus represent a genetic lesion inducing hormone-independent growth of transformed cells. Indeed, cyclin D1 overexpression has been found in up to 50% of primary breast cancers, and in about one-third of these cases, this is linked to amplification of the 11q13 chromosomal region, which also includes the CCND1 gene. These tumors are predominantly estrogen receptor-positive, and for this reason, these patients are often selected for adjuvant antiestrogen therapy. No information is available, however, as to whether cyclin D1 overexpression due to gene amplification might interfere with and reduce antiestrogen efficacy. This was investigated here by taking advantage of an experimental model that reproduces cyclin D1 overexpression resulting from increased CCND1 gene dosage in hormone-responsive human breast cancer cells. For this, MCF-7 cells stably transfected with a tet-inducible cyclin D1 expression vector were tested for their in vitro response to steroidal (ICI 182,780) and nonsteroidal (trans-4-hydroxytamoxifen) antiestrogens under condition of low (endogenous only) or high (exogenous) cyclin D1 levels. Results show that although cyclin D1 overexpression seems to interfere with the early cell cycle effects of antiestrogens, it does not prevent their cytostatic actions, so that growth of cyclin-overexpressing MCF-7 cells is still efficiently inhibited in vitro by these drugs.

Expression of neural cell adhesion molecule-related sialoglycoprotein in small cell lung cancer and neuroblastoma cell lines H69 and CHP-212.

Monoclonal antibodies (MAbs) 123C3 and 123A8 generated against a membrane preparation of a small cell lung carcinoma (SCLC) specimen recognize not only SCLC and bronchial carcinoids but also a significant portion of non-small cell lung carcinomas (non-SCLC) of various histological types. Together with 13 other monoclonal antibodies, which show preference for SCLC, they have been ranked as SCLC cluster 1 (SC-1) Mabs. In this study we show that SC-1 MAbs are directed against a restricted number of epitopes, and that SC-1 MAbs and a polyclonal antiserum directed against the neural cell adhesion molecule (NCAM) recognize identical glycoproteins, indicating that SC-1 antigens are closely related to or identical with NCAM. Long polysialic acid units composed of alpha-(2,8)-linked N-acetylneuraminic acid units, which in mammals are found exclusively on NCAM, were present on SC-1 antigens in SCLC. This provides further evidence that SC-1 MAbs recognize NCAM. The SC-1 antigens in the SCLC cell line H69 were present in two forms, NCAM-containing alpha-(2,8)-polysialic acid units identified by antiserum 735, the NCAM-H form, and the less sialylated NCAM-L form. The NCAM-H form consisted of diffusely migrating sialoglycoproteins with a molecular weight of 200,000-250,000, which resolved after neuraminidase treatment into two proteins with molecular weights of 140,000 and 180,000. Since the NCAM-H form is expressed in the lung tumor type with a poor prognosis, our results suggest that NCAM might be implicated in the invasive behavior of these NCAM-positive lung tumors.

Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMS1, within the amplified chromosome 11q13 region in human carcinomas.

Amplification of the chromosome 11q13 region is frequently found in human breast cancer and in squamous cell carcinomas of the head and neck, and has been associated with an unfavourable clinical course of disease. The known oncogenes within the amplified 11q13 region, INT2 and HSTF1, are rarely expressed in these tumours, indicating that another, hitherto unidentified, gene or genes confer(s) the biological (prognostic) significance to the amplification of the 11q13 region. To identify the gene or genes, we have constructed a cDNA library from a cell line with an 11q13 amplification and have performed differential cDNA cloning using [32P]dCTP-labelled cDNAs from human squamous cell carcinoma cell lines with and without an 11q13 amplification. We isolated two cDNA clones, U21B31 and U21C8, which recognize two genes amplified and overexpressed in cell lines harbouring an 11q13 amplification. In breast carcinomas and in squamous cell carcinomas amplification of both the U21B31 and the U21C8 gene was found in most tumours with an amplification of the 11q13 region, despite the large distance between both genes. Sequence analysis of the U21C8 cDNA clone revealed no homology to known genes; we call this gene EMS1. The U21B31 cDNA clone corresponded to the 3' end of the PRAD1 proto-oncogene, recently cloned from a parathyroid adenoma. Both gene products are of interest as potential markers to identify tumours with an 11q13 amplification.

D11S287, a putative oncogene on chromosome 11q13, is amplified and expressed in squamous cell and mammary carcinomas and linked to BCL-1.

Approximately 15 to 20% of primary breast cancers and an even higher proportion of squamous cell carcinomas of the head and neck show amplification of DNA markers on band q13 of human chromosome 11. However, known genes within the amplified region, such as the FGF-related oncogenes INT-2 and HST-1, are very rarely expressed in these tumors. Here we show that another candidate oncogene, designated D11S287, implicated in the pathogenesis of parathyroid adenomas, is also amplified in breast cancers. Significantly, it is consistently coamplified with INT-2 and HST-1 in 36 out of 202 primary tumors, including one case in which the amplified unit did not encompass the translocation breakpoint marker BCL-1. This implies that D11S287 is on the same side of the breakpoint as INT-2, and pulsed-field gel electrophoresis indicates that D11S287 is less than 250 kb from the BCL-1 marker. Since D11S287 RNA was present at elevated levels in a group of tumors and cell lines in which the 11q13 region is amplified, it may be the key oncogene on this amplified unit, and could also be activated by BCL-1 translocations.

