Gene regulatory networks: a new conceptual framework to analyse breast cancer behaviour.

The study of complex systems has clearly evidenced that a few overall behavioural properties cannot be inferred from the properties of their single parts and are rather determined by their architecture. Such an approach has been recently proposed in biology to understand genome functioning and in oncology to endeavour a more consistent explanation of the variegated cancer behaviours. In the present perspective, we summarise the basic concepts of the proposed global approach and then we reconsider, in this new context, tumour dormancy and primary tumour removal effects, which recently emerged as critical points for breast cancer understanding.

An immunohistochemically positive E-cadherin status is not always predictive for a good prognosis in human breast cancer.

BACKGROUND: in primary breast cancers dichotomic classification of E-cadherin expression, according to an arbitrary cutoff, may be inadequate and lead to loss of prognostic significance or contrasting prognostic indications. We aimed to assess the prognostic value of high and low E-cadherin levels in a consecutive case series (204 cases) of unilateral node-negative non-lobular breast cancer patients with a 8-year median follow-up and that did not receive any adjuvant therapy after surgery. METHODS: expression of E-cadherin was investigated by immunohistochemistry and assessed according to conventional score (0, 1+, 2+, 3+). Multiple correspondence analysis was used to visualise associations of both categorical and continuous variables. The impact of E-cadherin expression on patients outcome was evaluated in terms of event-free survival curves by the Kaplan-Meier method and proportional hazard Cox model. RESULTS: respect to intermediate E-cadherin expression values (2+), high (3+) or low (0 to 1+) E-cadherin expression levels had a negative prognostic impact. In fact, both patients with a low-to-nil (score 0 to 1+) expression level of E-cadherin and patients with a high E-cadherin expression level (score 3+) demonstrated an increased risk of failure (respectively, hazard ratio (HR)=1.71, confidence interval (CI)=0.72-4.06 and HR=4.22, CI=1.406-12.66) and an interesting association with young age. CONCLUSIONS: the findings support the evidence that high expression values of E-cadherin are not predictive for a good prognosis and may help to explain conflicting evidence on the prognostic impact of E-cadherin in breast cancer when assessed on dichotomic basis.

A prediction model for breast cancer recurrence after adjuvant hormone therapy.

Hormone therapy with tamoxifen has long been the established adjuvant treatment for node-positive, estrogen-receptor-positive breast cancer in postmenopausal women. Since 30-40% of these patients fail to respond, reliableoutcome prediction is necessary for successful treatment allocation. Using pathobiological variables (available in mostclinical records: tumor size, nodal involvement, estrogen and progesterone receptor content) from 596 patients recruitedat a comprehensive cancer center, we developed a prediction model which we validated in an independent cohort of 175patients recruited at a general hospital. Calculated at 3 and 4 years of follow-up, the discrimination indices were 0.716[confidence limits (CL) 0.641, 0.752] and 0.714 (CL 0.650, 0.750) for the training data, and 0.726 (CL 0.591, 0.769) and0.677 (CL 0.580, 0.745) for the testing data. Waiting for more effective approaches from genomic and proteomic studies, amodel based on consolidated pathobiological variables routinely assessed at relatively low costs may be considered as thereference for assessing the gain of new markers over traditional ones, thus substantially improving the conventional use ofprognostic criteria.

A novel retinoic/butyric hyaluronan ester for the treatment of acute promyelocytic leukemia: preliminary preclinical results.

All-trans retinoic acid (ATRA) represents the therapy of choice for patients with acute promyelocytic leukemia (APL). However, patients often relapse due to ATRA-resistance. The molecular basis of APL alterations indicates that addition of a histone deacetylase inhibitor to ATRA may restore the sensitivity to retinoids. We explored the in vitro and in vivo effects of a novel retinoic/butyric hyaluronan ester (HBR) on a retinoic acid (RA)-sensitive human myeloid cell line, NB4, and on its RA-resistant subclone, NB4.007/6. In vitro, HBR induced growth arrest and terminal differentiation in RA-sensitive NB4 cells (as confirmed by an increased expression of CD11 family members and nitroblue tetrazolium assay), whereas it inhibited the growth of RA-resistant cells by apoptosis, paralleled by an increase in the levels of caspase 3 and 7. In vivo, HBR treatment of NB4-inoculated severe combined immunodeficient mice resulted in a statistically significant increase in survival time (P<0.0001), comparable to that induced by a maximum tolerated dose of RA alone. Also on P388-inoculated mice, HBR was active in contrast to RA that was completely ineffective. Present findings suggest that, owing to the simultaneous presence of RA and an histone deacetylases inhibitor, HBR might be useful in controlling the proliferation of RA-resistant cells and the differentiation of RA-sensitive cells.

