Computational approaches for the design of peptides with anti-breast cancer properties

Background: Breast cancer is the most common cancer among women. Tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment, yet many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Therefore, scientists are searching for breast cancer drugs that have different molecular targets. Methodology: Recently, a computational approach was used to successfully design peptides that are new lead compounds against breast cancer. We used replica exchange molecular dynamics to predict the structure and dynamics of active peptides, leading to the discovery of smaller bioactive peptides. Conclusions: These analogs inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition. We outline the computational methods that were tried and used along with the experimental information that led to the successful completion of this research.

Negative regulation of NF-κB by the ING4 tumor suppressor in breast cancer

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.

Role of KCNMA1 in breast cancer

KCNMA1 encodes the α-subunit of the large conductance, voltage and Ca(2+)-activated (BK) potassium channel and has been reported as a target gene of genomic amplification at 10q22 in prostate cancer. To investigate the prevalence of the amplification in other human cancers, the copy number of KCNMA1 was analyzed by fluorescence-in-situ-hybridization (FISH) in 2,445 tumors across 118 different tumor types. Amplification of KCNMA1 was restricted to a small but distinct fraction of breast, ovarian and endometrial cancer with the highest prevalence in invasive ductal breast cancers and serous carcinoma of ovary and endometrium (3-7%). We performed an extensive analysis on breast cancer tissue microarrays (TMA) of 1,200 tumors linked to prognosis. KCNMA1 amplification was significantly associated with high tumor stage, high grade, high tumor cell proliferation, and poor prognosis. Immunofluorescence revealed moderate or strong KCNMA1 protein expression in 8 out of 9 human breast cancers and in the breast cancer cell line MFM223. KCNMA1-function in breast cancer cell lines was confirmed by whole-cell patch clamp recordings and proliferation assays, using siRNA-knockdown, BK channel activators such as 17ß-estradiol and the BK-channel blocker paxilline. Our findings revealed that enhanced expression of KCNMA1 correlates with and contributes to high proliferation rate and malignancy of breast cancer.

The multifaceted roles of Eph/ephrin signaling in breast cancer

http://www.ncbi.nlm.nih.gov/pubmed/22568950

Inhibition of the AKT/mTOR and erbB pathways by gefitinib, perifosine and analogs of gonadotropin-releasing hormone I and II to overcome tamoxifen resistance in breast cancer cells

Endocrine resistance in breast cancer remains a major clinical problem and is caused by crosstalk mechanisms of growth factor receptor cascades, such as the erbB and PI3K/AKT pathways. The possibilities a single breast cancer cell has to achieve resistance are manifold. We developed a model of 4-hydroxy-tamoxifen (OHT)‑resistant human breast cancer cell lines and compared their different expression patterns, activation of growth factor receptor pathways and compared cells by genomic hybridization (CGH). We also tested a panel of selective inhibitors of the erbB and AKT/mTOR pathways to overcome OHT resistance. OHT‑resistant MCF-7-TR and T47D-TR cells showed increased expression of HER2 and activation of AKT. T47D-TR cells showed EGFR expression and activated MAPK (ERK-1/2), whereas in resistant MCF-7-TR cells activated AKT was due to loss of CTMP expression. CGH analyses revealed remarkable aberrations in resistant sublines, which were predominantly depletions. Gefitinib inhibited erbB signalling and restored OHT sensitivity in T47D-TR cells. The AKT inhibitor perifosine restored OHT sensitivity in MCF-7-TR cells. All cell lines showed expression of receptors for gonadotropin-releasing hormone (GnRH) I and II, and analogs of GnRH-I/II restored OHT sensitivity in both resistant cell lines by inhibition of erbB and AKT signalling. In conclusion, mechanisms to escape endocrine treatment in breast cancer share similarities in expression profiling but are based on substantially different genetic aberrations. Evaluation of activated mediators of growth factor receptor cascades is helpful to predict response to specific inhibitors. Expression of GnRH-I/II receptors provides multi-targeting treatment strategies.

Multicycle dose-intensive chemotherapy for women with high-risk primary breast cancer: results of International Breast Cancer Study Group Trial 15-95

