Evaluating the impact of dose reductions and delays on progression-free survival in women with ovarian cancer treated with either three-weekly or dose-dense carboplatin and paclitaxel regimens in the national prospective OPAL cohort study.

OBJECTIVES: To determine the impact of chemotherapy dose reductions and dose delays on progression-free survival (PFS) in women with ovarian cancer receiving first line chemotherapy in a real world prospective cohort study. METHODS: Patients with newly diagnosed epithelial ovarian (or peritoneal, fallopian tube) cancer enrolled in a national Australian prospective study, OPAL, who commenced three-weekly carboplatin (AUC 5 or 6) and paclitaxel 175 mg/m2 (CP) or carboplatin (AUC 5 or 6) and dose-dense weekly paclitaxel 80 mg/m2 (DD-CP) were eligible. Primary endpoint was PFS. RESULTS: 634 evaluable patients, 309 commenced CP and 325 DD-CP. Patient's age was similar in the two groups (median 62 years, range 21-79). All planned chemotherapy doses were completed by 66% vs 40% (p < 0.001) in the CP and DD-CP groups respectively. There was at least one treatment delay in 28% vs 58% (p < 0.001) in the CP and DD-CP groups, respectively, and 29% vs 49% (p < 0.001), respectively, required at least a 15% dose reduction for either carboplatin or paclitaxel. Median PFS was 29.2 [22.9, 43.8] and 21.5 [19.4, 23.1] months in the CP and DD-CP groups respectively. Adjusting for age, histology and FIGO stage PFS did not differ between treatment groups. Median PFS was similar in patients irrespective of dose reduction or dose delay. CONCLUSION: Patients receiving DD-CP required more dose reductions and delays due to haematological toxicities and lower completion rates than CP without significant difference in median PFS between CP and DD-CP. Median PFS was similar in patients irrespective of dose reduction or dose delay.

The association between the inflammatory potential of diet and risk of developing, and survival following, a diagnosis of ovarian cancer.

PURPOSE: Inflammation has been implicated in ovarian carcinogenesis. This study evaluated two dietary indices: the Dietary Inflammatory Index (DII®) and the Empirical Dietary Inflammatory Pattern (EDIP), in relation to risk of developing, and survival following, a diagnosis of ovarian cancer. METHODS: Data came from the Australian Ovarian Cancer Study (1375 cases, 1415 population controls). DII and EDIP scores were computed from dietary information obtained using a semiquantitative food-frequency questionnaire. Logistic regression was used to assess the association between DII and EDIP scores and risk of ovarian cancer and proportional hazards models were used for survival analysis. RESULTS: A high DII score, reflecting a more pro-inflammatory diet, was associated with a modest increased risk of ovarian cancer [odds ratio (OR) DII scoreQ4 vs.Q1 = 1.31, 95% CI 1.06-1.63, ptrend = 0.014]. Likewise a high EDIP score was associated with an increase in risk of ovarian cancer [OR EDIP scoreQ4 vs.Q1 = 1.39, 95% confidence interval (CI) 1.12-1.73, ptrend = 0.002]. We found no association between DII or EDIP score and overall or ovarian cancer-specific survival. CONCLUSION: In conclusion, our results suggest that a pro-inflammatory diet modestly increases the risk of developing ovarian cancer.

Correction to: The association between the inflammatory potential of diet and risk of developing, and survival following, a diagnosis of ovarian cancer.

In the original publication of this article on page 6, paragraph "Discussion", line 4, 'In a U.S. population-based case-control study (n = 493 cases) Peres et al., reported a non-significant association between DII score and risk of developing ovarian cancer of similar magnitude (OR DII scoreQ4 vs. Q1 1.35, 95% CI 0.93-1.97) [20]'. It should read as 'In a U.S. population-based case-control study (n = 493 cases) Peres et al., reported a significant association between DII score and risk of developing ovarian cancer (OR DII scoreQ4 vs. Q1 1.72, 95% CI 1.18-2.51) [20]'.

Obesity and survival among women with ovarian cancer: results from the Ovarian Cancer Association Consortium.

