Adjuvant chemotherapy in elderly patients with pancreatic cancer.

BACKGROUND: Adjuvant chemotherapy improves survival for patients with resected pancreatic cancer. Elderly patients are under-represented in Phase III clinical trials, and as a consequence the efficacy of adjuvant therapy in older patients with pancreatic cancer is not clear. We aimed to assess the use and efficacy of adjuvant chemotherapy in older patients with pancreatic cancer. METHODS: We assessed a community cohort of 439 patients with a diagnosis of pancreatic ductal adenocarcinoma who underwent operative resection in centres associated with the Australian Pancreatic Cancer Genome Initiative. RESULTS: The median age of the cohort was 67 years. Overall only 47% of all patients received adjuvant therapy. Patients who received adjuvant chemotherapy were predominantly younger, had later stage disease, more lymph node involvement and more evidence of perineural invasion than the group that did not receive adjuvant treatment. Overall, adjuvant chemotherapy was associated with prolonged survival (median 22.1 vs 15.8 months; P<0.0001). Older patients (aged ≥70) were less likely to receive adjuvant chemotherapy (51.5% vs 29.8%; P<0.0001). Older patients had a particularly poor outcome when adjuvant therapy was not delivered (median survival=13.1 months; HR 1.89, 95% CI: 1.27-2.78, P=0.002). CONCLUSION: Patients aged ≥70 are less likely to receive adjuvant therapy although it is associated with improved outcome. Increased use of adjuvant therapy in older individuals is encouraged as they constitute a large proportion of patients with pancreatic cancer.

Role of endoplasmic reticulum stress induction by the plant toxin, persin, in overcoming resistance to the apoptotic effects of tamoxifen in human breast cancer cells.

BACKGROUND: Persin is a plant toxin that displays synergistic cytotoxicity with tamoxifen in human breast cancer cell lines. Here, we examined the ability of persin to circumvent tamoxifen resistance and delineated the intracellular signalling pathways involved. METHODS: The induction of apoptosis in tamoxifen-resistant and -sensitive breast cancer cells was measured by flow cytometry following treatment with persin±tamoxifen. Markers of endoplasmic reticulum stress (ERS) were analysed following treatment, and their causal role in mediating persin-induced apoptosis was determined using chemical inhibitors and RNA interference. RESULTS: Cells that were resistant to an apoptotic concentration of tamoxifen maintained an apoptotic response to persin. Persin-induced apoptosis was associated with an increase in markers of ERS, that is, CHOP expression and XBP-1 splicing and was decreased by CHOP siRNA. The CASP-4 inhibitor Z-YVAD-FMK markedly inhibited persin-induced apoptosis in both tamoxifen-sensitive and -resistant cells. CONCLUSION: The cytotoxic effects of persin are CASP-4 dependent and mediated by CHOP-dependent and -independent ERS signalling cascades. Increased ERS signalling contributes to persin-induced reversal of tamoxifen resistance.

The prognostic and predictive value of serum CA19.9 in pancreatic cancer.

BACKGROUND: Current staging methods for pancreatic cancer (PC) are inadequate, and biomarkers to aid clinical decision making are lacking. Despite the availability of the serum marker carbohydrate antigen 19.9 (CA19.9) for over two decades, its precise role in the management of PC is yet to be defined, and as a consequence, it is not widely used. METHODS: We assessed the relationship between perioperative serum CA19.9 levels, survival and adjuvant chemotherapeutic responsiveness in a cohort of 260 patients who underwent operative resection for PC. RESULTS: By specifically assessing the subgroup of patients with detectable CA19.9, we identified potential utility at key clinical decision points. Low postoperative CA19.9 at 3 months (median survival 25.6 vs 14.8 months, P=0.0052) and before adjuvant chemotherapy were independent prognostic factors. Patients with postoperative CA 19.9 levels>90 U/ml did not benefit from adjuvant chemotherapy (P=0.7194) compared with those with a CA19.9 of ≤90 U/ml (median 26.0 vs 16.7 months, P=0.0108). Normalization of CA19.9 within 6 months of resection was also an independent favorable prognostic factor (median 29.9 vs 14.8 months, P=0.0004) and normal perioperative CA19.9 levels identified a good prognostic group, which was associated with a 5-year survival of 42%. CONCLUSIONS: Perioperative serum CA19.9 measurements are informative in patients with detectable CA19.9 (defined by serum levels of >5 U/ml) and have potential clinical utility in predicting outcome and response to adjuvant chemotherapy. Future clinical trials should prioritize incorporation of CA19.9 measurement at key decision points to prospectively validate these findings and facilitate implementation.