Hypoxia and estrogen receptor profile influence the responsiveness of human breast cancer cells to estradiol and antiestrogens.

Angiogenesis activation mediated by vascular endothelial growth factor (VEGF) is one of the factors that can cause antiestrogen treatment failure in estrogen receptor (ER)?positive breast cancer patients. Since VEGF synthesis is modulated not only by hypoxia but also by steroid hormones, we investigated the relationship between hypoxic and estrogenic/antiestrogenic stimuli in two human breast cancer cell lines expressing both ER6alpha and ERbeta (MCF7) or only ERbeta (MDA-MB231). In both cell lines, the VEGF level was significantly influenced by hypoxic conditions and in antiestrogen-responsive MCF7 cells, this effect was not counteracted by tamoxifen or ICI 182780, thus providing an experimental explanation for the resistance to endocrine treatment observed in patients with ER-positive tumors. In MDA-MB231 cells, estradiol significantly reduced the VEGF level, suggesting that through the ERbeta isoform it may function as a negative modulator of VEGF synthesis under hypoxia, and providing evidence for a complex interplay of the estrogen-dependent and hypoxia-dependent pathways.

Prognosis in node-negative primary breast cancer: a neural network analysis of risk profiles using routinely assessed factors.

BACKGROUND: The present study investigated complex time-dependent effects of routinely assessed factors on the risk of breast cancer recurrence over follow-up time, with a partial logistic artificial neural network (PLANN) model. PATIENTS AND METHODS: PLANN was applied to data from 1793 patients with node-negative breast cancer, not submitted to any adjuvant treatment and with a minimal potential follow-up of 10 years. RESULTS: The shape of the hazard function changed according to histology, which showed a time-dependent effect, partly modulated by estrogen receptors (ERs). Age and progesterone receptors (PgR) showed protective effects; the latter was more evident for short follow-up and high ER values. Tumour size and ER content showed time-dependent unfavourable effects at early and long follow-up times, respectively. Predicted values of disease recurrence probability at 2 years of follow-up showed that low steroid-receptor content, young age and large tumour size were associated with the highest risk of relapse. Although the oldest patients with high ER content seem to be those most protected overall, high risk predictions tend to spread also to higher steroid-receptor contents, intermediate ages and small tumour size, with an increase in follow-up time. CONCLUSION: PLANN with suitable visualisation techniques provided thorough insights into the dynamics of breast cancer recurrence for improving individual risk staging of node-negative breast cancer patients.

Vascular endothelial growth factor in node-positive breast cancer patients treated with adjuvant tamoxifen.

In 212 postmenopausal women with node-positive oestrogen receptor-positive (ER(LBA)) breast cancer subjected to radical surgery and adjuvant tamoxifen, the risk of 6-year relapse increased with increasing values of intratumoral vascular endothelial growth factor (VEGF) in patients whose tumours had a low/intermediate ER(LBA) content compared to patients with high-ER(LBA) tumours. These findings indicate that tumour progression, activated or sustained by high VEGF levels, may be counteracted in high-ER(LBA) cancers by tamoxifen, which in contrast fails to contrast the metastatic potential in low-ER(LBA) tumours.

Infiltrating ductal and lobular breast carcinomas are characterised by different interrelationships among markers related to angiogenesis and hormone dependence.

To obtain a more integrated understanding of the different breast cancer phenotypes and to investigate whether bio-molecular profiles can distinguish between specific histotypes, we explored the interrelations among several biologic variables indicative of, or related to, hormone dependence, proliferation and apoptosis control, and angiogenesis in ductal and lobular carcinomas, the most common histotypes. Oestrogen and progesterone receptors, tumour proliferative activity, the expression of cyclin A, p16(ink4A), p27(kip1), p21(waf1), p53, bcl-2, and levels of vascular endothelial growth factor and hypoxia-inducible factor-1alpha (HIF-1alpha) were evaluated in 190 in ductal and 67 lobular carcinomas. Our findings support the hypothesis that in ductal and lobular carcinomas are two distinct, partially phenotypically unrelated entities, the latter being characterised by the presence of features indicative of differentiation such as oestrogen receptors, low proliferation and lack of p53 expression and associated with low vascular endothelial growth factor content compared to angiogenesis in ductal carcinomas. Conversely, no significant difference was found between lobular carcinomas and in ductal carcinomas considering the frequency distribution of PgR-positive cases, cyclin-dependent kinase inhibitors acting at the G1/S boundary, bcl-2 and HIF-1alpha protein expression. Although both generally defined as hormone responsive, in ductal and lobular carcinomas are also characterised by biologic patterns in which proteins related to hormone responsiveness, cell-cycle control, apoptosis and angiogenesis were differently associated. This finding suggests the need to refine breast cancer characterisation in order to provide detailed information about individual tumours, or subsets of tumours, that will help in defining optimal treatment approaches.