BACKGROUND: Observational studies have reported a modest association between obesity and risk of ovarian cancer; however, whether it is also associated with survival and whether this association varies for the different histologic subtypes are not clear. We undertook an international collaborative analysis to assess the association between body mass index (BMI), assessed shortly before diagnosis, progression-free survival (PFS), ovarian cancer-specific survival and overall survival (OS) among women with invasive ovarian cancer. METHODS: We used original data from 21 studies, which included 12 390 women with ovarian carcinoma. We combined study-specific adjusted hazard ratios (HRs) using random-effects models to estimate pooled HRs (pHR). We further explored associations by histologic subtype. RESULTS: Overall, 6715 (54%) deaths occurred during follow-up. A significant OS disadvantage was observed for women who were obese (BMI: 30-34.9, pHR: 1.10 (95% confidence intervals (CIs): 0.99-1.23); BMI: ⩾35, pHR: 1.12 (95% CI: 1.01-1.25)). Results were similar for PFS and ovarian cancer-specific survival. In analyses stratified by histologic subtype, associations were strongest for women with low-grade serous (pHR: 1.12 per 5 kg m(-2)) and endometrioid subtypes (pHR: 1.08 per 5 kg m(-2)), and more modest for the high-grade serous (pHR: 1.04 per 5 kg m(-2)) subtype, but only the association with high-grade serous cancers was significant. CONCLUSIONS: Higher BMI is associated with adverse survival among the majority of women with ovarian cancer.

Inhibition of ANKRD1 sensitizes human ovarian cancer cells to endoplasmic reticulum stress-induced apoptosis.

High expression of Ankyrin Repeat Domain 1 (ANKRD1) in ovarian carcinoma is associated with poor survival, and in ovarian cancer cell lines is associated with platinum resistance. Importantly, decreasing ANKRD1 expression using siRNA increases cisplatin sensitivity. In this study, we investigated possible mechanisms underlying the association of ANKRD1 with cisplatin response. We first demonstrated that cisplatin-induced apoptosis in ovarian cancer cell lines was associated with endoplasmic reticulum (ER) stress, evidenced by induction of Glucose-Regulated Protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153) and increased intracellular Ca(2+) release. The level of sensitivity to cisplatin-induced apoptosis was associated with ANKRD1 protein levels and poly (ADP-ribose) polymerase (PARP) cleavage. COLO 316 ovarian cancer cells, which express high ANKRD1 levels, were relatively resistant to cisplatin, and ER stress-induced apoptosis, whereas OAW42 and PEO14 cells, which express lower ANKRD1 levels, are more sensitive to ER stress-induced apoptosis. Furthermore, we show that overexpression of ANKRD1 attenuated cisplatin-induced cytotoxicity, and conversely siRNA knockdown of ANKRD1 sensitized ovarian cancer cells to cisplatin and ER stress-induced apoptosis associated with induction of GADD153, and downregulation of BCL2 and BCL-XL. Taken together, these results suggest that ANKRD1 has a significant role in the regulation of apoptosis in human ovarian cancer cells, and is a potential molecular target to enhance sensitivity of ovarian cancer to chemotherapy.

Evidence for a time-dependent association between FOLR1 expression and survival from ovarian carcinoma: implications for clinical testing. An Ovarian Tumour Tissue Analysis consortium study.