Prediction of outcome of early ER+ breast cancer is improved using a biomarker panel, which includes Ki-67 and p53.

BACKGROUND: The aim of this study is to determine whether immunohistochemical (IHC) assessment of Ki67 and p53 improves prognostication of oestrogen receptor-positive (ER+) breast cancer after breast-conserving therapy (BCT). In all, 498 patients with invasive breast cancer from a randomised trial of BCT with or without tumour bed radiation boost were assessed using IHC. METHODS: The ER+ tumours were classified as 'luminal A' (LA): ER+ and/or PR+, Ki-67 low, p53-, HER2- or 'luminal B' (LB): ER+ and/or PR+and/or Ki-67 high and/or p53+ and/or HER2+. Kaplan-Meier and Cox proportional hazards methodology were used to ascertain relationships to ispilateral breast tumour recurrence (IBTR), locoregional recurrence (LRR), distant metastasis-free survival (DMFS) and breast cancer-specific survival (BCSS). RESULTS: In all, 73 patients previously LA were re-classified as LB: a greater than four-fold increase (4.6-19.3%) compared with ER, PR, HER2 alone. In multivariate analysis, the LB signature independently predicted LRR (hazard ratio (HR) 3.612, 95% CI 1.555-8.340, P=0.003), DMFS (HR 3.023, 95% CI 1.501-6.087, P=0.002) and BCSS (HR 3.617, 95% CI 1.629-8.031, P=0.002) but not IBTR. CONCLUSION: The prognostic evaluation of ER+ breast cancer is improved using a marker panel, which includes Ki-67 and p53. This may help better define a group of poor prognosis ER+ patients with a greater probability of failure with endocrine therapy.

Collagen and calcium-binding EGF domains 1 is frequently inactivated in ovarian cancer by aberrant promoter hypermethylation and modulates cell migration and survival.

BACKGROUND: Collagen and calcium-binding EGF domains 1 (CCBE1) is an uncharacterised gene that has down-regulated expression in breast cancer. As CCBE1 maps to 18q21.32, a region frequently exhibiting loss of heterozygosity in ovarian cancer, the aim of this study was to determine the expression and function of CCBE1 in ovarian cancer. METHODS: Expression and methylation patterns of CCBE1 were determined in ovarian cancer cell lines and primary tumours. CCBE1 contains collagen repeats and an aspartic acid/asparagine hydroxylation/EGF-like domain, suggesting a function in extracellular matrix remodelling and migration, which was determined using small-interfering RNA (siRNA)-mediated knockdown and over-expression of CCBE1 in cell lines. RESULTS: CCBE1 is expressed in normal ovary, but is reduced in ovarian cancer cell lines and primary carcinomas. Pharmacological demethylation/deacetylation in ovarian cancer cell lines re-induced CCBE1 expression, indicating that epigenetic mechanisms contribute to its silencing in cancer. CCBE1 promoter hypermethylation was detected in 6/11 (55%) ovarian cancer cell lines and 38/81 (41%) ovarian carcinomas. siRNA-mediated knockdown of CCBE1 in ovarian cancer cell lines enhanced their migration; conversely, re-expression of CCBE1 reduced migration and survival. Hence, loss of CCBE1 expression may promote ovarian carcinogenesis by enhancing migration and cell survival. CONCLUSIONS: These data suggest that CCBE1 is a new candidate tumour suppressor in ovarian cancer.