PURPOSE: To compare adjuvant dose-intensive epirubicin and cyclophosphamide chemotherapy administered with filgrastim and progenitor cell support (DI-EC) with standard-dose anthracycline-based chemotherapy (SD-CT) for patients with early-stage breast cancer and a high risk of relapse, defined as stage II disease with 10 or more positive axillary nodes; or an estrogen receptor-negative or stage III tumor with five or more positive axillary nodes. PATIENTS AND METHODS: Three hundred forty-four patients were randomized after surgery to receive seven cycles of SD-CT over 22 weeks, or three cycles of DI-EC (epirubicin 200 mg/m2 plus cyclophosphamide 4 gm/m2 with filgrastim and progenitor cell support) over 6 weeks. All patients were assigned tamoxifen at the completion of chemotherapy. The primary end point was disease-free survival (DFS). RESULTS: After a median follow-up of 5.8 years (range, 3 to 8.4 years), 188 DFS events had occurred (DI-EC, 86 events; SD-CT, 102 events). The 5-year DFS was 52% for DI-EC and 43% for SD-CT, with hazard ratio of DI-EC compared with SD-CT of 0.77 (95% CI, 0.58 to 1.02; P = .07). The 5-year overall survival was 70% for DI-EC and 61% for SD-CT, with a hazard ratio of 0.79 (95% CI, 0.56 to 1.11; P = .17). There were eight cases (5%) of anthracycline-induced cardiomyopathy (two fatal) among those who received DI-EC. Women with hormone receptor-positive tumors benefited significantly from DI-EC. CONCLUSION: There was a trend in favor of DI-EC with respect to disease-free survival. A larger trial or meta-analysis will be required to reveal the true effect of dose-intensive therapy.

Quality of life and quality-adjusted survival (Q-TWiST) in patients receiving dose-intensive or standard dose chemotherapy for high-risk primary breast cancer

Quality of life (QL) is an important consideration when comparing adjuvant therapies for early breast cancer, especially if they differ substantially in toxicity. We evaluated QL and Q-TWiST among patients randomised to adjuvant dose-intensive epirubicin and cyclophosphamide administered with filgrastim and progenitor cell support (DI-EC) or standard-dose anthracycline-based chemotherapy (SD-CT). We estimated the duration of chemotherapy toxicity (TOX), time without disease symptoms and toxicity (TWiST), and time following relapse (REL). Patients scored QL indicators. Mean durations for the three transition times were weighted with patient reported utilities to obtain mean Q-TWiST. Patients receiving DI-EC reported worse QL during TOX, especially treatment burden (month 3: P<0.01), but a faster recovery 3 months following chemotherapy than patients receiving SD-CT, for example, less coping effort (P<0.01). Average Q-TWiST was 1.8 months longer for patients receiving DI-EC (95% CI, -2.5 to 6.1). Q-TWiST favoured DI-EC for most values of utilities attached to TOX and REL. Despite greater initial toxicity, quality-adjusted survival was similar or better with dose-intensive treatment as compared to standard treatment. Thus, QL considerations should not be prohibitive if future intensive therapies show superior efficacy.

The role of TRPV6 in breast carcinogenesis

TRPV6 is an endothelial calcium entry channel that is strongly expressed in breast adenocarcinoma tissue. In this study, we further confirmed this observation by analysis of breast cancer tissues, which indicated that TRPV6 mRNA expression was up-regulated between 2-fold and 15-fold compared with the average in normal breast tissue. Whereas TRPV6 is expressed in the cancer tissue, its role as a calcium channel in breast carcinogenesis is poorly understood. Therefore, we investigated how TRPV6 affects the viability, apoptosis, and calcium transport in the breast cancer cell line T47D. Hormones can also affect the tumor development; hence, we determined the effects of estradiol, progesterone, and 1,25-vitamin D on TRPV6 transcription. Interestingly, the estrogen receptor antagonist tamoxifen reduced expression of TRPV6 and is able to inhibit its calcium transport activity (IC(50), 7.5 micromol/L). The in vitro model showed that TRPV6 can be regulated by estrogen, progesterone, tamoxifen, and 1,25-vitamin D and has a large influence on breast cancer cell proliferation. Moreover, the effect of tamoxifen on cell viability was enhanced when TRPV6 expression was silenced with small interfering RNA. TRPV6 may be a novel target for the development of calcium channel inhibitors to treat breast adenocarcinoma expressing TRPV6.

Inhibition of ErbB2 by receptor tyrosine kinase inhibitors causes myofibrillar structural damage without cell death in adult rat cardiomyocytes

Inhibition of ErbB2 (HER2) with monoclonal antibodies, an effective therapy in some forms of breast cancer, is associated with cardiotoxicity, the pathophysiology of which is poorly understood. Recent data suggest, that dual inhibition of ErbB1 (EGFR) and ErbB2 signaling is more efficient in cancer therapy, however, cardiac safety of this therapeutic approach is unknown. We therefore tested an ErbB1-(CGP059326) and an ErbB1/ErbB2-(PKI166) tyrosine kinase inhibitor in an in-vitro system of adult rat ventricular cardiomyocytes and assessed their effects on 1. cell viability, 2. myofibrillar structure, 3. contractile function, and 4. MAPK- and Akt-signaling alone or in combination with Doxorubicin. Neither CGP nor PKI induced cardiomyocyte necrosis or apoptosis. PKI but not CGP caused myofibrillar structural damage that was additive to that induced by Doxorubicin at clinically relevant doses. These changes were associated with an inhibition of excitation-contraction coupling. PKI but not CGP decreased p-Erk1/2, suggesting a role for this MAP-kinase signaling pathway in the maintenance of myofibrils. These data indicate that the ErbB2 signaling pathway is critical for the maintenance of myofibrillar structure and function. Clinical studies using ErbB2-targeted inhibitors for the treatment of cancer should be designed to include careful monitoring for cardiac dysfunction.