BACKGROUND: Folate receptor 1 (FOLR1) is expressed in the majority of ovarian carcinomas (OvCa), making it an attractive target for therapy. However, clinical trials testing anti-FOLR1 therapies in OvCa show mixed results and require better understanding of the prognostic relevance of FOLR1 expression. We conducted a large study evaluating FOLR1 expression with survival in different histological types of OvCa. METHODS: Tissue microarrays composed of tumour samples from 2801 patients in the Ovarian Tumour Tissue Analysis (OTTA) consortium were assessed for FOLR1 expression by centralised immunohistochemistry. We estimated associations for overall (OS) and progression-free (PFS) survival using adjusted Cox regression models. High-grade serous ovarian carcinomas (HGSC) from The Cancer Genome Atlas (TCGA) were evaluated independently for association between FOLR1 mRNA upregulation and survival. RESULTS: FOLR1 expression ranged from 76% in HGSC to 11% in mucinous carcinomas in OTTA. For HGSC, the association between FOLR1 expression and OS changed significantly during the years following diagnosis in OTTA (Pinteraction=0.01, N=1422) and TCGA (Pinteraction=0.01, N=485). In OTTA, particularly for FIGO stage I/II tumours, patients with FOLR1-positive HGSC showed increased OS during the first 2 years only (hazard ratio=0.44, 95% confidence interval=0.20-0.96) and patients with FOLR1-positive clear cell carcinomas (CCC) showed decreased PFS independent of follow-up time (HR=1.89, 95% CI=1.10-3.25, N=259). In TCGA, FOLR1 mRNA upregulation in HGSC was also associated with increased OS during the first 2 years following diagnosis irrespective of tumour stage (HR: 0.48, 95% CI: 0.25-0.94). CONCLUSIONS: FOLR1-positive HGSC tumours were associated with an increased OS in the first 2 years following diagnosis. Patients with FOLR1-negative, poor prognosis HGSC would be unlikely to benefit from anti-FOLR1 therapies. In contrast, a decreased PFS interval was observed for FOLR1-positive CCC. The clinical efficacy of FOLR1-targeted interventions should therefore be evaluated according to histology, stage and time following diagnosis.

Dietary folate and related micronutrients, folate-metabolising genes, and ovarian cancer survival.

OBJECTIVE: Folate is essential for DNA synthesis and methylation and is implicated in tumour progression. Few studies have examined its role in ovarian cancer survival. Our objective was to determine relationships between intake of folate, related one-carbon nutrients, single nucleotide polymorphisms (SNPs) in folate-metabolising genes and survival following ovarian cancer diagnosis. METHODS: This analysis included 1270 women with invasive epithelial ovarian cancer diagnosed in 2002-2006. Pre-diagnostic and some post-diagnostic lifestyle, dietary, and sociodemographic information was collected via self-administered questionnaires. DNA samples were genotyped for SNPs in methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and methionine synthase reductase (MTRR) genes. Adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox regression. RESULTS: Multivariate analyses did not identify associations between higher pre-diagnostic intake of folate, folic acid, vitamins B2, B6, and B12, methionine, betaine or choline and survival overall. In stratified analyses, higher folic acid and folate intake was associated with significantly worse survival among women with mucinous tumours (HRs per 100 µg 1.30 and 1.43, respectively) and smokers (HRs per 100 µg 1.23 and 1.16 respectively). There was also a suggestion that higher supplemental folic acid use post-diagnosis was associated with worse survival (HR per 100 µg 1.03, 95%CI 1.00-1.05). MTHFR SNP rs2066470 was significantly associated with survival (per allele HR 0.81, 95%CI 0.67-0.98). CONCLUSIONS: Our data provide little evidence that folate intake affects ovarian cancer survival. However, combined effects with smoking, and findings within the mucinous subtype and for post-diagnosis folic acid, warrant further investigation.

Physical symptoms, coping styles and quality of life in recurrent ovarian cancer: a prospective population-based study over the last year of life.

OBJECTIVE: The aim of this study was to describe the trajectory of physical symptoms, coping styles and quality of life (QoL) and the relationship between coping and QoL over the last year of life in women with recurrent ovarian cancer. METHODS: The patient cohort were women recruited to the Australian Ovarian Cancer Study who subsequently experienced recurrent, invasive ovarian cancer and completed at least one psychosocial assessment (optimism, minimisation, hopelessness/helplessness, QoL) during the last year of life (n=217). RESULTS: QoL declined sharply from six months before death. Lack of energy was the most prevalent symptom over three measurement periods (67-92%) and also the most severe. Anorexia (36-55%), abdominal swelling (33-58%), nausea (26-47%) and pain (26-43%) all increased in prevalence and severity towards the end of life. Higher optimism (p=0.009), higher minimisation (p=0.003) and lower helplessness/hopelessness (p=0.03) at baseline were significant predictors of subsequent higher QoL. CONCLUSIONS: Progressive deterioration in quality of life may be an indicator of death within about six months and therefore should be an important consideration in decisions about subsequent treatment. Coping styles which independently predicted subsequent changes in QoL could potentially be targeted by interventions to minimise worsening QoL.