BAG-1 predicts patient outcome and tamoxifen responsiveness in ER-positive invasive ductal carcinoma of the breast.

BAG-1 (bcl-2-associated athanogene) enhances oestrogen receptor (ER) function and may influence outcome and response to endocrine therapy in breast cancer. We determined relationships between BAG-1 expression, molecular phenotype, response to tamoxifen therapy and outcome in a cohort of breast cancer patients and its influence on tamoxifen sensitivity in MCF-7 breast cancer cells in vitro. Publically available gene expression data sets were analysed to identify relationships between BAG-1 mRNA expression and patient outcome. BAG-1 protein expression was assessed using immunohistochemistry in 292 patients with invasive ductal carcinoma and correlated with clinicopathological variables, therapeutic response and disease outcome. BAG-1-overexpressing MCF-7 cells were treated with antioestrogens to assess its effects on cell proliferation. Gene expression data demonstrated a consistent association between high BAG-1 mRNA and improved survival. In ER+ cancer (n=189), a high nuclear BAG-1 expression independently predicted improved outcome for local recurrence (P=0.0464), distant metastases (P=0.0435), death from breast cancer (P=0.009, hazards ratio 0.29, 95% CI: 0.114-0.735) and improved outcome in tamoxifen-treated patients (n=107; P=0.0191). BAG-1 overexpression in MCF-7 cells augmented antioestrogen-induced growth arrest. A high BAG-1 expression predicts improved patient outcome in ER+ breast carcinoma. This may reflect both a better definition of the hormone-responsive phenotype and a concurrent increased sensitivity to tamoxifen.

The key hypoxia regulated gene CAIX is upregulated in basal-like breast tumours and is associated with resistance to chemotherapy.

Basal-like tumours account for 15% of invasive breast carcinomas and are associated with a poorer prognosis and resistance to therapy. We hypothesised that this aggressive phenotype is because of an intrinsically elevated hypoxic response. Microarrayed tumours from 188 patients were stained for hypoxia-inducible factor (HIF)-1alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and factor inhibiting HIF (FIH)-1, and carbonic anhydrase (CA) IX stained in 456 breast tumours. Tumour subtypes were correlated with standard clincopathological parameters as well as hypoxic markers. Out of 456 tumours 62 (14%) tumours were basal-like. These tumours were positively correlated with high tumour grade (P<0.001) and were associated with a significantly worse disease-free survival compared with luminal tumours (P<0.001). Fifty percent of basal-like tumours expressed HIF-1alpha, and more than half expressed at least one of the PHD enzymes and FIH-1. Basal-like tumours were nine times more likely to be associated with CAIX expression (P<0.001) in a multivariate analysis. Carbonic anhydrase IX expression was positively correlated with tumour size (P=0.005), tumour grade (P<0.001) and oestrogen receptor (ER) negativity (P<0.001). Patients with any CAIX-positive breast tumour phenotype and in the basal tumour group had a significantly worse prognosis than CAIX-negative tumours when treated with chemotherapy (P<0.001 and P=0.03, respectively). The association between basal phenotype and CAIX suggests that the more aggressive behaviour of these tumours is partly due to an enhanced hypoxic response. Further, the association with chemoresistance in CAIX-positive breast tumours and basal-like tumours in particular raises the possibility that targeted therapy against HIF pathway or downstream genes such as CAs may be an approach to investigate for these patients.

Promoter methylation of the mutated in colorectal cancer gene is a frequent early event in colorectal cancer.