The effect of endocrine responsiveness on high-risk breast cancer treated with dose-intensive chemotherapy: results of International Breast Cancer Study Group Trial 15-95 after prolonged follow-up

BACKGROUND: The role of adjuvant dose-intensive chemotherapy and its efficacy according to baseline features has not yet been established. PATIENTS AND METHODS: Three hundred and forty-four patients were randomized to receive seven courses of standard-dose chemotherapy (SD-CT) or three cycles of dose-intensive epirubicin and cyclophosphamide (epirubicin 200 mg/m(2) plus cyclophosphamide 4 mg/m(2) with filgrastim and progenitor cell support). All patients were assigned tamoxifen at the completion of chemotherapy. The primary end point was disease-free survival (DFS). This paper updates the results and explores patterns of recurrence according to predicting baseline features. RESULTS: At 8.3-years median follow-up, patients assigned DI-EC had a significantly better DFS compared with those assigned SD-CT [8-year DFS percent 47% and 37%, respectively, hazard ratio (HR) 0.76; 95% confidence interval 0.58-1.00; P = 0.05]. Only patients with estrogen receptor (ER)-positive disease benefited from the DI-EC (HR 0.61; 95% confidence interval 0.39, 0.95; P = 0.03). CONCLUSIONS: After prolonged follow-up, DI-EC significantly improved DFS, but the effect was observed only in patients with ER-positive disease, leading to the hypothesis that efficacy of DI-EC may relate to its endocrine effects. Further studies designed to confirm the importance of endocrine responsiveness in patients treated with dose-intensive chemotherapy are encouraged.

Switching of vascular phenotypes within a murine breast cancer model induced by angiopoietin-2

Sustained growth of solid tumours can rely on both the formation of new and the co-option of existing blood vessels. Current models suggest that binding of angiopoietin-2 (Ang-2) to its endothelial Tie2 receptor prevents receptor phosphorylation, destabilizes blood vessels, and promotes vascular permeability. In contrast, binding of angiopoietin-1 (Ang-1) induces Tie2 receptor activation and supports the formation of mature blood vessels covered by pericytes. Despite the intense research to decipher the role of angiopoietins during physiological neovascularization and tumour angiogenesis, a mechanistic understanding of angiopoietin function on vascular integrity and remodelling is still incomplete. We therefore assessed the vascular morphology of two mouse mammary carcinoma xenotransplants (M6378 and M6363) which differ in their natural angiopoietin expression. M6378 displayed Ang-1 in tumour cells but no Ang-2 in tumour endothelial cells in vivo. In contrast, M6363 tumours expressed Ang-2 in the tumour vasculature, whereas no Ang-1 expression was present in tumour cells. We stably transfected M6378 mouse mammary carcinoma cells with human Ang-1 or Ang-2 and investigated the consequences on the host vasculature, including ultrastructural morphology. Interestingly, M6378/Ang-2 and M6363 tumours displayed a similar vascular morphology, with intratumoural haemorrhage and non-functional and abnormal blood vessels. Pericyte loss was prominent in these tumours and was accompanied by increased endothelial cell apoptosis. Thus, overexpression of Ang-2 converted the vascular phenotype of M6378 tumours into a phenotype similar to M6363 tumours. Our results support the hypothesis that Ang-1/Tie2 signalling is essential for vessel stabilization and endothelial cell/pericyte interaction, and suggest that Ang-2 is able to induce a switch of vascular phenotypes within tumours.

Cathepsin D specifically cleaves the chemokines macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, and SLC that are expressed in human breast cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

Does subjective burden of early breast cancer and its treatment affect immune measures during adjuvant therapy?