Patterns of chemotherapy treatment for women with invasive epithelial ovarian cancer--a population-based study.

OBJECTIVE: Ovarian cancer five-year survival is poor at <40%. In the absence of effective screening or new treatments, ensuring all women receive optimal treatment is one avenue to improve survival. There is little population-based information regarding the primary chemotherapy treatment that women with epithelial ovarian cancer receive. This information is essential to identify potential gaps in care. METHODS: Cancer registries identified all women diagnosed with invasive epithelial ovarian cancer in Australia in 2005 (n=1192). Histopathology, chemotherapy and comorbidity information was abstracted from medical records. Multivariable logistic regression was used to identify factors associated with chemotherapy commencement, regimen, and completion. RESULTS: Women >70 years (p<0.0001), those with high-grade, stage IA/IB cancers (vs. stages IC-IV, p=0.003) and those with mucinous cancers (p=0.0002) were less likely to start chemotherapy. Most treated women received platinum-based drugs (97%), but only 68% received combination carboplatin-paclitaxel and only half completed six cycles without treatment modification/delay. Approximately 19% received single-agent carboplatin: mostly those aged >70 (p<0.0001) and/or with co-morbidities (p<0.0001). Age was the strongest predictor of completing six cycles of combination therapy. CONCLUSIONS: For specific patient groups, particularly older women, there is notable variation from standard treatment. Understanding how treatment variations affect survival and determining optimal regimens for these groups are research priorities.

The E3 ubiquitin ligase EDD is an adverse prognostic factor for serous epithelial ovarian cancer and modulates cisplatin resistance in vitro.

Despite a high initial response rate to first-line platinum/paclitaxel chemotherapy, most women with epithelial ovarian cancer relapse with recurrent disease that becomes refractory to further cytotoxic treatment. We have previously shown that the E3 ubiquitin ligase, EDD, a regulator of DNA damage responses, is amplified and overexpressed in serous ovarian carcinoma. Given that DNA damage pathways are linked to platinum resistance, the aim of this study was to determine if EDD expression was associated with disease recurrence and platinum sensitivity in serous ovarian cancer. High nuclear EDD expression, as determined by immunohistochemistry in a cohort of 151 women with serous ovarian carcinoma, was associated with an approximately two-fold increased risk of disease recurrence and death in patients who initially responded to first-line chemotherapy, independently of disease stage and suboptimal debulking. Although EDD expression was not directly correlated with relative cisplatin sensitivity of ovarian cancer cell lines, sensitivity to cisplatin was partially restored in platinum-resistant A2780-cp70 ovarian cancer cells following siRNA-mediated knockdown of EDD expression. These results identify EDD as a new independent prognostic marker for outcome in serous ovarian cancer, and suggest that pathways involving EDD, including DNA damage responses, may represent new therapeutic targets for chemoresistant ovarian cancer.

Carboplatin and paclitaxel interact antagonistically in a megakaryoblast cell line--a potential mechanism for paclitaxel-mediated sparing of carboplatin-induced thrombocytopenia.