The mutated in colorectal cancer (MCC) gene is in close linkage with the adenomatous polyposis coli (APC) gene on chromosome 5, in a region of frequent loss of heterozygosity in colorectal cancer. The role of MCC in carcinogenesis, however, has not been extensively analysed, and functional studies are emerging, which implicate it as a candidate tumor suppressor gene. The aim of this study was to examine loss of MCC expression due to promoter hypermethylation and its clinicopathologic significance in colorectal cancer. Correspondence of MCC methylation with gene silencing was demonstrated using bisulfite sequencing, reverse transcription-polymerase chain reaction and Western blotting. MCC methylation was detected in 45-52% of 187 primary colorectal cancers. There was a striking association with CDKN2A methylation (P<0.0001), the CpG island methylator phenotype (P<0.0001) and the BRAF V600E mutation (P<0.0001). MCC methylation was also more common (P=0.0084) in serrated polyps than in adenomas. In contrast, there was no association with APC methylation or KRAS mutations. This study demonstrates for the first time that MCC methylation is a frequent change during colorectal carcinogenesis. Furthermore, MCC methylation is significantly associated with a distinct spectrum of precursor lesions, which are suggested to give rise to cancers via the serrated neoplasia pathway.

DLEC1 and MLH1 promoter methylation are associated with poor prognosis in non-small cell lung carcinoma.

The significance of chromosome 3p gene alterations in lung cancer is poorly understood. This study set out to investigate promoter methylation in the deleted in lung and oesophageal cancer 1 (DLEC1), MLH1 and other 3p genes in 239 non-small cell lung carcinomas (NSCLC). DLEC1 was methylated in 38.7%, MLH1 in 35.7%, RARbeta in 51.7%, RASSF1A in 32.4% and BLU in 35.3% of tumours. Any two of the gene alterations were associated with each other except RARbeta. DLEC1 methylation was an independent marker of poor survival in the whole cohort (P=0.025) and in squamous cell carcinoma (P=0.041). MLH1 methylation was also prognostic, particularly in large cell cancer (P=0.006). Concordant methylation of DLEC1/MLH1 was the strongest independent indicator of poor prognosis in the whole cohort (P=0.009). However, microsatellite instability and loss of MLH1 expression was rare, suggesting that MLH1 promoter methylation does not usually lead to gene silencing in lung cancer. This is the first study describing the prognostic value of DLEC1 and MLH1 methylation in NSCLC. The concordant methylation is possibly a consequence of a long-range epigenetic effect in this region of chromosome 3p, which has recently been described in other cancers.

Prognostic significance of DNA repair proteins MLH1, MSH2 and MGMT expression in non-small-cell lung cancer and precursor lesions.

AIMS: To investigate the role of DNA repair proteins and their prognostic significance in non-small-cell lung cancer (NSCLC). METHODS AND RESULTS: A retrospective analysis of 108 cases of stage I-II NSCLC was undertaken. Immunohistochemical expression of DNA repair proteins MLH1, MSH2 and MGMT was assessed using tissue microarrays of paraffin-embedded samples of invasive carcinoma and precursor lesions. Results were analysed in relation to clinicopathological parameters and patient survival. Reduced expression of MLH1 was found in 58.5% of tumours and occurred less frequently in poorly differentiated tumours (P = 0.044) and large cell carcinomas (P = 0.004). MSH2 and MGMT expression was reduced in 18.1% and 77.8% of cases, respectively. There was an inverse relationship between MLH1 and MSH2 expression (P = 0.012). Normal expression of MLH1, MSH2 and MGMT was found in all cases of squamous metaplasia and squamous dysplasia. Only a single case of carcinoma in situ (12.5%) showed reduced MLH1, none showed reduced MSH2 and 25% showed reduced MGMT. Survival analyses showed no prognostic significance based on expression of MLH1 (P = 0.92), MSH2 (P = 0.78) or MGMT (P = 0.57). CONCLUSIONS: Reduction in expression of DNA repair proteins MLH1, MSH2 and MGMT is relatively common in NSCLC, appears to be a late event in the development of invasive malignancy and does not influence survival in this patient cohort.