Structure, stability, methylation, expression and glucocorticoid induction of endogenous and transfected proviral genes of mouse mammary tumor virus in mouse fibroblasts.

A sequence of mouse genomic DNA containing an endogenous mouse mammary tumor virus (MMTV) provirus, which was isolated by molecular cloning in lambda vector phage, has been reintroduced into cultured mouse L cells, using the thymidine-kinase cotransfection technique. Individual cell clones that acquired the transfected MMTV proviral DNA have been isolated. The transfected DNA remains stable in these cells and does not become methylated. It is transcribed into MMTV RNA, And the transcription is stimulated by glucocorticoid hormones.

Constitutive active GTPases Rac and Cdc42 are associated with endoreplication in PAE cells.

The Rho-like guanine triphosphate (GTP)ases become activated by extracellular ligands and regulate a wide variety of biological processes, including cell motility, spreading of cells, cytoskeletal organisation and transcriptional activity. We studied the effect of expression of WtRac and Cdc42 and of their constitutive active V12 variants on cell cycle transition using the isopropylthiogalactoside (IPTG) inducible Rac and Cdc42 transfectants of porcine aortic endothelial (PAE) cells. Expression of V12Rac or V12Cdc42 resulted initially in an enrichment of cells in G2/M, followed by the appearance of multinucleated cells with some of the nuclei still being able to incorporate bromodeoxyuridine (BrdU). By fluorescent activated cell sorter (FACS) analysis, these cells appeared as polyploid cells. Prolonged activation of V12Rac or V12Cdc42 resulted in genomic instability and these cells finally detached from the culture plate. These findings indicate that induction of the constitutive active V12 forms of Rac and Cdc42 results in 'mitotic slippage', where endoreplication takes place irrespective of the exit from cytokinesis.

Neural cell adhesion molecule expression, neuroendocrine differentiation and prognosis in lung carcinoma.

We investigated the expression of the neural cell adhesion molecule (NCAM) in a series of surgically resected lung carcinomas of various histological subtypes by means of a panel of monoclonal antibodies recognising different N-CAM epitopes. In a subgroup of 56 tumours, the results of immunostaining with MAb 123C3--the antibody studied most extensively in our material--were compared to the ultrastructure, and in 231 radically resected non-small cell carcinomas, with histological tumour type and with clinical follow-up data. N-CAM expression was not limited to neuroendocrine tumours, as assessed ultrastructurally. Non-small cell lung carcinomas positive for MAb 123C3 showed post-operative overall and disease-free survival times significantly shorter than 123C3-negative non-small cell carcinomas.

Neural cell adhesion molecules (NCAM) and NCAM-PSA expression in neuroendocrine lung tumors.

Neural cell adhesion molecules (NCAM) represent specific markers of neuroendocrine (NE) differentiation in lung cancer. Because the polysialic acid form (NCAM-PSA) has reduced adhesion properties, we hypothesized that NCAM-PSA expression could favor metastatic spread. Immunostaining of NCAM and NCAM-PSA were therefore compared in 120 NE lung tumors, including 17 typical carcinoids, 3 atypical carcinoids, 30 large cell NE carcinomas and 70 small cell lung carcinomas, as compared with 25 adenocarcinomas and 25 squamous cell carcinomas. Neural cell adhesion molecules were negative in adenocarcinomas and squamous cell carcinomas but were constantly expressed in all NE tumors from typical carcinoids to small cell lung carcinomas. NCAM-PSA expression was significantly more frequent in high-grade tumors, with 24 of 30 positive cases in large cell NE carcinomas and 65 of 70 positive cases in small cell lung carcinoma, than in carcinoids with 10 of 17 and 2 of 3 positive cases in typical carcinoids and atypical carcinoids, respectively. The neural cell adhesion molecule-polysialic acid form scores of staining were significantly higher in high-grade as compared with low-grade tumors (p = 0.002), and were correlated with nodal spread (p = 0.04) and metastasis (p = 0.016) across histologic classes but not in individual tumor type. We conclude that NCAM-PSA connotes poor differentiation and aggressive clinical behavior in the spectrum of NE lung tumors, but cannot be regarded as a prognostic factor in individual tumor classes.

The prognostic value of NCAM, p53 and cyclin D1 in resected non-small cell lung cancer.

Expression of the neural cell adhesion molecule, NCAM, in frozen sections has been associated with decreased postoperative survival in non-small cell lung carcinoma. Of the various isoforms of NCAM described, the highly sialylated isoform plays a role in the migration of embryonal cells from the neural crest and is expressed by highly malignant tumours such as small cell lung carcinomas. We investigated the clinical significance of expression of this NCAM isoform as a prognostic factor in a series of 96 non-small cell lung carcinomas resected with curative intent. We also evaluated the effect of microwave pre-treatment of formalin-fixed, paraffin-embedded sections on the NCAM immunostaining and related the outcome to the postoperative clinical course of disease. In addition, in an attempt to extend our search for possible molecular markers of unfavourable prognosis in lung cancer, we evaluated increased immunostaining for p53 and cyclin D1 in the same series. We did not find a significant relation between expression of NCAM or its highly sialylated isoform and the length of postoperative survival. The numbers of positive cases (9 and 14, respectively) were relatively low. Increased p53 and cyclin D1 immunostaining (50 and 55 of the 96 tumours) failed to show a significant relation with postoperative survival. In our material, tumour stage was the only significant prognostic factor.