PURPOSE: Clinical observation has shown that paclitaxel ameliorates the antiplatelet toxicity of carboplatin when the two drugs are combined, although antitumour activity and white cell toxicity are at least additive. We hypothesized that this is due to an interaction between the two drugs at the level of the platelet precursor. METHODS: We measured inhibition of growth of the megakaryoblast cell line MEG-01 following exposure to paclitaxel and carboplatin singly or combined. Drug interaction was assessed by median effect analysis. RESULTS: An antagonistic interaction was observed, and this was most marked at drug concentrations giving a low level of growth inhibition (P < 0.002, sign test). The interaction was not sequence-dependent. There was no significant difference in whole-cell accumulation of platinum or the amount of platinum adducts on DNA following combined treatment in comparison with carboplatin alone. CONCLUSIONS: These results provide the first evidence of an antagonistic interaction between paclitaxel and carboplatin in a platelet precursor and provide an explanation for the platelet-sparing effect of the combination of these chemotherapeutic agents. While the mechanisms underlying the interaction described in this report are yet to be fully elucidated, this study provides evidence that the antagonism between paclitaxel and carboplatin in MEG-01 cells is not due to reduced platination of DNA.

Relative expression of progesterone receptors A and B in endometrioid cancers of the endometrium.

The nuclear receptor for the female hormone progesterone (PR) is widely expressed in uterine cancer. PR is expressed as two proteins (PRA and PRB) with different functions, and in vitro evidence reveals PRA to inhibit PRB function, so the cellular ratio of PRA:PRB is likely to be an important determinant of progesterone action. The relative expression of PRA and B and their involvement in the pathogenesis of endometrial cancer is not known. The aims of this study were to determine PRA and B expression by dual immunofluorescent histochemistry in endometrial adenocarcinomas compared with expression in normal and hyperplastic glands, and to correlate expression in tumors with clinical features including grade. Significantly lower PR levels were found in tumors compared with normal glands and areas of complex atypical hyperplasia within the same specimen. The normal glands expressed both of the isoforms at similar levels, whereas there was increased predominance of one isoform in hyperplastic areas and in tumors, which suggested that the loss of coordinated expression of PR isoforms was an early event in tumor progression. The majority of tumors [27 (58%) of 46] expressed only one PR isoform, and the proportion expressing either PRA or B was the same [14 (30%) of 46, and 13 (28%) of 46, respectively]. One-half of all tumors ([23 (50%) of 46] expressed either PRA only or a predominance of PRA, and a few tumors [10 (22%) of 46] expressed comparable levels of PRA and B. Similar levels of PRA and B were noted only in FIGO grade 1 tumors, whereas higher grades (2 and 3) were associated with a predominance of one isoform. In summary, expression of only one PR isoform was common in endometrial cancers, which indicates that the decreased PR levels observed in these cancers arise from the loss of one PR isoform. Expression of a single PR isoform was associated with higher clinical grade, which suggests a relationship between the loss of PR isoform expression and features of poorer prognosis. Disruption of relative PR isoform expression was observed in complex atypical hyperplasia, which suggests that early alterations in the ratio of PRA:PRB may precede and/or be implicated in the development of endometrial adenocarcinoma. Alterations in the ratio of PR isoform expression are likely to cause disordered regulation of target genes, resulting in altered progestin action in the uterus, and this may be involved in the pathogenesis of endometrial cancer.

Histone deacetylase inhibitors decrease proliferation and modulate cell cycle gene expression in normal mammary epithelial cells.

Full-term pregnancy early in reproductive life is protective against breast cancer in women. The protective effects of parity have variously been attributed to the differentiation that accompanies pregnancy and lactation, alterations in ovarian hormone receptor levels, and altered sensitivity to ovarian hormones. Butyrate, a short-chain fatty acid, induces differentiation in breast cancer cell lines and decreases hormone receptor expression. Butyrate also inhibits proliferation in breast cancer cell lines and modulates expression of key cell cycle-regulatory proteins including cyclin D1. Given these properties, butyrate could be considered a promising agent for breast cancer prevention. Therefore, this study aimed to determine the effects of butyrate on normal human breast epithelial cells and to compare the effects of two stable butyrate derivatives with more favorable pharmacological properties: phenylacetate and its p.o. active precursor phenylbutyrate. Treatment with each agent resulted in concentration-dependent growth inhibition in a normal breast epithelial cell line and two breast cancer cell lines (MCF-7 and MDA-MB-231). Phenylbutyrate and butyrate inhibited proliferation to a similar extent, but phenylacetate was less effective in all of the cell lines. All three of the agents induced differentiation (accumulation of lipid droplets) in normal as well as in breast cancer cells and caused a decrease in estrogen receptor (ER) mRNA in MCF-7 cells. The butyrates decreased expression of cyclin D1, increased expression of p21(Waf1/Cip1), and hypophosphorylated pRB in the normal mammary epithelial cells. The effects on cyclin D1 expression correlated with the effects on cell proliferation, which suggests that modulation of cyclin D1 expression may underpin the antiproliferative effects of butyrates. We have shown that butyrate and butyrate-like agents are able to decrease proliferation and induce differentiation in normal breast cells as well as in malignant breast cells (ER-positive and ER-negative) and, as such, may be considered as candidate chemopreventative agents for women at high risk of developing breast cancer.