Progestins both stimulate and inhibit breast cancer cell cycle progression while increasing expression of transforming growth factor alpha, epidermal growth factor receptor, c-fos, and c-myc genes.

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.

Regulation of the functional interaction between cyclin D1 and the estrogen receptor.

We report that the functional interaction between cyclin D1 and the estrogen receptor (ER) is regulated by a signal transduction pathway involving the second messenger, cyclic AMP (cAMP). The cell-permeable cAMP analogue 8-bromo-cAMP caused a concentration-dependent enhancement of cyclin D1-ER complex formation, as judged both by coimmunoprecipitation and mammalian two-hybrid analysis. This effect was paralleled by increases in ligand-independent ER-mediated transcription from an estrogen response element containing reporter construct. These effects of 8-bromo-cAMP were antagonized by a specific protein kinase A (PKA) inhibitor, indicating that the signaling pathway involved was PKA dependent. Further, we show that culture of MCF-7 cells on a cellular substratum of murine preadipocytes also enhanced the functional interaction between cyclin D1 and ER in a PKA-dependent manner. These findings demonstrate a collaboration between cAMP signaling and cyclin D1 in the ligand-independent activation of ER-mediated transcription in mammary epithelial cells and show that the functional associations of cyclin D1 are regulated as a function of cellular context.

Cooperation of p27(Kip1) and p18(INK4c) in progestin-mediated cell cycle arrest in T-47D breast cancer cells.

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G(1) cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27(Kip1) among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27(Kip1) and few were bound to p21(Cip1). In vitro, recombinant His(6)-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His(6)-p27 in vitro or p27(Kip1) in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18(INK4c) and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18(INK4c) led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27(Kip1) and p18(INK4c) cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor beta and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation.

Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression.

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.

Mechanisms of cyclin-dependent kinase inactivation by progestins.

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. In breast cancer cells the predominant effect of synthetic progestins is long-term growth inhibition and arrest in G1 phase. Progestin-mediated growth arrest of T-47D breast cancer cells was preceded by inhibition of cyclin D1-Cdk4, cyclin D3-Cdk4, and cyclin E-Cdk2 kinase activities in vitro and reduced phosphorylation of pRB and p107. This was accompanied by decreases in the expression of cyclins D1, D3, and E, decreased abundance of cyclin D1- and cyclin D3-Cdk4 complexes, increased association of the cyclin-dependent kinase (CDK) inhibitor p27 with the remaining Cdk4 complexes, and changes in the molecular masses and compositions of cyclin E complexes. In control cells cyclin E eluted from Superdex 200 as two peaks of approximately 120 and approximately 200 kDa, with the 120-kDa peak displaying greater cyclin E-associated kinase activity. Following progestin treatment, almost all of the cyclin E was in the 200-kDa, low-activity form, which was associated with the CDK inhibitors p21 and p27; this change preceded the inhibition of cell cycle progression. These data suggest preferential formation of this higher-molecular-weight, CDK inhibitor-bound form and a reduced number of cyclin E-Cdk2 complexes as mechanisms for the decreased cyclin E-associated kinase activity following progestin treatment. Ectopic expression of cyclin D1 in progestin-inhibited cells led to the reappearance of the 120-kDa active form of cyclin E-Cdk2 preceding the resumption of cell cycle progression. Thus, decreased cyclin expression and consequent increased CDK inhibitor association are likely to mediate the decreases in CDK activity accompanying progestin-mediated growth inhibition.

c-Myc or cyclin D1 mimics estrogen effects on cyclin E-Cdk2 activation and cell cycle reentry.

Estrogen-induced progression through G1 phase of the cell cycle is preceded by increased expression of the G1-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, we derived clonal MCF-7 breast cancer cell lines in which c-Myc or cyclin D1 was expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 was sufficient for S-phase entry in cells previously arrested in G1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression was not accompanied by increased cyclin D1 expression or Cdk4 activation, nor was cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 was sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21, as has been described, following estrogen treatment. Interestingly, this was accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130, identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21.