Psychosocial factors have been described as affecting cellular immune measures in healthy subjects. In patients with early breast cancer we explored bi-directional psycho-immune effects to determine whether subjective burden has an impact on immune measures, and vice versa. Patients (n = 239) operated for early breast cancer and randomized into International Breast Cancer Study Group (IBCSG) adjuvant clinical trials were assessed immediately before the beginning of adjuvant treatment (baseline) and 3 and 6 months thereafter, at the beginning of the corresponding treatment cycle. Cellular immune measures (leukocytes, lymphocytes, lymphocyte subset counts), markers of activation of the cellular immune system (beta2-microglobulin, soluble interleukin-2 receptor serum levels), and self-report subjective burden (global indicators of physical well-being, mood, coping effort) were assessed concurrently. The relationship between subjective burden and gradients of immune measures was investigated with regression analyses controlling for adjuvant treatment. There was a pattern of small negative associations between all variables assessing subjective burden before the beginning of adjuvant therapy with the gradients of the markers of activation of the cellular immune system and NK cell counts. In particular, better mood predicted a decline in the course of beta2-microglobulin and IL-2r at months 3 and 6. The gradient of beta2-microglobulin was associated with mood and coping effort at month 3. However, the effect sizes were very small. In conclusion, in this explorative investigation, there was an indication for subjective burden affecting and being affected by markers of activation of the cellular immune system during the first 3 and 6 months of adjuvant therapy. The question of clinical significance remains unanswered. These associations have to be investigated with refined assessment tools and schedules.

AMR-Me inhibits PI3K/Akt signaling in hormone-dependent MCF-7 breast cancer cells and inactivates NF-κB in hormone-independent MDA-MB-231 cells

AMR-Me, a C-28 methylester derivative of triterpenoid compound Amooranin isolated from Amoora rohituka stem bark and the plant has been reported to possess multitude of medicinal properties. Our previous studies have shown that AMR-Me can induce apoptosis through mitochondrial apoptotic and MAPK signaling pathways by regulating the expression of apoptosis related genes in human breast cancer MCF-7 cells. However, the molecular mechanism of AMR-Me induced apoptotic cell death remains unclear. Our results showed that AMR-Me dose-dependently inhibited the proliferation of MCF-7 and MDA-MB-231 cells under serum-free conditions supplemented with 1 nM estrogen (E2) with an IC50 value of 0.15 µM, 0.45 µM, respectively. AMR-Me had minimal effects on human normal breast epithelial MCF-10A + ras and MCF-10A cells with IC50 value of 6 and 6.5 µM, respectively. AMR-Me downregulated PI3K p85, Akt1, and p-Akt in an ERα-independent manner in MCF-7 cells and no change in expression levels of PI3K p85 and Akt were observed in MDA-MB-231 cells treated under similar conditions. The PI3K inhibitor LY294002 suppressed Akt activation similar to AMR-Me and potentiated AMR-Me induced apoptosis in MCF-7 cells. EMSA revealed that AMR-Me inhibited nuclear factor-kappaB (NF-κB) DNA binding activity in MDA-MB-231 cells in a time-dependent manner and abrogated EGF induced NF-κB activation. From these studies we conclude that AMR-Me decreased ERα expression and effectively inhibited Akt phosphorylation in MCF-7 cells and inactivate constitutive nuclear NF-κB and its regulated proteins in MDA-MB-231 cells. Due to this multifactorial effect in hormone-dependent and independent breast cancer cells AMR-Me deserves attention for use in breast cancer prevention and therapy

Mitotic figure counts are significantly overestimated in resection specimens of invasive breast carcinomas

Several authors have demonstrated an increased number of mitotic figures in breast cancer resection specimen when compared with biopsy material. This has been ascribed to a sampling artifact where biopsies are (i) either too small to allow formal mitotic figure counting or (ii) not necessarily taken form the proliferating tumor periphery. Herein, we propose a different explanation for this phenomenon. Biopsy and resection material of 52 invasive ductal carcinomas was studied. We counted mitotic figures in 10 representative high power fields and quantified MIB-1 immunohistochemistry by visual estimation, counting and image analysis. We found that mitotic figures were elevated by more than three-fold on average in resection specimen over biopsy material from the same tumors (20±6 vs 6±2 mitoses per 10 high power fields, P=0.008), and that this resulted in a relative diminution of post-metaphase figures (anaphase/telophase), which made up 7% of all mitotic figures in biopsies but only 3% in resection specimen (P<0.005). At the same time, the percentages of MIB-1 immunostained tumor cells among total tumor cells were comparable in biopsy and resection material, irrespective of the mode of MIB-1 quantification. Finally, we found no association between the size of the biopsy material and the relative increase of mitotic figures in resection specimen. We propose that the increase in mitotic figures in resection specimen and the significant shift towards metaphase figures is not due to a sampling artifact, but reflects ongoing cell cycle activity in the resected tumor tissue due to fixation delay. The dwindling energy supply will eventually arrest tumor cells in metaphase, where they are readily identified by the diagnostic pathologist. Taken together, we suggest that the rapidly fixed biopsy material better represents true tumor biology and should be privileged as predictive marker of putative response to cytotoxic chemotherapy.