Expression of c-erbB receptors, heregulin and oestrogen receptor in human breast cell lines.

Members of the c-erbB family have been implicated in poor prognosis in breast cancer. Given the propensity for heterodimerisation within the erbB family, the pattern of co-expression of these receptors is likely to be as functionally important as aberrant expression of any given receptor alone. Therefore, the patterns of expression of the receptors, epidermal growth factor receptor (EGF-R), c-erbB-2, c-erbB-3, c-erbB-4, and one of the erbB ligands, heregulin (HRG), were examined in normal and malignant breast cell lines and compared with expression of oestrogen receptor (ER), a classical indicator of good prognosis. There was an inverse correlation between ER and EGF-R mRNA levels, as previously described, but no correlation between either of these receptors and c-erbB-2. c-erbB-3 expression was positively correlated with ER. In contrast, HRG expression was inversely related to ER. Expression of antisense-ER resulted in increased EGF-R mRNA, demonstrating a functional link between the expression of these 2 genes, however, there was no significant change in c-erbB-2 or c-erbB-3 mRNA, suggesting that ER is not directly involved in control of expression of these genes. A comparison of individual erbB receptors and HRG revealed that the majority of lines expressing increased levels of c-erbB-2 also expressed elevated levels of c-erbB-3 mRNA, and none of the cell lines that expressed both c-erbB-2 and either c-erbB-3 or c-erbB-4 expressed the ligand HRG. In summary, the levels of expression of c-erbB-1, -2, -3, and -4 varied in this series of breast cell lines, and the pattern of expression and the relationship of each growth factor receptor to the expression of ER was quite distinct. The lack of expression of HRG in cell lines that express receptors may be indicative of paracrine interactions between erbB ligands and their cognate receptors and may suggest that the ligand and receptors are expressed in different subtypes of breast epithelial cells from which the cell lines are derived.

Chronic carbon monoxide exposure: a clinical syndrome detected by neuropsychological tests.

Chronic exposure to carbon monoxide produces a clinical syndrome that is often overlooked because of obscure symptomatology, a range of presentations, and lack of awareness of the problem. To help physicians recognize and treat the chronic carbon monoxide exposure syndrome, the authors present its objective symptomatology, an approach to diagnosis emphasizing neuropsychological tests, a treatment protocol, and theoretical considerations for the mechanism of hyperbaric oxygen's therapeutic action. For elucidation, eight patients treated in the hyperbaric chamber at a tertiary care facility are described. Diagnosis can be facilitated by recognizing the syndrome based on the patient's history, as well as physical and neuropsychological examinations, with emphasis on identifying potential sources of carbon monoxide exposure. The evaluation and treatment protocols presented, though still experimental, show promise for improving functional, cognitive, and psychiatric capacities.

Mechanisms determining sensitivity to cisplatin in three mutant Chinese hamster ovary cell lines.