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. III. Hormone receptor status and responsiveness.

PMC42 is a new human breast carcinoma cell line. In this report the content of estrogen, progesterone, and glucocorticoid receptors in PMC42 has been determined. The estrogen receptor content (1,750 cytoplasmic sites and 350 nuclear sites) was lower than that described for MCF7 and T47D. The cells would not proliferate in serum-free medium without the addition of beta-estradiol (optimum concentration 10(-8) M) or progesterone (5 X 10(-8) M). The addition of both hormones induced a more than additive increase in proliferation (P less than .005, n = 18). Similarly, addition of insulin or hydrocortisone induced proliferation; however, in this case, the effect of the hormones together was only additive. The addition of tamoxifen (10(-6) M) led to a significant decrease in cell numbers and inhibited the stimulatory effects of 10(-8) M beta-estradiol.

Effects of pharmacological concentrations of estrogens on proliferation and cell cycle kinetics of human breast cancer cell lines in vitro.

High dose estrogen therapy has been used effectively in the treatment of human breast cancer. To understand the mechanisms involved, the effects of high concentrations (5-100 microM) of estrogens were studied in estrogen receptor (ER) positive (T-47D and MCF-7) and ER negative (MDA-MB-330) human breast cancer cell lines in vitro. Inhibition of cellular proliferation was seen with the synthetic estrogen diethylstilbestrol (DES) at concentrations greater than 10 microM in each of the three cell lines. In T-47D cells DES was shown by clonogenic survival assays to be cytotoxic. This effect was evident in both plateau phase and exponentially growing cultures, in contrast to the effects of the antiestrogen tamoxifen, which has minimal effects on plateau phase cells. The effects of DES on the proliferation of exponentially growing cultures were accompanied by changes in cell cycle parameters which included an increase in the percentages of S-phase, G2 + M, and polyploid cells and a corresponding decrease in the percentage of G0-G1 cells. These changes, which contrasted with the known effects of tamoxifen, were not seen in the non- or slowly cycling plateau phase T-47D cells. Such results are consistent with two mechanisms of action of high dose estrogen in vitro: a cell cycle phase-specific effect and cell cycle-independent cytotoxicity. The stereoisomers 17 alpha-estradiol and 17 beta-estradiol had similar potency to DES in inhibiting cell proliferation and inducing these changes in cell cycle parameters in both MCF-7 and MDA-MB-330 cells. The high-dose estrogen effect was ligand specific in that estrone and estriol were less potent than DES, 17 alpha-estradiol and 17 beta-estradiol in inhibiting cell proliferation, and the characteristic cell cycle changes were produced only by concentrations of estriol greater than 75 microM and not at all by estrone at concentrations up to 100 microM. The androgens testosterone and dihydrotestosterone were similar in effect to estrone. The cell cycle changes associated with estrogen-induced growth inhibition in vitro are identical to those observed during regression of ER positive but not ER negative human tumor xenografts in nude mice. However, the role of ER in mediating estrogen-induced regression of ER positive tumors in vivo remains undefined.

Regulation of epidermal growth factor receptor levels by 1,25-dihydroxyvitamin D3 in human breast cancer cells.