The antiproliferative effects of Uncaria tomentosa extracts and fractions on the growth of breast cancer cell line.

Uncaria tomentosa, also known as "Uña de gato", is a Rubiaceae species widely used in South-American folk medicine for the treatment of cancer, arthritis, gastritis and epidemic diseases. Extracts of the plant have been shown to possess cytostatic and anti-inflammatory activity as well as mutagenic and antimutagenic properties. However, to date no studies have been carried out to verify the direct antitumor activity of the extracts. The present study investigates the effects of some extracts and their chromatographic fractions from the bark of U. tomentosa on the growth of a human breast cancer cell line (MCF7). Our data indicated that, in addition to the antimutagenic activity, U. tomentosa extracts and fractions exert a direct antiproliferative activity on MCF7. The bioassay-directed fractionation from barks and leaves resulted in the isolation of two active fractions, which displayed an IC50 of 10 mg/ml and 20 mg/ml, respectively and an antiproliferative effect, with about 90% of inhibition at a concentration of 100 mg/ml.

Contribution of vascular endothelial growth factor to the Nottingham prognostic index in node-negative breast cancer.

The prognostic contribution of intratumour VEGF, the most important factor in tumour-induced angiogenesis, to NPI was evaluated by using flexible modelling in a series of 226 N-primary breast cancer patients in which steroid receptors and cell proliferation were also accounted for. VEGF provided an additional prognostic contribution to NPI mainly within ER-poor tumours.

The effect of formulation and concentration of cholesteryl butyrate solid lipid nanospheres (SLN) on NIH-H460 cell proliferation.

Experimental factorial design was used to evaluate the influence of two factors involved in producing cholesteryl butyrate (chol-but) solid lipid nanospheres (SLN), microemulsion formulation and microemulsion/water ratio, on the effect of the SLN on the proliferation of NIH-H460, a non-small-cell lung carcinoma; six experimental settings were tested. The cells were treated with scalar concentrations of cholesteryl butyrate (from 0.008 to 1.000 mM) for each experimental condition; NIH-H460 cell growth was inhibited in all cases. The best experimental setting provided complete inhibition at 0.125 mM chol-but, while at the same concentration sodium butyrate provided only 38% inhibition.

Modulation of cell cycle-related protein expression by sodium butyrate in human non-small cell lung cancer cell lines.

To elucidate the mechanism of action of sodium butyrate (NaB), we examined its effect on the expression of some cell cycle-related proteins (cyclins D1 and E, p16(ink4), p21(waf1), p27(kip1)) in 2 human non-small cell lung cancer cell lines (NCI-460 and NCI-H23) characterized by wild- type and mutant TP53, respectively. The growth of both cell lines was inhibited in a dose-dependent manner and this process was accompanied by a modulation of cell cycle-related proteins. In NCI-H460, the p27(kip1) and p16(ink4) protein levels were markedly increased following NaB treatment, whereas p21(waf1) was only slightly elevated, with a peak at 2 mM NaB, and p53 was unaffected by any concentration. By contrast, in NCI-H23, a marked increase in p21(waf1) protein was paralleled by decreased p53 levels, whereas all the other investigated proteins remained stable. The results suggest that NaB blocks the growth of both cell lines by induction of cyclin-dependent kinase inhibitors (in particular, p21(waf1) in NCI-H23 and p27(kip1) and p16(ink4) in NCI-H460) through a p53-dependent or p53-independent mechanism, and open up interesting perspectives for the use of NaB as an alternative or additional strategy in the treatment of non-small cell lung carcinoma.

Sodium butyrate modulates cell cycle-related proteins in HT29 human colonic adenocarcinoma cells.