To gain insight into factors determining the response of tumours to cisplatin, we studied pathways involved in resistance to cisplatin: drug uptake, cytoplasmic detoxification and DNA repair, in three cisplatin-sensitive Chinese hamster ovary (CHO)2 mutant cell lines. The mutant lines, CHO-MMC6, CHO-MMC1, CHO-MMS2, displayed inherent sensitivity to cisplatin (2.2, 4.1 and 10.6-fold, respectively) compared to the CHO-K1 line from which they were derived. CHO-MMS2 was the only mutant to show sensitivity to UV and this was slight (< 2-fold). None of the mutants displayed increased sensitivity to X-irradiation. The CHO-MMS2 cell line appeared to have multiple mechanisms involved in its sensitivity to cisplatin, including increased drug accumulation, decreased levels of glutathione and a decreased capacity for DNA repair. The CHO-MMC1 mutant demonstrated reduced ability for DNA repair in a host cell reactivation assay, but no difference in drug accumulation or glutathione levels compared to the parent. The CHO-MMC6 cell line was not defective in any of the mechanisms studied. These three mutant cell lines demonstrate that similar mechanisms may account for inherent sensitivity or resistance to cisplatin, and suggest that multiple mechanisms may determine the sensitivity of human tumours to cisplatin.

Antisense estrogen receptor RNA expression increases epidermal growth factor receptor gene expression in breast cancer cells.

In human breast cancer, progression to a more malignant phenotype is often accompanied by decreased expression of estrogen receptor (ER) and increased expression of epidermal growth factor receptor (EGFR). Higher levels of this receptor tyrosine kinase are found in tumors lacking ER, and a quantitative, inverse relationship exists between the level of ER and EGFR mRNA in human breast cell lines. Antisense ER (ASER) RNA was used to evaluate the consequence of decreased ER expression in breast cancer cells, specifically to determine whether ER is involved in the regulation of EGFR gene expression. ER-positive MCF-7 human breast cancer cells were transfected with ASER, and clones constitutively expressing ASER RNA had decreased ER and up to a 3-fold increase in the expression of EGFR mRNA. To confirm that this observation was a direct consequence of ASER expression, a metal-inducible ASER expression construct was transfected into MCF-7 cells, and transfected clones were isolated and characterized. Northern analysis revealed an induction of ASER RNA within 1 h of the addition of zinc, which was followed by a 4-fold increase in EGFR mRNA levels, maximal at 6-12 h. The basal level of expression of the glucocorticoid receptor is also inversely related to that of ER among breast cancer cell lines, but neither constitutive nor inducible expression of ASER affected the expression of glucocorticoid receptor. These data support the hypothesis that the level of expression of ER specifically influences the expression of EGFR in human breast cancer cells and provides a potential link between loss of steroid sensitivity and the acquisition of autonomous growth.

Expression and tyrosine phosphorylation of EMS1 in human breast cancer cell lines.

The EMS1 gene encodes an 80/85 kDa c-src substrate and localises with the CCND1 gene to chromosome 11q13. This locus is amplified in approximately 13% of human breast cancers. EMS1 gene amplification and expression were characterised in a panel of human breast cancer cell lines to determine at what levels expression is regulated. The degree of tyrosine phosphorylation of EMS1 protein was also determined and compared with the activity of src-family kinases. The EMS1 gene was amplified in 6 of 20 cell lines investigated: MDA-MB-134, -157, -175, -453, ZR-75-1 and MCF-7. In the MDA-MB-157 and MCF-7 cell lines, EMS1 was amplified in the absence of CCND1 gene amplification. EMS1 protein levels were increased relative to normal breast epithelial cells in 6 cell lines (ZR-75-1, MDA-MB-134, -175, 453, MCF-7 and BT-474). Of these, BT-474 is the only cell line that does not exhibit EMS1 amplification or increased EMS1 mRNA levels. EMS1 tyrosine phosphorylation was 3-fold higher in BT-474 and T-47D cells, which exhibited relatively high total src activity coupled with expression of both c-fyn and c-yes, than in MDA-MB-453 cells, which expressed only c-yes. Our results therefore demonstrate gene amplification to be the predominant mechanism underlying EMS1 over-expression in human breast cancer cell lines and identify tyrosine phosphorylation as a further level at which regulation of this protein may be perturbed.

Inverse regulation of oestrogen receptor and epidermal growth factor receptor gene expression in MCF-7 breast cancer cells treated with phorbol ester.