Specific, high affinity receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been demonstrated in human breast cancer cells. In addition, 1,25-(OH)2D3 has been shown to inhibit replication in some human breast cancer cell lines, although the mechanism(s) of this anti-tumor activity remain undefined. There is currently considerable interest in the role of autocrine growth factors in the control of breast cancer cell proliferation and the effects of steroid hormones on their production, receptor binding, and action. Since the epidermal growth factor (EGF) receptor mediates the effects of both EGF and the autocrine growth factor, alpha-transforming growth factor, we investigated the effect of 1,25-(OH)2D3 on EGF receptor levels in several human breast cancer cell lines. Preincubation of T-47D cells with 1,25-(OH)2D3 for 24 h resulted in a significant concentration-dependent decline in the specific binding of [125I]EGF. The effect was observed when EGF binding was assayed at either 0 or 37 degrees C, both before and after treatment with acid to remove receptor bound endogenous ligand. This indicated that the effect on [125I]-EGF binding was not due to effects of 1,25-(OH)2D3 on receptor internalization and degradation or receptor occupancy. The half-maximal inhibitory concentration of 1,25-(OH)2D3 was approximately 2 nM. The decrease in EGF binding was due to a decrease in receptor number from 2,900 sites/cell in control cultures to 2,330 and 1,730 sites/cell in cells treated for 24 h with 10(-8) and 10(-6) M 1,25-(OH)2D3, respectively. There was no change in the affinity of the receptor for EGF following treatment with 1,25-(OH)2D3 [Kd = 0.075 +/- 0.006 nM (+/- SEM) for control and Kd = 0.083 +/- 0.004 nM for treated cells]. Decreased EGF receptor levels were also achieved with a number of analogues of 1,25-(OH)2D3 in accordance with their affinities for the 1,25-(OH)2D3 receptor, i.e., potencies for decreasing EGF binding in T-47D cells were in the order: 1,25-(OH)2D3 greater than 1,24,25-trihydroxyvitamin D3 greater than 1,25,26-trihydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3 greater than or equal to 25-hydroxyvitamin D3. Specific, saturable EGF binding to MCF-7 cells was also reduced by 1,25-(OH)2D3 while binding to BT-20 and HBL-100 cells was unaffected by this treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of the growth-inhibitory effects of progestins and the antiestrogen hydroxyclomiphene on human breast cancer cells by epidermal growth factor and insulin.

The molecular basis of the growth-inhibitory effects of progestins or antiestrogens in human breast cancer has not been fully elucidated. Both direct actions and indirect actions, where the growth inhibition results from modulation of the production of, and/or the response to, growth factors, have been proposed. In this study the ability of some growth factors to modulate progestin-induced inhibition of cell proliferation was investigated in vitro, using T-47D human breast cancer cells. When T-47D cells grown in insulin-containing medium were treated for 4 to 5 days with the synthetic progestin, ORG 2058, at a concentration of 10 nM, cell numbers were reduced to 10 to 20% of control. Simultaneous treatment with epidermal growth factor (EGF) and ORG 2058 led to a partial reversal of the growth-inhibitory effect of the progestin. The magnitude of the effect of EGF was concentration dependent, being half-maximal at 0.48 ng/ml (0.08 nM) and maximal at concentrations greater than 5 ng/ml (greater than 0.8 nM), where cell numbers were increased by 50% compared to those in the presence of ORG 2058 alone. ORG 2058 was no more potent in the absence of insulin, and, after several passages in insulin-free medium, addition of insulin failed to modulate the effect of ORG 2058. However, when maximal concentrations of insulin (5 micrograms/ml) and EGF (10 ng/ml) were administered together with ORG 2058, insulin and EGF appeared to act synergistically to reduce the ORG 2058-induced inhibition of proliferation. In similar experiments in which cells were treated with hydroxyclomiphene, a potent antiestrogen, insulin was shown to partially reverse the growth-inhibitory effects of hydroxyclomiphene. Significant increases in cell number above hydroxyclomiphene-treated controls were apparent at insulin concentrations greater than 50 ng/ml, and at 5 micrograms/ml the increase was approximately 2-fold. In contrast to the situation with progestins, simultaneous treatment with EGF and insulin had only an additive effect in reversing the growth-inhibitory effect of the antiestrogen. The results are compatible with the hypothesis that part of the growth-inhibitory effects of progestin and antiestrogen on human breast cancer cell proliferation is mediated by inhibition of autocrine growth factor production. However, they do not exclude more direct mechanisms involving modulation of progesterone and/or estrogen receptors by EGF and/or insulin.(ABSTRACT TRUNCATED AT 400 WORDS)