Sodium butyrate (NaB), a product of colonic fermentation of dietary fibre, has been shown to inhibit cell proliferation by blocking cells in the G0/G1 phase of the cell cycle through a mechanism of action still not completely understood. We investigated the effect of NaB on the level of some G1 phase-related proteins in a colon carcinoma cell line (HT29). In particular, we addressed our attention to cyclin D1 (the key regulator of G1S progression), p21waf1/cip1 (the main inactivator of the cyclin D/cdk complex), and p53 (the most important regulator of p21waf1/cip1 gene transcription). At inhibitory concentrations (higher than 1 mM) NaB reduced cyclin D1 and p53 level in a dose-dependent manner and sustained the synthesis of p21waf1/cip1, probably in a p53-independent way, accounting for the G0/G1 block observed by flow cytometry. Present results provide further evidence on the molecular mechanism at the basis of the physiological role of NaB and support the hypothesis that an unbalanced diet, poor in carbohydrates and therefore in NaB, could result in functional alterations with clinical and carcinogenic implications.

Time-dependent relevance of steroid receptors in breast cancer.

PURPOSE: To analyze the time-dependent prognostic role of the investigated variables, considered, when appropriate, on a continuous scale, for the purpose of evaluating and describing the interrelationships between clinically relevant patient and tumor characteristics (age, size and histology, and estrogen receptor [ER] and progesterone receptor content) and the risk of new disease manifestation. PATIENTS AND METHODS: We applied a flexible statistical model to a case series of 1,793 patients with axillary lymph node-negative breast cancer with a minimal potential follow-up of 10 years. To avoid a potential confounding effect of adjuvant treatment, only patients given local-regional therapy until relapse were considered. RESULTS: ER content and tumor size (adjusted for all the other covariates) showed a time-dependent relationship with the risk of new disease manifestations. In particular, ER content failed to show a prognostic effect within the first years of follow-up; thereafter, a positive association with risk of relapse was observed. For tumor size, within the first years of follow-up, the risk of relapse was directly related to size for only tumors up to 2.5 cm in diameter; thereafter, the impact on prognosis progressively decreased. CONCLUSION: The availability of a long follow-up on a large breast cancer series, as well as the use of innovative statistical approaches, allowed us to explore the functional relation between steroid receptors and clinical outcome and to generate a hypothesis on the involvement of ER in favoring long-term metastasis development.

Biological markers as indicators of response to primary and adjuvant chemotherapy in breast cancer.

Interest in translational studies on breast cancer is presently devoted to identify biological predictors of treatment response. In patients with operable breast cancer, subjected to primary and adjuvant chemotherapy, we analyzed the predictivity on objective clinical response and relapse-free survival of biological markers related to different cellular aspects and functions. Tumour proliferative rate (evaluated as the (3)H-thymidine-labelling index, TLI), oestrogen and progesterone receptors (ER and PgR, evaluated by the dextran-coated-charcoal method), nuclear DNA ploidy and the immunocytochemical expression of p53, bcl-2 and bax proteins were determined before primary treatment, at the time of diagnosis, and after primary chemotherapy, at surgery. Objective clinical response was significantly related only to pre-treatment p53 expression or PgR status, with a higher rate for tumours not expressing than for those expressing p53 (94% vs. 72%), as well as for PgR-negative (PgR(-)) than for PgR-positive (PgR(+)) tumours (86% vs. 68%). In the overall series, 8-year clinical outcome was significantly related only to post-treatment steroid receptors. In particular, higher 8-year relapse-free survival rate was observed for patients with ER(-) or PgR(-) than for those with ER(+) (64% vs. 38%) or PgR(+) (53% vs. 37%) tumours. Such findings held true even within the sub-set of patients who received adjuvant post-operative chemotherapy, i.e., those with node-positive (N(+)) or ER(-)/node-negative (N(-)) tumours, among whom also rapid proliferation or the presence of apoptosis-favouring markers (bcl-2(-) or bax(+), singly and in association) on surgical specimens identified a sub-set of women who benefited from systemic treatment. The different biological markers were variously indicative of clinical outcome, with a predictivity on tumour shrinkage for p53 and PgR, detected before primary chemotherapy, and on long-term follow-up for ER, PgR and, to a lesser extent, TLI and apoptosis-modulating markers. Int. J. Cancer (Pred. Oncol.) 84:580-586, 1999.

Relationship between steroid receptors (as continuous variables) and response to adjuvant treatments in postmenopausal women with node-positive breast cancer.