In human breast cancer cell lines, an inverse relationship exists between the basal levels of oestrogen receptor (ER) and epidermal growth factor receptor (EGF-R) gene expression. In addition, the tumour-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) inhibits ER and stimulates EGF-R expression in MCF-7 breast cancer cells. This study aimed to define further the potential mechanisms involved in the modulation of ER and EGF-R gene expression by TPA. ER mRNA levels were reduced after 3 h and declined to 30% of control between 12 and 72 h after exposure to 10 nM TPA. This decrease in mRNA levels was preceded by an apparent fall in ER transcription rate. There was no effect on the stability of ER mRNA following pretreatment for 3-24 h with TPA, supporting the conclusion that the fall in ER mRNA levels was predominantly due to a decrease in ER transcription rate. Levels of EGF-R mRNA increased 10-fold by 12 h due predominantly to an increased transcription rate. The TPA-induced decrease in ER mRNA was unaffected by the simultaneous administration of the protein synthesis inhibitor cycloheximide, whereas the increase in EGF-R mRNA was inhibited by co-incubation with cycloheximide. These data indicate a requirement for continuing protein synthesis for the TPA effect on EGF-R but not on ER mRNA levels. Because the modulation of ER and EGF-R gene expression by TPA is likely to involve the protein kinase C (PKC) signal transduction pathway, the effects of other known activators of PKC were investigated. The non-phorboid tumour promoter mezerein modulated ER (an 80% decrease) and EGF-R (a 20-fold increase) mRNA levels in a similar manner to TPA. In contrast, neither 1,2-dioctanoyl-sn-glycerol (DiC8) nor 1-oleoyl-2-acetyl-sn-glycerol (OAG), both permeant analogues of the endogenous physiological activators of PKC, affected ER and EGF-R mRNA levels. These latter results were not due to a lack of efficacy because a single administration of DiC8 was as effective as TPA in inducing c-fos mRNA at 30 min. However DiC8 was less active in the later induction of c-myc mRNA. These data demonstrate reciprocal regulation of ER and EGF-R gene expression by TPA, involving effects on transcriptional events, which appear to be mediated by sustained activation of PKC.

Antiestrogen inhibition of cell cycle progression in breast cancer cells in associated with inhibition of cyclin-dependent kinase activity and decreased retinoblastoma protein phosphorylation.

To define the mechanisms by which antiestrogens inhibit breast cancer cell proliferation, the effects of the antiestrogen ICI 182780 on G1 cyclins and their cyclin-dependent kinase (CDK) partners were investigated in MCF-7 cells. Inhibition of entry into S phase became evident 9 h after treatment, with the proportion of cells in S phase reaching a minimum by 24 h. ICI 182780 increased the proportion of the hypophosphorylated, growth inhibitory form of the retinoblastoma protein (pRB). This change began at 4-6 h, preceding effects on S phase. This suggests that there are early effects on the activities of CDKs that target pRB that are not merely a consequence of changes in cell cycle progression. The kinase activity of Cdk2 decreased to low levels at 18-24 h when changes in S phase and pRB phosphorylation were well advanced. An earlier effect was seen on kinase activity associated with immunoprecipitated cyclin D1, which was reduced approximately 40% by 12 h, with further decreases at 18-24 h. Cdk2 and Cdk4 protein levels remained constant over 24 h. Cyclin D1 messenger RNA and protein were down-regulated by ICI 182780 from 2 h, with levels halved at 8 h. ICI 182780 also increased the expression of the CDK inhibitors p27KIP1 and p21WAF1/CIP1 at later times. These observations are compatible with the hypothesis that antiestrogens block entry of cells into S phase and inhibit cell proliferation as the consequence of an early decline in pRB phosphorylation contributed to by reduced cyclin D1/Cdk4 activity. At later times, increased CDK inhibitor abundance may act to repress Cdk2 and Cdk4 activities, causing additional reductions in pRB phosphorylation, thus maintaining the antiestrogen blockade of cell cycle progression.