In current clinical practice for breast cancer patients, estrogen (ER) and progesterone receptor (PgR) concentrations, quantified by the dextran-coated charcoal assay, are categorized by an arbitrary cutoff into a negative or positive status. However, although the results obtained with this approach are easy to interpret, such a representation could oversimplify the relationship between ER and PgR content and patient outcome and imply an assumption of monotonicity, which is generally expected but rarely proven. We evaluated the relationship between ER and PgR content (considered on a continuous scale) and clinical outcome, using a flexible statistical model, in a group of postmenopausal patients with N-positive operable tumors who were submitted to surgery and different adjuvant treatments (tamoxifen or CMF). Univariate analysis indicated that in the tamoxifen-treated group, ER level, number of metastatic nodes (pN) and age, but not PgR, were significant indicators of clinical outcome (p = 0.032, p = 0.021 and p = 0.029, respectively). Multivariate analysis indicated that in this group of patients there was no interaction between variables, and in the final model for disease-free survival (DFS) only ER and pN were retained with an overall predictive ability of the regression model of 0.723, as evaluated by Harrell's c. However, pN markedly contributed to the predictive ability of the model with respect to ER, since a marked decrease in Harrell's c statistic (c = 0.582) was observed when pN was removed from the model. In the CMF-treated group, only pN affected clinical outcome. When the estimated DFS curves obtained from the final Cox regression models were plotted according to four values of ER (in the tamoxifen-treated group) or three values of pN (in the CMF-treated group) we observed that in the tamoxifen-treated group patients with an ER concentration equal to 0 fmol/mg cytosol protein had the worst prognosis, whereas a marked improvement of the expected DFS was observed for patients with a low but detectable ER level (generally classified as ER-negative because falling below the conventional cutoff value of 10 fmol/mg cytosol protein). Our results seem to suggest that the use of steroid receptor concentrations on a continuous scale, instead of dichotomous "status", is to be preferred in the choice of adequate therapeutic strategies.

Hyaluronic acid as drug delivery for sodium butyrate: improvement of the anti-proliferative activity on a breast-cancer cell line.

The potential clinical utility of sodium butyrate, a natural compound known to inhibit tumor-cell growth, is hampered by the difficulty of achieving effective in-vivo concentrations. The short half-life (about 5 minutes) of sodium butyrate results in rapid metabolism and excretion. To increase the availability of sodium butyrate over a longer period of time, we co-valently linked it to hyaluronic acid (a component of the extracellular matrix). Its major advantages as a drug carrier consist in its high biocompatibility and its ability to bind CD44, a specific membrane receptor frequently over-expressed on the tumor-cell surface. The degree of substitution of hyaluronic acid with butyrate residues ranged from d.s.=0.10 to d.s.=2.24 (1.8-28.4% w/w). The biological activity of hyaluronic-acid-butyric-ester derivatives was evaluated in terms of the inhibition of the growth of the MCF7 cell line and compared with that of sodium butyrate. After 6 days of treatment, we observed a progressive improvement of the anti-proliferative activity up to d.s.=0.20; thereafter, the anti-proliferative effect of the ester derivatives decreased. Fluorescence microscopy showed that after 2 hr of treatment fluorescein-labelled compounds appeared to be almost completely internalized into MCF7 cells, expressing CD44 standard and variant isoforms. These findings indicate that hyaluronic acid could offer an important advantage in drug delivery, in addition to its biocompatibility: the ability to bind to CD44, which are known to be frequently over-expressed on the tumor-cell surface.

Cholesteryl butyrate in solid lipid nanospheres as an alternative approach for butyric acid delivery.

BACKGROUND: Cholesteryl-butyrate chosen as lipid matrix of solid lipid nanospheres (SLNs) could be a suitable pro-drug to deliver butyric acid and overcome one of the most limiting disadvantages of the compound: the short half-life due to a rapid metabolism. METHODS: We evaluated the antiproliferative effect, with respect to that of sodium butyrate, of four SLNs (SLN1, SLN2, SLN3 and SLN4) characterized by a different concentration of cholesteryl-butyrate (range, 1.7-30 mM) on NIH-H460, a non-small cell lung carcinoma cell line. RESULTS: After 6 days of treatment, all SLN preparations induced a dose-dependent inhibition of NHI-H460 cell growth: the most effective SLN preparation (SLN1) was able to induce a complete growth inhibition already at 0.25 mM, a concentration at which sodium butyrate induced only a 55% inhibition. Fluorescence microscopy showed that 6-coumarin-tagged SLNs were almost completely internalized by cells after 5 min of treatment. CONCLUSIONS: The present results indicate that owing to their peculiar characteristics, SLNs could be an interesting alternative approach for butyric acid delivery into tumor cells.